Handbook of Plant Breeding

Volume 10

Editor-in-chief
Jaime Prohens-Tomás
Universidad de Politecnica de Valencia Dep. Biotecnologia, Valencia, Spain
Fernando Nuez
Polytechnic University COMAV-UPV, Valencia, Spain
Marcelo J. Carena
Fargo, North Dakota, USA
The field of plant breeding covers a broad range of different species and categories
of plants. While there are many techniques and issues that are similar across these
species, many more are unique to each genus.
The Handbook of Plant Breeding is organized by major crop categories and includes
the most up-to-date molecular techniques being used. It will serve as a resource for
plant breeding laboratories in both the university and industrial setting.

More information about this series at http://www.springer.com/series/7290

Antonio M. De Ron
Editor

Grain Legumes

1  3
Editor
Antonio M. De Ron
Misión Biológica de Galicia (MBG)
Spanish National Research Council (CSIC)
Pontevedra
Spain

ISSN 2363-8478          ISSN 2363-8486 (electronic)

Handbook of Plant Breeding
ISBN 978-1-4939-2796-8    ISBN 978-1-4939-2797-5 (eBook)
DOI 10.1007/978-1-4939-2797-5

Library of Congress Control Number: 2015942916

Springer New York Heidelberg Dordrecht London

© Springer Science+Business Media New York 2015
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Preface

Legume species belong to the Fabaceae family and are characterized by their fruit,
usually called pods. Several species of this family were domesticated by humans,
such as soybean, beans, faba bean, pea, chickpea, lentil, peanut, lupine, pigeon pea,
mung bean, peanut, or cowpea and many of them are of great relevance as human
food and animal feed. Food legumes are typically consumed as dry seeds, which
have high protein content, and in some cases as immature seeds or pods.
Members of the legume family, the Fabaceae or Leguminosae, fill critical niches
in most terrestrial biomes. This is one of the few plant families whose species are
capable of “fixing” nitrogen from the air, through association with specialized soil
bacteria, for use as a natural fertilizer, thus reducing fertilizer requirements. The
family has traditionally been divided into three subfamilies: Caesalpinioideae,
Mimosoideae and Papilionoideae, this latter subfamily contains most of the major
food and feed legumes.
Several grain legume crops are crucial elements of global agriculture and
nutrition, both as food and feed since they are major sources of plant protein.
Legumes contribute to the sustainable improvement of the environment when
grown in agricultural rotations due to their ability of biological nitrogen fixation
and their effects on the soil, and yield of the next crop, and the services given to
other components of agroecosystems such as pollinators. Legumes play a key role
in the diversification and sustainable intensification of agriculture, particularly in
light of new and urgent challenges such as climate change. The overall objective
is to increase the sustainability of the food and feed chain at all its steps, meet the
requirements of citizens for safe, healthy and affordable food via the nutritional
prevention of diet-related diseases and assure food quality and authenticity.
Reducing energy and water consumption and optimizing process control contribute
to making food processing and distribution more sustainable and the food sector
more competitive.
The demand for plant proteins for human nutrition has increased over the past
few decades in many countries due to: (i) demographic growth and urbanization,
(ii) the limited land areas which can be used for production of food crops while
farming systems are changing towards specialized cereal and oilseed production,
(iii) a decrease in animal protein production due to shortage of irrigation and/

v
vi Preface

or rainfall especially, and (iv) deliberate reduction in red meat consumption for
health reasons. Because of the high protein content of their seeds, grain legumes
are attractive candidates to overcome the deficiency in plant protein production.
However, in comparison to cereals, limited improvement in farming practices has
been achieved over the past few decades to enhance the production of important grain
legumes. A number of limiting factors affect legume yield, with water deficiency
in quantity or quality being among the key ones, to obtain more stable and more
reliable production. Even though these constraints have become structural in many
agrosystems, very limited research and development efforts have been devoted to
strategies to improve grain legume production under stress conditions to contribute
to the development of sustainable agriculture worldwide.
Further, the decrease in legume cropping is linked to a heavier use of chemical
fertilizers, pesticides and herbicides than in the past and an overall simplification
of agricultural systems. This has reduced the level of above-and below-ground
biodiversity in terms of macro- and microorganisms living in the agroecosystem
and has caused an increased pollution of the environment, impairing the beneficial
effects biodiversity has on crop productivity and the maintenance of agroecosystem
services for future generations. In addition, the decrease in legume cropping in
some agricultural areas urgently needs to be reversed as nitrogen fertilizers costs
are increasing with rising energy costs, leading to high production costs for farmers,
and substantial greenhouse gas emissions linked to the use of nitrogen fertilizers.
Also social and scientific issues should be considered. Interest in legumes has
been decreasing among many farmers, breeders, processing sector entrepreneurs and
scientists. Most worrying is the fact that knowledge on grain legumes with regard
to growing legumes in rotations, appropriate harvesting, storage and preparation
of the seed for further reproduction or processing have progressively been lost.
In addition, the use of legumes in human diet is decreasing in many developed
countries and knowledge on how to use legumes in food preparations is being
lost, despite continued calls by the medical professions to include a wider range
of plant proteins in the diet. To reverse these current trends, actions must be taken,
to promote wider use of legumes in crop production that will enable significant
benefits in economic, environmental and climate change spheres.
Approaches aimed at the improvement and exploitation of legume nutritional
and technological qualities are needed and can be expected to drive consumers and
farmers towards new, diverse, healthier and more sustainable choices. To contribute
to the development of sustainable agriculture, special attention has to be paid to the
factors limiting legume yield to obtain more consistent production and to fill the
knowledge and development gap on strategies to improve grain legume production
under stress conditions.
The decrease in manufacture of inorganic N fertilizers will result in reducing
the emission of greenhouse gas. Nitrous oxide (N2O) is produced naturally in the
soil during the microbial processes of nitrification and denitrification; considered
over a 100-year period, N2O is a greenhouse gas with tremendous global warming
potential (GWP) when compared to carbon dioxide (CO2) since it has 310 times
the ability per molecule of that gas to trap heat in the atmosphere. The decline of
Preface vii

soil fertility with loss of organic matter, the excessive use of chemical fertilizers,
the inappropriate use of the scarce water resources and the increase in soil acidity
and salinity, particularly in dry regions, all pose real threats to economic, social and
environmental sustainability. Agricultural systems involving legumes represent a
cheaper and more sustainable alternative to conventional practices by symbiotically
capturing atmospheric N2, thus reducing the use of industrially produced nitrogen
in the production of field crops. Improved N management is needed not only to
optimize economic returns to farmers but also to minimize environmental concerns
associated with N use, namely leaching problems and water pollution.
Intercropping or crop rotation including legumes is a promising strategy for more
sustainable crop production in many agricultural systems through the N transfer
and N release from legume residue. In crop rotation, legume crops can be used in
between of cereals or other cash crops (e.g., vegetables). The final contribution of
fixed N2 to the soil depends upon the legume species N balance, environmental
conditions and agricultural practices.
Globally, grain legumes are the most relevant source of plant protein, especially
in many countries in Asia, Africa, and Latin America, but there are some constraints
in their production, such as poor adaptation, pests and diseases, and unstable yield.
Current research trends in legumes are focused on new methodologies involving
genetic and -omic studies, as well as new approaches to the genetic improvement of
these species, including the relationships with their symbiotic rhizobia.
The book on grain legumes includes two parts. The first one consists of eight
crop-specific chapters devoted to the most produced and consumed worldwide grain
legume crops covering the whole range of topics related to breeding: origin and
evolution, genetic resources, breeding achievements, specific goals and techniques,
including the potential and actual integration of new technologies. The second part
includes five cross chapters covering topics that relate to the different crops of the
general chapters. All the chapters have been written by outstanding breeders and
scientists with wide experience in their crops and topics. This handbook contains
all the basic and updated information on the state of the art of breeding grain
legumes. The vast amount of knowledge collected in this volume should not only
serve breeders but also researchers, students and academicians. It may be regarded
as a scientific knowledge platform that provides practical plant breeders with new
scientific information, but also to make molecular biologists more familiar with the
peculiarities of breeding of the main grain legume species.

Pontevedra, Spain Antonio M. De Ron

Acknowledgement

The editor acknowledges the excellent contributions of all the authors, as well as the
support by the Springer Editorial team: Kenneth K. Teng, Hanna Smith, Elizabeth
Orthmann and Megha Koirala. Special thanks to the Editors-in-Chief of this series
Handbook of Plant Breeding, Jaime Prohens, Fernando Nuez and Marcelo Carena,
for giving me the great opportunity to edit this Handbook on Grain Legumes.

ix
Contents

1  Common Bean������������������������������������������������������������������������������������������   1

Antonio M. De Ron, Roberto Papa, Elena Bitocchi, Ana M. González,
Daniel G. Debouck, Mark A. Brick, Deidré Fourie, Frédéric Marsolais,
James Beaver, Valérie Geffroy, Phillip McClean, Marta Santalla,
Rafael Lozano, Fernando Juan Yuste-Lisbona and Pedro A. Casquero

2 Pea�������������������������������������������������������������������������������������������������������������   37
Thomas D. Warkentin, Petr Smýkal, Clarice J. Coyne, Norman Weeden,
Claire Domoney, Deng-Jin Bing, Antonio Leonforte, Zong Xuxiao,
Girish Prasad Dixit, Lech Boros, Kevin E. McPhee, Rebecca J. McGee,
Judith Burstin and Thomas Henry Noel Ellis

3 Chickpea���������������������������������������������������������������������������������������������������   85
Teresa Millán, Eva Madrid, José I. Cubero, Moez Amri,
Patricia Castro and Josefa Rubio

4 Lentil��������������������������������������������������������������������������������������������������������   111
Thomas R. Stefaniak and Kevin E. McPhee

5  Faba Bean�������������������������������������������������������������������������������������������������  141

Gérard Duc, Jelena M. Aleksić, Pascal Marget, Aleksandar Mikić,
Jeffrey Paull, Robert J. Redden, Olaf Sass, Frederick L. Stoddard,
Albert Vandenberg, Margarita Vishnyakova and Ana M. Torres

6 Lupins�������������������������������������������������������������������������������������������������������  179
Wojciech Święcicki, Magdalena Kroc and Katarzyna Anna Kamel

7 Cowpea������������������������������������������������������������������������������������������������������  219
Ousmane Boukar, Christian A. Fatokun, Philip A. Roberts,
Michael Abberton, Bao Lam Huynh, Timothy J. Close,
Stephen Kyei-Boahen, Thomas J.V. Higgins and Jeffrey D. Ehlers

xi
xii Contents

8  Grass Pea��������������������������������������������������������������������������������������������������  251

Nuno Felipe Almeida, Diego Rubiales and Maria Carlota Vaz Patto

9  The Legume–Rhizobia Symbiosis�����������������������������������������������������������  267

Jean-Jacques Drevon, Nora Alkama, Adnane Bargaz, A. Paula Rodiño,
Kiriya Sungthongwises and Mainassara Zaman-Allah

10  Nutritional Value��������������������������������������������������������������������������������������  291

Francesca Sparvoli, Roberto Bollini and Eleonora Cominelli

11  Seed Physiology and Germination of Grain Legumes��������������������������  327

Jaime Kigel, Leah Rosental and Aaron Fait

12  Reproductive Biology of Grain Legumes�����������������������������������������������  365

María José Suso, Penelope J. Bebeli and Reid G. Palmer

13  Grain Legume Cropping Systems in Temperate Climates�������������������  401

Thomas F. Döring

Index����������������������������������������������������������������������������������������������������������������  435
Contributors

Michael Abberton  International Institute of Tropical Agriculture, Ibadan, Nigeria

Jelena M. Aleksić  Laboratory for Plant Molecular Biology, Institute of Molecular
Genetics and Genetic Engineering (IMGGE), University of Belgrade, Belgrade,
Serbia
Nora Alkama  Department des Sciences Agronomiques, Universite de Mouloud
Mammeri, Tizi Ouzou, Algeria
Nuno Felipe Almeida  Instituto de Tecnologia Química e Biológica António Xavier
(ITQB), Oeiras, Portugal
Moez Amri  Regional Field Crop Research Center of Beja (CRRGC), Beja, Tunisia
Adnane Bargaz  Biosystems and Technology, Swedish University of Agricultural
Sciences, Alnarp, Sweden
James Beaver  Department of Crop and Agro-Environmental Science, University
of Puerto Rico, Mayaguez, PR, USA
Penelope J. Bebeli  Department of Crop Science, Laboratory of Plant Breeding and
Biometry, Agricultural University of Athens, Athens, Greece
Deng-Jin Bing  Agriculture and Agri-Food Canada, Lacombe, AB, Canada
Elena Bitocchi Dipartimento di Scienze Agrarie, Food and Environmental
Sciences, Università Politecnica delle Marche, Ancona, Italy
Roberto Bollini  CNR Institute of Agricultural Biology and Biotechnology, Milan,
Italy
Lech Boros Department of Seed Science and Technology, Institute of Plant
Breeding and Acclimatization—National Research Institute, Radzików, Poland
Ousmane Boukar International Institute of Tropical Agriculture, Cowpea
Breeding Unit, Kano, Kano, Nigeria

xiii
xiv Contributors

Mark A. Brick  Department of Soil and Crop Sciences, Colorado State University,
Fort Collins, CO, USA
Judith Burstin  INRA, UMR1347 Agroecology, Dijon, France
Pedro A. Casquero  Engineering and Agricultural Sciences, University of León,
León, Spain
Patricia Castro  Genetic Improvement of Fruits and Vegetables Lab, Beltsville,
MD, USA
Timothy J. Close Department of Botany and Plant Sciences, University of
California, Riverside, CA, USA
Eleonora Cominelli CNR Institute of Agricultural Biology and Biotechnology,
Milan, Italy
Clarice J. Coyne  USDA, Washington State University, Pullman, WA, USA
José I. Cubero  Departmento de Genetica, Universidad de Córdoba, Córdoba, Spain
Antonio M. De Ron  Biology of Agrosystems, Misión Biológica de Galicia (MBG),
Spanish National Research Council (CSIC), Pontevedra, Spain
Daniel G. Debouck  Genetic Resources Program, International Center for Tropical
Agriculture (CIAT), Cali, Valle del Cauca, Colombia
Girish Prasad Dixit  Indian Institute of Pulses Research, Kanpur, Uttar Pradesh,
India
Claire Domoney  Department of Metabolic Biology, John Innes Centre, Norwich
Research Park, Norwich, UK
Thomas F. Döring Department of Agronomy and Crop Science, Humboldt
University Berlin, Berlin, Germany
Jean-Jacques Drevon  Eco&Sols, INRA, Montpellier, France
Gérard Duc  UMR1347 Agroecologie, INRA, Dijon, France
Jeffrey D. Ehlers Department of Agricultural Development, Bill and Melinda
Gates Foundation, Seattle, WA, USA
Thomas Henry Noel Ellis CGIAR Research Program on Grain Legumes,
Patancheru, Telangana, India
Aaron Fait Blaustein Institutes for Desert Research, Ben Gurion University,
Midreshet Ben-Gurion, Israel
Christian A. Fatokun International Institute ot Tropical Agriculture (IITA),
Ibadan, Oyo State, Nigeria
Deidré Fourie  Plant Breeding, ARC-Grain Crops Institute, Potchefstroom, South
Africa
Contributors xv

Valérie Geffroy  Université Paris Sud, Orsay, France

Ana M. González  Biology of Agrosystems, Misión Biológica de Galicia (MBG),
Spanish National Research Council (CSIC), Pontevedra, Spain
Thomas J.V. Higgins  CSIRO Agriculture Flagship, Canberra, ACT, Australia
Bao Lam Huynh  Department of Nematology, University of California—Riverside,
Riverside, CA, USA
Katarzyna Anna Kamel  Department of Genomics, Institute of Plant Genetics,
Polish Academy of Sciences, Poznan, Poland
Jaime Kigel  Hebrew University of Jerusalem, Rehovot, Israel
Magdalena Kroc Department of Genomics, Institute of Plant Genetics, Polish
Academy of Sciences, Poznan, Poland
Stephen Kyei-Boahen International Institute of Tropical Agriculture (IITA),
Nampula, Mozambique
Antonio Leonforte  Nuseed Innovation Centre, Horsham, VC, Australia
Rafael Lozano  Department of Biology (Genetics), Research Center on Agricultural
and Food Biotechnology (BITAL), University of Almería, Almería, Spain
Eva Madrid  Institute for Sustainable Agriculture, CSIC, Córdoba, Spain
Pascal Marget  UMR1347 Agroecologie, INRA, Dijon, France
Frédéric Marsolais Agriculture and Agri-Food Canada, Genomics and
Biotechnology, Southern Crop Protection and Food Research Centre, London, ON,
Canada
Phillip McClean Department of Plant Science, North Dakota State University,
Fargo, ND, USA
Rebecca J. McGee  USDA-ARS, Grain Legume Genetics and Physiology Research
Unit, Washington State University, Pullman, WA, USA
Kevin E. McPhee  Department of Plant Sciences, North Dakota State University,
Fargo, ND, USA
Aleksandar Mikić  Forage Crops Department, Institute of Field and Vegetable
Crops, Novi Sad, Serbia
Teresa Millán  Department of Genetics, Campus de Rabanales, Córdoba, Spain
Reid G. Palmer Department of Agronomy, Iowa State University, Ames, IA,
USA
Roberto Papa  Dipartimento di Scienze Agrarie, Food and Environmental Sciences,
Università Politecnica delle Marche, Ancona, Italy
xvi Contributors

Jeffrey Paull  School of Agriculture, Food and Wine, The University of Adelaide,
Glen Osmond, SA, Australia
Robert J. Redden  Agriculture Productivity. Department of Economic
Development, Jobs, Transport & Resources, Australian Temperate Field Crops
Collection Horsham, VIC, Australia
Philip A. Roberts  Department of Nematology, University of California—Riverside,
Riverside, CA, USA
A. Paula Rodiño  Biology of Agrosystems, Misión Biológica de Galicia (MBG),
Spanish National Research Council (CSIC), Pontevedra, Spain
Leah Rosental French Associates Institute for Agriculture & Biotechnology of
Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of
the Negev, Beer Sheva, Israel
Diego Rubiales  Institute for Sustainable Agriculture, CSIC, Córdoba, Spain
Josefa Rubio Area de Mejora y Biotecnologia, IFAPA Centro “Alameda del
Obispo”, Córdoba, Spain
Marta Santalla Biology of Agrosystems, Misión Biológica de Galicia (MBG),
Spanish National Research Council (CSIC), Pontevedra, Spain
Olaf Sass Norddeutsche Pflanzenzucht Hans-Georg Lembke KG, Holtsee,
Germany
Petr Smýkal  Department of Botany, Palacky University in Olomouc, Olomouc,
Czech Republic
Francesca Sparvoli CNR Institute of Agricultural Biology and Biotechnology,
Milan, Italy
Thomas R. Stefaniak  North Central Research Extension Center, North Dakota
State University, Minot, ND, USA
Frederick L. Stoddard Department of Agricultural Sciences, University of
Helsinki, Helsinki, Finland
Kiriya Sungthongwises Faculty of Agriculture, Khon Kaen University, Khon
Kaen, Thailand
María José Suso Plant Breeding. Instituto de Agricultura Sostenible (CSIC),
Córdoba, Spain
Wojciech Święcicki  Department of Genomics, Institute of Plant Genetics, Polish
Academy of Sciences, Poznan, Poland
Ana M. Torres Area de Mejora y Biotecnología, IFAPA Centro Alameda del
Obispo, Córdoba, Spain
Albert Vandenberg Department of Plant Sciences/Crop Development Centre,
University of Saskatchewan, Saskatoon, SK, Canada
Contributors xvii

Maria Carlota Vaz Patto  Instituto de Tecnologia Química e Biológica António

Xavier (ITQB), Oeiras, Portugal
Margarita Vishnyakova  Genetic Resources of Grain Legumes, Vavilov Institute
of Plant Industry, Saint-Petersburg, Russian Federation
Thomas D. Warkentin  Crop Development Centre, University of Saskatchewan,
Saskatoon, SK, Canada
Norman Weeden  Department of Plant Sciences and Plant Pathology, Montana
State University, Bozeman, MT, USA
Zong Xuxiao Chinese Academy of Agricultural Sciences (CAAS), Institute of
Crop Science (ICS), Beijing, Beijing, China
Fernando Juan Yuste-Lisbona Department of Biology (Genetics), Research
Center on Agricultural and Food Biotechnology (BITAL), University of Almería,
Almería, Spain
Mainassara Zaman-Allah  CIMMYT, Southern Africa Regional Office, Harare,
Zimbabwe
About the Author

Antonio M. De Ron is Research Professor at the Misión Biológica de Galicia

(MBG), Spanish National Research Council (CSIC) in Pontevedra, Spain. He re-
ceived both his graduate and doctorate in Biology from the University of Santia-
go de Compostela (USC, Spain). Initially he worked as a postgraduate student in
plant protection at the National Institute for Agricultural Research (INIA, Spain).
In 1988, Prof De Ron gained a staff position as scientist at the MBG working on
legume genetics, germplasm and breeding and he is currently the leader of the Bi-
ology of Agrosystems Research Group. He was also part-time lecturer of genetics
and breeding at the USC. Currently he is also serving as Leader of the Protein
Crops Working Group of the European Association for Research on Plant Breeding
(EUCARPIA, The Netherlands). He has published many scientific articles, books
and monographs, as well as several educational publications. Prof De Ron has won
awards for his work and is well recognized in the international scientific community
for his achievements.

xix
Chapter 1
Common Bean

Antonio M. De Ron, Roberto Papa, Elena Bitocchi, Ana M. González,

Daniel G. Debouck, Mark A. Brick, Deidré Fourie, Frédéric Marsolais,
James Beaver, Valérie Geffroy, Phillip McClean, Marta Santalla,
Rafael Lozano, Fernando Juan Yuste-Lisbona and Pedro A. Casquero

1 Introduction

The common bean ( Phaseolus vulgaris L.) is a diploid annual species and is pre-
dominantly self-pollinating. Common bean consists of two major gene pools, Me-
soamerican and Andean, characterized by partial reproductive isolation, that include
wild populations and cultivated varieties. The common bean is the third most im-
portant food legume crop worldwide, surpassed only by soybean ( Glycine max (L.)
Merr.) and peanut ( Arachis hypogea L.). Among the main food crops, the common
bean shows the greatest variation in growth habit, seed characteristics (size, shape
and colour) and maturation time. This variability enables its production in a wide
range of cropping systems and environments as diverse as the Americas, Africa, the
Middle East, China and Europe (Blair et al. 2010). Despite being cultivated for its
fresh pods and grains, beans are produced and consumed mainly as dry grain.

A. M. De Ron () · A. M. González · M. Santalla

Biology of Agrosystems, Misión Biológica de Galicia (MBG), Spanish National Research
Council (CSIC), El Palacio-Salcedo, 36143 Pontevedra, Spain
e-mail: [email protected]
A. M. González
e-mail: [email protected]
M. Santalla
e-mail: [email protected]
R. Papa · E. Bitocchi
Dipartimento di Scienze Agrarie, Food and Environmental Sciences, Università Politecnica delle
Marche, Via Brecce Bianche, 60131 Ancona, Italy
e-mail: [email protected]
E. Bitocchi
e-mail: [email protected]
© Springer Science+Business Media New York 2015 1
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_1
2 A. M. De Ron et al.

The common bean has lately gained attention as a functional food due to its
health benefits and human disease prevention. In fact, its inclusion in diets is linked
to reduce risk of obesity, diabetes, cardiovascular diseases, and colon, prostate and
breast cancer (Correa 1981; Hangen and Bennink 2003; Thompson et al. 2009).
These health benefits may be attributed to its important fibre and starch content,
ability to regulate glycaemia and gastrointestinal function, as well as to its antioxi-
dant properties provided by the presence of phenolic compounds and proteins.

D. G. Debouck
Genetic Resources Program, International Center for Tropical Agriculture (CIAT), Km 17 recta a
Palmira, Cali, Valle del Cauca AA 6713, Colombia
e-mail: [email protected]
M. A. Brick
Department of Soil and Crop Sciences, Colorado State University, 307 University Avenue, Fort
Collins, CO 80532, USA
e-mail: [email protected]
D. Fourie
Plant Breeding, ARC-Grain Crops Institute, 114 Chris Hani Street, Private Bag X 1251,
Potchefstroom, South Africa
e-mail: [email protected]
F. Marsolais
Agriculture and Agri-Food Canada, Genomics and Biotechnology, Southern Crop Protection and
Food Research Centre, 1391 Sandford St., London, ON N5V 4T3, Canada
e-mail: [email protected]
J. Beaver
Department of Crop and Agro-Environmental Science, University of Puerto Rico, Call Box 9000,
Mayaguez, PR 00681-9000, USA
e-mail: [email protected]
V. Geffroy
Batiment 630, Université Paris Sud, Orsay, France
e-mail: [email protected]
P. McClean
Department of Plant Science, North Dakota State University, Fargo, ND, USA
e-mail: [email protected]
R. Lozano
Department of Biology (Genetics), Research Center on Agricultural and Food Biotechnology
(BITAL), University of Almería, Almería, Spain
e-mail: [email protected]
F. J. Yuste-Lisbona
Department of Biology (Genetics), Research Center on Agricultural and Food Biotechnology
(BITAL), University of Almería, Almería, Spain
e-mail: [email protected]
P. A. Casquero
Engineering and Agricultural Sciences, University of León, León, Spain
e-mail: [email protected]
1  Common Bean 3

For centuries, farmers have maintained their heirloom varieties and have
exchanged their seeds with surrounding areas, mainly in local markets. It is not
always easy to know the use given by farmers to their old landraces, and it must be
assumed that snap and dry beans have probably been selected under dissimilar crite-
ria and pressure. This results in a very different set of characteristics for size, shape,
tenderness and cooking quality of the edible parts of plant. Therefore, the traditional
varieties are a valuable source of well-adapted germplasm of common bean. The
current common bean germplasm collections show a wide variation of phenotypes,
although in many developed countries where landraces are being replaced by elite
cultivars the genetic erosion is affecting the species. Also the traditional intercrop-
ping with maize in many countries is into abeyance, and sole cropping of bean may
become unsustainable in some environments as the soil is eroded and the pressure
of pests and diseases builds up (Davis and Woolley 1993).
The current integration of genomic data into gene bank documentation systems
and its combination with genetic, taxonomic, agronomic, phenotypic and ecologi-
cal data will usher in a new era for the valorization of the common bean genetic
resources.

2  Origin and Systematics

2.1 Phylogeny

Most of the Phaseolus species are geographically distributed in Mesoamerica, and

for this reason the genus is considered to have originated in Mesoamerica (Freytag
and Debouck 2002; Delgado-Salinas et al. 2006) between 6 and 4 million years
(Ma) ago (Delgado-Salinas et al. 2006). This indicates that the Phaseolus genus
originated after the late Miocene (ca. 7 Ma ago, Coates et al. 2004) when the clo-
sure of the Isthmus of Panama allowed the connection of Mesoamerica and South
America through a land bridge. Eight well-supported crown clades characterize the
Phaseolus genus, with an average age of ca. 2 Ma, thus indicating that most of the
diversity came into existence after the formation of the actual geographical and
geological form of Mexico (ca. 5 Ma ago; Delgado-Salinas et al. 2006). Among the
eight Phaseolus clades, the vulgaris group is the oldest, at ~ 4 Ma. Along with P.
vulgaris, there are three other domesticated Phaseolus species that belong to this
group (P. dumosus, P. coccineus, P. acutifolius), with the most closely related spe-
cies to P. vulgaris being P. coccineus and P. dumosus. Gepts et al. (1999) suggested
that P. vulgaris diverged from P. dumosus and P. coccineus some 2 Ma ago, through
an analysis of the sequence data of the α-amylase inhibitor gene. The other domes-
ticated species, P. lunatus, is most distantly related to P. vulgaris (Delgado-Salinas
et al. 2006).
4 A. M. De Ron et al.

2.2 Origin

Wild forms of P. vulgaris occur from northern Mexico to northwestern Argentina,

and they are characterized by three distinct gene pools (Fig. 1.1): Mesoamerica, the
Andes and northern Peru–Ecuador (Debouck et al. 1993; Kami et al. 1995). The
Mesoamerican and Andean are the two main gene pools, where the geographical
structure is evident also for the domesticated forms, as it has been demonstrated

Fig. 1.1   Common bean gene pools

1  Common Bean 5

through studies based on P. vulgaris morphology, seed proteins, allozymes, multi-

locus molecular markers and nucleotide data (Bellucci et al. 2014a). The third gene
pool is constituted by wild populations that grow in a small geographical area on the
western slopes of the Andes, the distinctiveness of which is the specific phaseolin
(main seed-storage protein), type I (‘Inca’, Kami et al. 1995). This phaseolin type
has not been found in the other two gene pools.
Until recently, the most credited origin of the species was the northern Peru–Ec-
uador hypothesis, as suggested by Kami et al. (1995) who sequenced a portion of
the gene coding for phaseolin and reported that the phaseolin type I gene does not
have the tandem direct repeats that are, instead, characteristic of the Mesoamerican
and Andean phaseolin types. Considering that duplications, which generate tandem
directs repeats, are more likely to occur than deletions, which specifically eliminate
a member of a tandem direct repeat, Kami et al. (1995) suggested that P. vulgaris
originated from the wild populations of northern Peru and Ecuador and subsequent-
ly spread northwards (from Colombia to northern Mexico) and southwards (from
southern Peru to Argentina).
The alternative hypothesis describes a Mesoamerican origin for P. vulgaris. Bitoc-
chi et al. (2012) investigated the nucleotide diversity at five gene fragments across
a wide sample of wild P. vulgaris accessions that were representative of the entire
geographical distribution of the species. In particular, three main outcomes supported
a Mesoamerican origin of the common bean. (i) A strong reduction in the genetic
diversity (90 %) of the Andean compared to Mesoamerican wild forms, indicating the
occurrence of a bottleneck in the Andean gene pool that predates its domestication.
(ii) A clear population structure is highlighted in Mesoamerica, with four different ge-
netic groups (B1, B2, B3 and B4) that characterize the accessions from this gene pool.
The B1 group included accessions distributed across all the Mesoamerica, while the
other three groups were characterized by only Mexican accessions; in particular, the
B2 group spread from central to southern Mexico, and the B3 and B4 being character-
istic of a wide area of central Mexico. Such a population structure had not been iden-
tified before in previous studies, the main reason for which was probably related to
the nature of the markers used; indeed, compared with multilocus molecular markers,
sequence data are less prone to homoplasy (e.g. Wright et al. 2005; Morrell and Clegg
2007), and the assumption of no recombination is less likely to be violated, and thus,
these sequence data were very useful to address evolutionary issues (Bitocchi et al.
2012, 2013). (iii) There is no clear distinction between the Mesoamerican and Andean
wild gene pools, which was indicated by the phylogenetic relationships between the
four different Mesoamerican genetic groups with the South American gene pools.
Considering all of these data, Bitocchi et al. (2012) suggested the Mesoamerican
origin of P. vulgaris, with Mexico being the more likely cradle of diversity of this
species, where all of the four different genetic groups are present. Moreover, they
suggested that the wild common bean that grows in northern Peru and Ecuador rep-
resents a relict population that only includes a fraction of the genetic diversity of the
ancestral population, with phaseolin type I appearing to be extinct in Mesoamerica.
This result was recently confirmed by the resequencing of 60 wild P. vulgaris geno-
types (Schmutz et al. 2014).
6 A. M. De Ron et al.

2.3 Domestication

The domesticated forms of P. vulgaris have important traits that distinguish them
from the wild forms, such as reduced and loss of the dissemination mechanisms, loss
of seed dormancy and photoperiod insensitivity, greater seed size and determinate
growth habit. The main effect of domestication was a reduction in the genetic diver-
sity in the domesticated forms that was imposed by founder effect (i.e. genetic drift)
and selection at loci controlling domestication traits. This reduction has been clearly
identified in the Mesoamerican domesticated gene pool in several studies (Papa et al.
2005; Papa et al. 2007; Rossi et al. 2009; Kwak and Gepts 2009; Nanni et al. 2011;
Bitocchi et al. 2013). The same studies have shown that, in contrast, in the Andean
gene pool, the bottleneck of domestication was less evident; in particular, Bitocchi
et al. (2013) showed a reduction in the diversity that was threefold greater in Meso-
america as compared with the Andes.
Bellucci et al. (2014b) applied next-generation sequencing technology (RNA-
Seq) to investigate, not only at nucleotide but also at transcriptome level, the do-
mestication process in Mesoamerica. They analysed nucleotide polymorphism and
gene differential expression in wild and domesticated forms at 27,243 contigs, each
representing a putative single gene. Their results showed that domestication not
only led to a drastic reduction of diversity (~ 60 %) but also decreased diversity of
gene expression (~ 18 %). Another important outcome of this study was the detec-
tion of ~ 9 % of contigs being affected by selection during domestication (directly
targets of selection or physically linked to the selected genes). The findings indi-
cated that positive selection was the rule, even if, in a few cases, selection increased
the nucleotide diversity in the domesticated forms at target loci associated with
abiotic stress responses, flowering time and morphology.
A still open debate concerns the occurrence of single or multiple domestications
within the two main gene pools, with studies suggesting both single (Papa and Gepts
2003; Kwak and Gepts 2009; Kwak et al. 2009; Rossi et al. 2009) and multiple (Singh
et al. 1991a, b, c; Chacón et al. 2005) events. However, the most recent studies support
a single domestication, in both Mesoamerica and the Andes (Bitocchi et al. 2013).
Mamidi et al. (2011) analysed sequence data from 13 loci and dated the do-
mestication bottlenecks to ca. 8000 and ca. 7000 years before the present for the
Mesoamerican and Andean gene pools, respectively. In Mesoamerica, two different
domestication geographical areas have been suggested recently: Rio Lerma–Rio
Grande de Santiago basin in west-central Mexico (Kwak et al. 2009) and in Oaxaca
Valley (Bitocchi et al. 2013). Similarly, in the Andes, Chacón et al. (2005) indicated
central-southern Peru as the geographical area where P. vulgaris was domesticated;
in contrast, other studies have suggested Bolivia and northern Argentina (Beebe
et al. 2001; Bitocchi et al. 2013).

2.4 Diffusion and Evolution Out of the Americas

The diffusion of P. vulgaris out of the American domestication centres appears to

have been very complex and to have involved numerous introductions into different
1  Common Bean 7

continents and countries. Several of these have been proposed as secondary centres
of diversification, such as Europe (Santalla et al. 2002; Angioi et al. 2010; Gioia et al.
2013), central-eastern and southern Africa, Brazil and China (Bellucci et al. 2014a).
In particular, P. vulgaris from Europe is characterized by a higher frequency of the
Andean (ca. 70 %) as compared to Mesoamerican types (Gepts and Bliss 1988; Gil
and De Ron 1992; Logozzo et al. 2007; Angioi et al. 2010). In Brazil, Burle et al.
(2010) reported that the Mesoamerican types are fourfold more frequent than the
Andean. In Africa, there is an equal frequency of the two types (Gepts and Bliss
1988; Asfaw et al. 2009; Blair et al. 2010), while China shows a predominance of
the Mesoamerican types (Zhang et al. 2008).
Moreover, once out of the Americas, the spatial isolation between the Meso-
american and Andean gene pools was not maintained, which provided increased
potential for their hybridization and introgression. In Europe, this aspect is very
important for breeding; indeed, their hybridization has led to the recombination of
the Mesoamerican and Andean traits that has resulted in the production of novel and
useful genotypes and phenotypes (i.e. resistance to biotic and abiotic stress; Rodiño
et al. 2006; Angioi et al. 2010; Blair et al. 2010; Santalla et al. 2010). However, vari-
ous studies suggest that in other continents, the introgression between these gene
pools appears not to be as relevant as it has been in Europe.

3 Genetic Resources and Utilization

Somewhere in Central America during the Pliocene and for 4 Ma (Delgado-Salinas
et al. 2006), a group of legumes evolved in what is today the section Phaseoli of the
Phaseolus genus (Freytag and Debouck 2002). One of them, P. vulgaris L., migrat-
ed northwards and to the Andes and has survived as wild in montane forests to this
date. When humans crossing Beringia during the last Ice Age colonized the Ameri-
cas, they found common beans growing wild from Mexico down to Argentina. Ge-
netic studies with the help of molecular markers have shown these beans to be di-
verse though grouped in 2–3 pools (Tohme et al. 1996). For reasons possibly linked
to food shortages, about 8000 years ago (Mamidi et al. 2011), Amerindians started
planting beans, that is, initiated a domestication process. This happened indepen-
dently in western Mexico (Kwak et al. 2009) and in the central Andes (Chacón et al.
2005), possibly at the same time or slightly earlier in the Andes. Beans planted with
corn were a basic staple for all New World civilizations from the Carolinas (USA)
down to Jujuy (Argentina). In 1493, the Spanish galleons brought common beans to
the Old World where new processes of selection and recombination resumed. Not
surprisingly, new landraces and some recombinants occurred in these new lands of
adoption such as Spain, Italy, eastern Africa and China. If time correlates with the
piling up of genetic diversity, useful sources are clearly in the secondary gene pool
of Phaseoli and in the wild forms (Porch et al. 2013).
Bean breeding has often focused first on transfer of resistances to diseases and
pests because of the imperative to secure the ‘meat of the poor’ throughout Latin
America and Africa (where the highest consumption per capita is registered). Yield
per se, tolerance to drought, adaptation to low phosphorus soils and nutritional qual-
8 A. M. De Ron et al.

ity are priorities of bean breeders since the 1990s (Broughton et al. 2003). Although
not the entire germplasm has been collected nor evaluated, many interesting traits
have been disclosed in ex situ collections (Table 1.1) and have been used to get
yield gain close to 20 % over the past 50 years (Singh et al. 2007). While many
landraces were topping at 400 kg/ha, yields of 2900 kg/ha are no longer the excep-
tion. Growth habit from a vine liana has been ‘domesticated’ too, namely with the
selection of type II, for mechanical harvesting, and changing the original poor root
system is coming into the horizon, by using the secondary gene pool (Porch et al.
2013). Although current ex situ collections harbour diversity (Table 1.2, where the
top five gene banks have 34 % of total accessions worldwide), wild species and
secondary gene pools are not yet fully represented nor evaluated, an obvious and
timely priority.

Table 1.1   Some bean germplasm used to overcome limiting factors in bean production
Trait looked for Material useda
Abiotic stresses
Aluminium toxicity G35346 ( P. coccineus, from Oaxaca)
Drought Common red Mexican G11212; G21212 land-
race from Colombia
Low phosphorus G19227A; Chaucha Chuga G19833
Diseases
Angular leaf spot Interspecific hybrids with P. coccineus; Boliv-
ian G8719; Mexican G2726
Anthracnose Aliya G02333; Kaboon G1588; Cornell
49–242 G5694
Anthracnose Interspecific hybrids with P. coccineus
Ascochyta blight P. dumosus G35182 from Guatemala
BGYMV Royal Red G04450; coccineus G35172 from
Rwanda
BCMV Porillo Sintético G4495, Royal Red G04450
Beet curly top virus California Pink G06222, Red Mexican
G05507
Beet curly top virus Porillo Sintético G04495, Burtner, Tio Canela
75
Common bacterial blight Interspecific hybrids with P. acutifolius VAX4,
MBE7
Common bacterial blight Montana No. 5; PI 207262
Halo blight Montcalm G06416, ICA Tundama G14016
Halo blight Pinto US 14 G18105
Halo blight Wis HBR 72 G03954
Fusarium root rot Porillo Sintético G04495; wild P. vulgaris
G12947
Pythium root rot PI 311987 G02323
1  Common Bean 9

Table 1.1  (continued)

Trait looked for Material useda
Rhizoctonia solani N203 G00881
Rot
Rust Compuesto Negro Chimaltenango G05711
Rust Redlands Pioneer G05747
Rust PI 260418
Web blight BAT 93; Flor de Mayo G14241
White mould Interspecific hybrids with P. coccineus
G35172
White mould Interspecific hybrids with P. costaricensis
G40604
Pests
Acanthoscelides weevil Wild P. vulgaris from western Mexico
G12952, G2771
Apion godmani pod weevil Amarillo 154 G03982; G03578
Empoasca leafhoppers California dark red kidney, from USA G17638
Ophiomyia bean fly P. coccineus G35023 and G35075, and inter-
specific hybrids
Whiteflies Aleyrodidae DOR 303
Zabrotes weevil Wild P. vulgaris from Chiapas, Mexico
G24582
Nitrogen fixation
N2 fixation under low P Bituyano from Cajamarca, Peru, G19348
Yield
Favourable QTLs Wild P. vulgaris from Colombia G24423
Favourable QTLs Wild P. vulgaris from Colombia G24404
Nutritional traits
Seed protein quantity PI 229815
High zinc content Peruvian landrace G23823
High iron content Peruvian landrace G23823
Polyphenols Wild P. vulgaris from Mexico G11025
a
G numbers refer to the International Center for Tropical Agriculture (CIAT) genebank, while PI
numbers refer to the Western Regional Plant Introduction Station at Pullman, Washington, USA
BCMV bean common mosaic virus, BGYMV bean golden yellow mosaic virus, QTLs quantitative
trait loci

Table 1.2   Major germplasm collections of Phaseolus beans, and type of accessions. FAO (2010)
Gene bank Accessions (%) Landraces (%) Wild species (%)
CIAT, Colombia 35,891 (14) 30,507 (85) 2153 (6)
USDA, USA 14,674 (6) 9832 (67) 880 (6)
Embrapa, Brazil 14,460 (6) 5784 (40) –
INIFAP, Mexico 12,752 (5) 7014 (55) 2168 (17)
IPK, Germany 8680 (3) 5729 (66) 87 (1)
10 A. M. De Ron et al.

4 Varietal Groups: Market Classes

Bean consumers of different countries and regions show specific preferences for
various combinations of seed size, shape, colour, cooking time, broth appearance
and storability (De Ron et al. 2000). Therefore, a classification often used for
common bean is the one into commercial types, which is based predominantly on
characteristics of grain colour and size, and is related to market preferences. The
wide range of seed characteristics has been formalized in the bean world into dis-
tinct commercial or market classes. Among the bean varieties grown in the world,
62 dry bean market classes are recognized (Santalla et al. 2001; FAO 2002) ac-
cording to consumer preferences, production and market price (Fig. 1.2). Dry bean
market classes are produced under recommended agronomic practices and traded
according to the defined class attributes. Thus, classes must be segregated through-
out production and distribution.
Increased diversity of commercial market classes has been achieved to meet
market and consumer interests. Among the Durango beans, the most important mar-
ket classes are ‘great northern’ and ‘pinto’. The most abundant market classes that
represent race ‘Nueva Granada’ are ‘dark red kidney’, ‘white kidney’, ‘calima’ and
‘large cranberry’ beans. Regarding the race Mesoamerica, the most popular bean
market classes are ‘navy’, ‘small white’, ‘mulatinho’, ‘carioca’ and the classes of
small black seed. The Chilean market classes most accepted and consumed are ‘tór-
tola’ and ‘coscorrón’. In addition, other minor market classes, such as ‘manteca’,
‘sapito’ and ‘cuyano’, are also consumed in more specific areas. Race Peru is char-

Fig. 1.2   Common bean international market classes. a Favada Pinto (race Nueva Granada). b Red
Caparron (race Peru). c Hook (race Durango). d Small Yellow (race Mesoamerica)
1  Common Bean 11

acterized by large seeds which are often round or oval but can also be elongated. Its
most popular types are ‘yellow canario’ and ‘azufrado’ beans.
Market classes usually include improved germplasm and thus tend to show a low
level of variability. However, the range of commercially available bean cultivars
and varieties in different market classes is constantly changing. New cultivars are
released for their increased yield potential, pest and disease resistance, full-season
and early double-cropped growth potential and improved market quality. Public
and private plant breeders develop new varieties by adding desirable features to
old cultivars or create new and better cultivars by recombining the best traits from
available germplasm.
The polymorphism of common bean is so great that, in each region, and even in
each locality, different varieties with similar characteristics correspond to different
names. There are several ethnic varieties or ‘heirloom’ varieties, which are charac-
teristic of an area or region, and they can be designated with different names. These
landraces evolved from ancient types by conscious or unconscious selection and are
currently well adapted to the agroecological conditions under which they have been
grown for centuries. In Europe, the high appreciation by consumers of these ‘heir-
loom’ varieties is recognized by the attribution of the protected geographical indica-
tion (PGI), one of the European Union marks attributable to traditional foods. With
the increased interest in ‘heirloom’ varieties (seeds passed down from generation to
generation), many fine old-fashioned varieties have been reintroduced recently by
various seed companies.

5 Major Breeding Achievements and Specific Goals

in Current Breeding

5.1 Achievements in Dry Bean Breeding in the USA

Along with corn ( Zea mays L.) and squash ( Cucurbita spp.), dry bean was among
the earliest crops domesticated in the Americas (Kaplan 1956). Native Americans
commonly grew beans as a companion crop with corn and squash in what is termed
the ‘three sisters’ or milpa method that originated in Mesoamerica and spread
northward into Mexico and the southwestern USA. Some of the old landraces were
eventually selected and produced by the New World settlers for local consumption.
Beans were also introduced into Europe from the New World as early as AD 1500
by the early explorers (Zeven 1997). Subsequently, they were reintroduced into the
eastern USA by Europeans that migrated from Europe to the USA. The first large-
scale commercial production of dry edible beans in the USA occurred in Orleans
County, New York in 1839. New York became one of the first important producers
of dry beans and maintained its dominance until the early 1900s when Michigan
became the leading producer.
A significant change in dry bean production occurred in 1917, when seed pro-
duction began shifting from the central and the eastern USA to the semiarid west-
ern USA, where today most commercial bean seed is produced (Brick and Lowry
12 A. M. De Ron et al.

2000). This shift initially occurred because seed-borne pathogens, such as anthrac-
nose (ANT; Colletotrichum lindemuthianum (Sacc et Magn.) Scrib.) and common
bacterial blight ( Xanthomonas axonopodis pv. phaseoli (Smith) Dye), became seri-
ous problems in commercial production fields (Adams 1996). Idaho was among
the first states to produce large quantities of commercial dry bean seed and still
produces more certified bean seed than any other state.

5.2 Genetic Improvement

Several books have been published that address dry bean improvement, production
challenges and genetic resources in the USA and the Europe: Genetic Resources of
Phaseolus Beans by P. Gepts (ed) in 1988; Common Bean Production Problems in
the Tropics by H. F. Schwartz and T. Pastor-Corrales (eds) in 1989; Common Beans:
Common Beans: Research for Crop Improvement by A. van Schoonhoven and O.
Voysest (eds) in 1991; Phaseolus spp: Bean Science by R. Maiti (ed) in 1997; Com-
mon Bean Improvement in the Twenty-First Century by S. P. Singh (ed) in 1999; Cata-
logue of Bean Genetic Resources by J. M. Amurrio, M. Santalla and A. M. De Ron
(eds) in 2001; Handbook on Evaluation of Phaseolus Germplasm by C. De la Cuadra,
A. M. De Ron and R. Schachl (eds) in 2001; and Compendium of Bean Diseases (2nd
edn) by H. F. Schwartz, J. R. Steadman, R. Hall and R. L. Forster (eds) in 2005.
Early breeding efforts primarily focused on improved disease resistance and ad-
aptation to local environments, later efforts also focused on improved seed quality,
improved plant architecture and breeding for yield. Among the early bean research-
ers, R. A. Emerson, renowned for his research on maize genetics, worked on beans
at the University of Nebraska from 1898 until 1912. The Michigan Agricultural
College (currently Michigan State University) was among the first institutions in
the USA to employ a full-time dry bean breeder in 1906 followed by the University
of Idaho in 1925 (Singh et al. 2007). Michigan State University released the first
USA navy bean cultivar ‘Robust’ in 1915 as a selection from locally grown landra-
ces. In the early twentieth century, breeding programmes at Cornell University and
Michigan Agricultural College focused on disease resistance, primarily resistance
to ANT (Burkholder 1930) and common bacterial blight (Adams 1996). Additional
research in the western USA focused on developing resistance to a range of patho-
gens, including rust ( Uromyces appendiculatus Pers: Unger.), white mould (caused
by Sclerotinia sclerotiorum (Lib.) DeBary), bacterial blights, viruses, root patho-
gens and beet curly top virus (BCTV) transmitted by the beet leafhopper (Circulifer
tenellus (Baker)).

5.3 Seed Yield

Many review papers and chapters have been published that summarize breeding strate-
gies to increase yield in dry bean (Beaver 1999; Brick and Grafton 1999; Singh 1999a,
1  Common Bean 13

1999b; Urrea and Singh 1994; Kelly 2004; Kelly and Cichy 2012; Vandemark et al.
2014). Some strategies employed by dry bean breeders to improve yield include in-
terracial and interspecific crosses, gamete selection, early generation testing, recurrent
selection, ideotype breeding and selection for physiological efficiency.
To ensure that breeding programmes have optimum genetic diversity for yield
improvement, Kelly et al. (1998) suggested a ‘three-tiered’ pyramidal breeding
strategy to manage germplasm in a breeding programme. The three tiers were com-
posed of three levels of germplasm improvement/advancement in the breeding pro-
gramme and included types of crossing protocols to use in each tier. The apex of the
pyramid consisted of elite, agronomically acceptable germplasm within the target
market class and the use of single-seed descent to advanced lines and testing of
advanced lines. Germplasm in this tier would be used to develop cultivars that are
commercially acceptable to the industry and have high yield. The intermediate tier
of the pyramid has diverse germplasm outside of the market class and includes the
use of interracial material, and pedigree and inbred backcross breeding methods.
The base tier places no restrictions on germplasm, including interspecific and inter-
racial material, and no restriction on breeding methods employed including gamete
selection, congruity backcrosses and conical crossing. This system would advance
germplasm up the tiers or maintain them as they became more adapted to optimize
improvement at each tier of the breeding pyramid.
Improvements in yield have also been achieved in some cases by selection for
yield components. However, because seed size is a descriptor of market class, only
the yield components pod number and seed number can be exploited to increase
yield. Selection of hybrid populations was especially relevant to crosses between
small-seeded Mesoamerican and large-seeded Andean germplasm because it pre-
vented breeders from combining the high pod load potential of small-seeded navy
beans with very large seed size of a kidney bean (White and Gonzales 1990), even
though maximum genetic diversity could be attained by crosses between the Middle
American and the Andean gene pools (Becerra-Velásquez and Gepts 1994). Stud-
ies with interracial crosses have shown mixed results to improve yield (Singh and
Urrea 1994; Singh et al. 2002; González et al. 2009). Interracial hybridization be-
tween beans from races Durango and Mesoamerica has been used to improve pinto,
great northern, small red and pink beans (Singh et al. 1993). Urrea and Singh (1994)
compared breeding methods in interracial crosses for beans and suggested that early
generation testing and selection should be used to more efficiently manage popula-
tions from interracial crosses. Singh and Urrea (1994) made crosses between races
of Andean and Middle American origin and found that on average mean yield was
higher in the interracial crosses that within race crosses. It is known that epistasis
can play a role in the performance of progeny that result from interracial crosses
(Johnson and Gepts 2002; Moreto et al. 2012).
During the early development of some market classes, yield gains were achieved
by selection for a more vigorous vine that produced higher biomass than traditional
landraces. However, cultivars developed by selection for more vigorous vine growth
had increased risk of white mould disease due to denser plant canopies that retained
canopy humidity. Subsequently, breeders developed cultivars with semi-vine habit
14 A. M. De Ron et al.

to enhance disease avoidance mechanisms, allow the plant canopy to dry faster
and allow the plant material to dry in the field after undercutting during harvest.
Yield gains that have occurred over the past 30 years appear to be linear over time
and should continue increasing due to improved plant architecture, disease resis-
tance and avoidance, drought and water-use efficiency and other traits. Today, ap-
proaches using molecular markers have enhanced the process of breeding for yield;
however, yield potential has not been reached in most market classes evidenced by
the linear relationship between seed yield over time, with no indication of a yield
plateau. Vandemark et al. (2014) reported that genetic gain in dry bean during the
past 30 years based on common trials was 13.9 kg ha−1 year−1 (0.77 % year−1) and
17.4 kg ha−1 year−1 (0.85 % year−1) for navy and pinto bean cultivars, respectively.
Vandemark et al. (2014) concluded that continued introgression of germplasm from
other races of common bean should provide new sources of genetic diversity to
enhance yield in the future.

5.4 Plant Architecture

The concept of an ideal plant type or ‘ideotype’ was first proposed in cereal crops
and later suggested for common bean by Adams (1982). Adams proposed the strat-
egy of ideotype breeding to improve yield potential and stability of the crop; today,
that concept is highly associated with upright or type II architecture. Based on the
success of ideotype breeding in small-seeded ‘navy’ and ‘black’ beans, Kelly et al.
(1990) used recurrent selection to develop and released the first upright type II pinto
cultivar ‘Sierra’. Breeding for upright type II plant architecture has continued to be
an important component of breeding some bean market classes in the western USA
and Canada since 1990. Upright architecture also provides a level of disease avoid-
ance for some fungal pathogens such as white mould, enables the crop to be direct
harvested thus reducing seed loss during cutting and field curing and facilitates the
use of furrow irrigation.

5.5 Disease Resistance

Diseases are one of the most important factors limiting bean production globally.
Recent reviews summarizing the current state of breeding for disease resistance in
dry bean were reported by Schwartz et al. (2005), Miklas et al. (2006), Terán et al.
(2009), Schwartz and Singh (2013) and Tryphone et al. (2013).
Common bacterial blight ( CBB; Fig. 1.3), caused by Xanthomonas axonopodis
pv. phaseoli (Xap) (Smith) Vauterin, Hoste, Kosters and Swings and its fuscans
variant, Xanthomonas axonopodis pv. phaseoli var. Fuscans, is considered one of
the most important production constraints worldwide. Singh and Muñoz (1999) and
Yu et al. (2000) recently published reviews on breeding for CBB resistance. The
identification of 22 quantitative trait locus (QTL) across all 11 chromosomes il-
1  Common Bean 15

Fig. 1.3   Common bacterial

blight in common bean pod
and seeds. (Courtesy HF
Schwartz)

lustrates the complexity of breeding for resistance to this disease. Resistance is

quantitatively inherited with largely additive effects, and the heritability is low to
moderately high. The combination of resistance sources from the primary, second-
ary and tertiary gene pools has resulted in the development of improved CBB resis-
tant lines such as XAN 159, OAC 88-1, Wilk 2 and VAX 1 through VAX 6 (Miklas
et al. 2006; Singh and Schwartz 2010; Viteri et al. 2013).
Halo blight (HB), caused by the bacterium Pseudomonas savastanoi pv. pha-
seolicola (Burkh.) Gardan et al., is a major disease of dry beans produced in moder-
ate to cool production areas. Pathogenic variation within the pathogen population
exists with nine races reported worldwide (Taylor et al. 1996a). Five putative R
genes (R1, R2, R3, R4 and R5) were tentatively identified by Teverson (1991) and
Taylor et al. (1996b). The resistant alleles were renamed Pse-1 to Pse-5 to standard-
ized naming by the Bean Improvement Cooperative Genetics Committee (Bassett
and Myers 1999). Races 7 and 8 were found to be most prevalent in Spain and South
Africa, respectively (Rico et al. 2003; Fourie 1998), and race 6 was the most widely
distributed in East Africa and the USA (Lamppa et al. 2002; Taylor et al. 1996a). A
new gene Pse-6 was found located on chromosome Pv04 within a well-described
R-gene cluster that conditions resistance to ANT and rust that conditions resistance
to races 1, 5, 7 and 9 (Miklas et al. 2014).
Liebenberg and Pretorius (2010) recently published a comprehensive review on
the pathology and control of common bean rust, caused by Uromyces appendicu-
latus (Pers.Pers.) Unger. High variability exists within the rust pathogen which co-
evolved with Andean and Middle American beans. High levels of resistance have
been identified from the Andean and Middle American gene pools, and nine major
resistance genes, Ur-3, Ur-4, Ur-5, Ur6, Ur-7, Ur-9, Ur-11, Ur-12 and Ur-13, have
been identified, tagged and mapped by several authors as reviewed by Singh and
Schwartz (2010) and Miklas et al. (2006). Except for Ur-12, that conditions adult
plant resistance, all these genes are race specific (Miklas et al. 2006). Designation
of a new gene, Ur-14, characterized from the cultivar ‘Ouro Negro’ was recently
proposed by de Souza et al. (2011). PI 310762 was reported to have high levels of
resistance and is considered an important resistance source to use in developing
cultivars with broad resistance (Pastor-Corrales et al. 2012).
Angular leaf spot (ALS), caused by Pseudocercospora griseola (Sacc.) Crous
and Braun, is a serious disease in South America, Central America and eastern Af-
rica. The pathogen is highly variable, and distinct Andean and Middle American
16 A. M. De Ron et al.

Fig. 1.4   Anthracnose on

bean leaves

races exist as a result of coevolution with the bean host (Singh and Schwartz 2010).
Despite this variability, high levels of resistance have been identified by a number
of authors in both gene pools (Singh and Schwartz 2010; Miklas et al. 2006). Re-
sistance has been reported from the secondary gene pool ( P. coccineus and P. du-
mosus) by Mahuku et al. (2003). ALS resistance is controlled by single dominant as
well as recessive genes, and a number of sequence characterized amplified regions
(SCAR) markers linked to these resistance genes have been developed (Singh and
Schwartz 2010; Miklas et al. 2006).
ANT (Fig. 1.4), caused by Collectotrichum lindemuthianum ((Sacc. and Magnus)
Briosi and Cavara), is a widespread fungal disease of common bean that causes
significant yield losses worldwide. The pathogen is highly variable, and a num-
ber of Andean and Middle American races have been identified (Kelly and Vallejo
2004; Singh and Schwartz 2010). High levels of resistance in both the primary and
secondary gene pools have been reported globally, including interspecific breeding
lines derived between the two gene pools (Singh and Schwartz 2010). Resistance
has been reported to be controlled by both single and multiple gene models (Singh
and Schwartz 2010) with at least 15 named Co-genes identified. Designation of a
new gene, Co-15, characterized from the cultivar Corinthiano was recently pro-
posed. Most of the resistance genes have been mapped to the integrated bean link-
age map, and molecular markers have been developed for use in marker-assisted
selection (MAS; Miklas et al. 2006; Singh and Schwartz 2010).
In 2013, Schwartz and Singh (2013) published a comprehensive review on
breeding for resistance to white mould disease, caused by Sclerotinia sclerotiorum
((Lib.) deBary), in common bean. White mould is a problem in many dry bean
production areas worldwide (Steadman 1983; Schwartz and Steadman 1989). Plant
architecture and genetic resistance have been identified as mechanisms for control
of white mould. Germplasm from race Durango does not possess adequate levels of
resistance to white mould; consequently, breeders have incorporated upright plant
1  Common Bean 17

architecture from race Mesoamerican to provide avoidance to the disease (Kelly

et al. 1990; Miklas et al. 2013; Kolkman and Kelly 2003; Osorno et al. 2010; Brick
et al. 2011). In addition, resistant QTLs from Andean germplasm have been intro-
gressed into breeding material (Singh et al. 2007, 2014; Griffiths 2009; Soule et al.
2011). Resistant QTL from the secondary gene pool has also been identified (Mik-
las et al. 1998; Singh et al. 2009a, b, 2013).
Root rots, caused by Fusarium, Rhizoctonia and Pythium species, are common
in most bean production areas. A primary source of resistance to root pathogens is
the plant introduction from Mexico, ‘N203’ (PI 203958) collected by Oliver Norvell
(Wallace and Wilkinson 1965). Resistance genes acquired from N203 were intro-
gressed into many market classes of bean. Despite extensive information on Middle
American Fusarium resistance sources, the transfer of resistance into Andean bean
cultivars has been limited (Román-Avilés and Kelly 2005).
The development of germplasm and cultivars with multiple disease and pest re-
sistance has become a common achievement in bean breeding programmes world-
wide. The releases BelDakMik-RMR 14 to BelDakMik-RMR 23 carry pyramided
resistance to all known races of rust in the USA and resistance to all strains of bean
common mosaic virus (BCMV) and bean common mosaic necrotic virus (BCMNV;
Terán et al. 2009). It is common to combine the recessive bc alleles with the domi-
nant I gene, termed ‘protectedIgene’, to provide resistance to all known strains of
BCMV or BCMNV (Brick and Grafton 1999). Markers linked to most resistant al-
leles have been published (Haley et al. 1994; Kelly et al. 1995; Miklas et al. 1996;
Johnson et al. 1997).

5.6 Protein Quality and Metabolites

Protein quality in common bean is suboptimal, like in other grain legumes, and
limited by the low concentration of sulphur amino acids, methionine and cysteine.
The sum of methionine and cysteine is considered as a nutritionally relevant param-
eter when assessing protein quality (FAO 2013). Protein quality improvement was
a major focus of breeding research in the 1970s and 1980s. The goals were both
to increase seed protein concentration and to balance the composition of essential
amino acids (Bliss and Brown 1983). This work relied on a microbiological assay
for bioavailable methionine (Kelly and Bliss 1975). There was good correlation
between bioavailable methionine measured with this assay and total methionine, as
well as the sum of total methionine and cystine.
The 7S globulin phaseolin is abundant in seed, accounting for up to 50 % of
total protein in cultivated varieties (Vitale and Bollini 1995). Lectins account for
5–10 % of total protein, and there are only low levels of the 11S globulin legumin
(Mühling et al. 1997). Efforts to improve protein quality relied on the availability
of alleles conferring a deficiency in phaseolin and erythroagglutinating phytohe-
magglutinin, from P. coccineus Mexican Red Runner, and Great Northern 1140 or
Pinto 111, respectively (Gepts and Bliss 1984; Osborn and Bliss 1985; Voelker et al.
1986). From the similar pattern of DNA hybridization observed between a group
18 A. M. De Ron et al.

of accessions of different geographical origin, Bollini et al. (1985) concluded that

there could be a unique genetic source for phytohemagglutinin deficiency. While
the 7S globulins from legume crops are relatively poor in sulphur amino acids, it
was found that methionine is actually positively correlated with phaseolin levels
(Gepts and Bliss 1984). This is in agreement with biochemical data on protein frac-
tions indicating that most of the methionine is associated with phaseolin, whereas
cysteine is found in non-phaseolin proteins (Chagas and Santoro 1997). Removing
phytohemagglutinin resulted in increased phaseolin concentration, probably as a
compensatory mechanism to maintain a stable seed protein concentration (Osborn
and Bliss 1985). By contrast, introducing arcelin-1 from a wild accession makes
this lectin the most abundant seed protein and lowers phaseolin concentration by
approximately threefold (Romero Andreas et al. 1986). Given the positive relation-
ship between phaseolin and total available methionine, research was conducted
to increase the percentage of phaseolin (Delaney and Bliss 1991a, b). A second-
ary aspect of this research concerns the removal of anti-nutritional proteins. Since
erythroagglutinating phytohemagglutinin is toxic when consumed in raw bean, re-
search has been performed, leading to the release of a phytohemagglutinin-deficient
cultivar (Campion et al. 2009a).
More recently, research on nutritional improvement has shifted to micronutri-
ents and the removal of anti-nutritional factors. Recognizing that deficiencies in
zinc and iron are associated with stunted growth, decreased immune function and
anaemia, the Consultative Group on International Agricultural Research (CGIAR)
has undertaken a major initiative towards the bio-fortification of common bean with
high concentration of these minerals, under the Harvest Plus programme. This work
has been covered in several recent reviews (Beebe 2012; Blair 2013; Kelly and
Cichy 2012). Screening germplasm identified a range of concentrations, with some
intergene pool landraces of the northern Andes and Phaseolus dumosus accessions
having over twofold the average mineral concentrations. The high concentrations
in these intergene pool landraces are likely a result of transgressive segregation,
and this has been confirmed in recombinant inbred populations (Blair et al. 2009).
The enhanced materials have been used to breed cultivars with intermediate to high
concentrations for Latin America and eastern Africa, including the so-called gorilla
beans, with further breeding on the way.
Phytic acid is negatively associated with mineral bioavailability, due to the forma-
tion of insoluble complexes. An ethyl methanesulphonate (EMS)-induced lpa mutant
has been isolated, having low phytic acid concentration (Campion et al. 2009b). This
mutant is affected in an ABC transporter gene, Pvmrp1, required for phytic acid and
raffinose oligosaccharide accumulation (Panzeri et al. 2011). Genotypes having low
phytic acid concentration have also been isolated by conventional breeding. A QTL
responsible for population variation in phytic acid concentration has been associated
with a member of the gene family of d-myo-inositol-3-phosphate synthases, involved
in the first committed step of myo-inositol biosynthesis, located on linkage group
(LG)b01 (Blair et al. 2012). Recently, it was shown that the lpa mutation enhances the
bioavailability of iron in women (Petry et al. 2013), which indicates that reduction of
phytic acid is a promising approach for biofortification.
1  Common Bean 19

A trait widely disseminated in recent years is slow darkening in pinto beans

(Junk-Knievel et al. 2008; Sanchez-Valdez et al. 2004; Singh et al. 2006). Slow
darkening is controlled by a single, recessive gene, sd, distinct from j, associated
with non-darkening (Elsadr et al. 2011). Although the sd gene has been mapped
(Felicetti et al. 2012), its nature and function are currently unknown. It is thought
that the slow darkening trait is related to the biosynthesis of tannins, which are
associated with reduced bioavailability of micronutrients. It will be interesting to
understand the molecular and biochemical phenotype of this trait and its possible
relationship with nutritional quality.
The work involving modification of protein composition has been continued,
taking advantage of genetically related lines integrating the same alleles discussed
above, conferring a deficiency in phaseolin and phytohemagglutinin (Osborn et al.
2003). The parental background is the Sanilac cultivar, and the wild-type line,
SARC1, integrates the lectin arcelin-1 as its major seed protein. As compared with
SARC1, the absence of phaseolin and phytohemagglutinin in SMARC1N-PN1 is
associated with a 10 % increase in methionine and 70 % increase in cysteine, mostly
at the expense of the abundant non-protein amino acid, S-methylcysteine (Taylor
et al. 2008). The combined concentration of methionine and cysteine, expressed
in mg per g protein, is increased by approximately 40 %, slightly exceeding the
Food and Agriculture Organization (FAO) guidelines. In this genotype, the con-
centration of another essential amino acid, leucine, may be slightly limiting (House
and Marsolais unpublished results). These changes are associated with an increased
concentration of legumin and several other sulphur-rich proteins, including albu-
min-2, defensin D1, Bowman–Birk-type proteinase inhibitor 2 and albumin-1A and
albumin-1B (Marsolais et al. 2010; Yin et al. 2011). Two proteins of 56 and 54 kDa
had been shown by Burow et al. (1993) to be elevated following suppression of
phaseolin and lectins. They likely correspond to group 3 late embryogenesis abun-
dant protein and the α-subunit of legumin, respectively (Yin et al. 2011). Profiling
in developing seeds using a high-density Combimatrix 90K microarray identified
transcripts coding for additional sulphur-rich protein types likely to be elevated in
the absence of phaseolin and lectins, including the basic 7S globulin and Kunitz
trypsin protease inhibitor (Liao et al. 2012). Genetic research is currently underway
to evaluate whether the increased cysteine concentration observed in SMARC1N-
PN1 could be used for protein quality improvement. In a subset of lines derived
from a cross between the cultivar Morden003 and SMARC1N-PN1, the combined
molar concentration of methionine and cysteine could be improved by 18–30 % as
compared with parental values (Hou et al. 2014).
Since dry bean is primarily a source of protein and fibre in nutrition, and that
there are significant health benefits from dietary fibre intake, there is significant in-
terest in breeding for improved dietary fibre concentration, while reducing raffinose
oligosaccharides, responsible for flatulence, an important trait related to consumer
preference. From an initial assessment of genetic variability, there are excellent
prospects for breeding genetic materials with improved dietary fibre and reduced
raffinose oligosaccharide concentrations (Brick et al. 2014).
20 A. M. De Ron et al.

6 Breeding Methods and Specific Techniques

Although common beans generally have a low outcrossing proportion (Brunner and
Beaver 1989), environmental factors may affect the level of outcrossing (Ibarra-
Pérez et al. 1997). The breeding objectives should identify the set of traits that
improved cultivars must possess for the target environment and identify other traits
that would be desirable for improved cultivars to possess. Kelly et al. (1998) noted
that the most appropriate breeding method may vary within an integrated genetic
improvement programme, depending on breeding objectives. In developing coun-
tries, the lack of a formal system for the release, multiplication and dissemina-
tion of seed of improved bean cultivars may require the use of more participatory,
community-based breeding methods.
The pedigree method is the most common procedure used to improve dry beans
(Kelly and Cichy 2012). Pedigree selection may be more useful in populations de-
rived from crosses between elite breeding lines. These populations are more likely
to produce progeny in early generations that possess the desired combination of
traits. MAS with codominant markers could be used in combination with pedigree
selection in earlier generation to identify bean breeding lines that possess desired
alleles of traits. Pedigree selection can be accelerated with the use of off-season
nurseries and the simultaneous screening of breeding lines for specific traits such as
disease resistance in greenhouse trials or with MAS (Osorno et al. 2010).
If the objective is rapid generation advance, single-seed or single-pod selection
can be employed in greenhouses or winter nurseries, which are not necessarily rep-
resentative of the target environment. Urrea and Singh (1994) proposed the modi-
fied pedigree (single-seed descent) method as a means to maintain genetic variabil-
ity and rapidly advance lines to more advanced generations where greater progress
can be made in the selection of quantitative traits such as seed yield. The multiple-
seed procedure or harvesting pods rather than single seed from individual plants
should generate sufficient genetic variability to select advanced lines for quantita-
tively inherited traits such as seed yield. Backcrossing is useful for the introgression
of simply inherited traits from different races or gene pools of the common bean
into cultivars or elite bean breeding lines.
Gamete selection may be an effective breeding method for the selection of mul-
tiple traits in populations derived from crosses involving more than two parents
(Singh 1994, 1999a, b). Gamete selection proved to be successful in the develop-
ment of breeding lines with multiple disease resistance, in part, because dominant
genes conferred resistance to several of the diseases under selection (Terán et al.
2009). The availability of codominant markers for recessive traits such as the bc-3
allele for BCMNV resistance (Drijfhout 1978) and the bgm gene for bean golden
yellow mosaic virus (BGYMV) resistance (Velez et al. 1998) would enhance the
effectiveness of gamete selection.
The combination of all favourable alleles for a quantitative trait into a single
genotype is highly unlikely. Therefore, multiple cycles of selection and recombina-
tion may be needed to improve quantitative traits such as seed yield and resistance
to abiotic stress.
1  Common Bean 21

In many ways, Andean beans are often more difficult to breed than smaller-
seeded beans from the Middle American gene pool. Andean beans have less genetic
variability that can be exploited by plant breeders (Beebe et al. 2001). Movement
of genes between gene pools is impeded by hybrid dwarfism in F1 plants caused by
the complementary dominant genes Dl1 (Middle American) and Dl2 (Andean; Singh
and Gutiérrez 1984). Nevertheless, bean breeders have been successful in transfer-
ring between gene pools specific genes for disease resistance such as BGYMV,
ANT and rust (Pastor-Corrales et al. 2007; Pastor-Corrales 2003). Due to the co-
evolution of beans and pathogens within the Andean and Middle American gene
pools, plant breeders and pathologists have found pyramiding resistance genes from
both gene pools can result in more durable resistance to diseases such as rust, ANT
and ALS (Kelly and Miklas 1998).
An important challenge facing contemporary plant breeders is the need to be
proficient in an ever-widening range of knowledge and techniques. How can a bean
breeder use these tools in an efficient and cost-effective manner? The cost of con-
ducting molecular tests from large commercial laboratories may continue to decline
due to gains in efficiency from economy of scale. Outsourcing these services may
be more cost-effective, reliable and more rapid than trying to maintain a state of the
art molecular breeding laboratory.

7 Integration of New Biotechnologies in Breeding

Programmes

7.1 Plant Breeding, Molecular Markers and Genetic Maps

The goal of plant breeding is to increase the number of favourable alleles needed
for a specific production condition. The quicker these alleles can be introduced and
stabilized in a breeding programme, the faster the rate of gain. A new technology,
MAS, emerged in the late 1990s and early 2000s that aided breeding programmes.
Molecular markers became available that tagged specific genes such as Ur-3 and
Co-2 (Geffroy et al. 1998), and programmes were able to track those genes in their
programmes which lessened, but did not eliminate the need for phenotypic test-
ing. Yet, the development of the molecular markers, while not labour intensive,
was time-consuming. New technologies built around the recently released common
bean genome sequence (Schmutz et al. 2014) are now becoming available.
Genetic linkage maps are highly valuable tools for the identification of genomic
regions carrying major genes and QTLs controlling agronomical traits as well as for
comparative genome analyses. In common bean, the first molecular marker-based
genetic maps were developed 20 years ago and were built mainly with restriction
fragment length polymorphisms (RFLPs) and random amplified polymorphic
DNAs (RAPDs) and contained only about hundreds of markers (Vallejos et al.
1992; Freyre et al. 1998).
22 A. M. De Ron et al.

In recent years, the establishment of genetic maps has benefited from the de-
velopment of new types of molecular markers which take advantage of automated
sequencing technologies, in particular the single-nucleotide polymorphisms (SNPs)
markers. The existence of high-throughput methods for assaying SNP is continually
reducing the cost of genotyping, and millions of SNP markers are now available in
common bean (Hyten et al. 2010; Felicetti et al. 2012; Souza et al. 2012; Blair et al.
2013; Goretti et al. 2014; Zou et al. 2014). The resulting dense genetic maps are
very useful for precisely localizing major genes and QTL involved in agronomic
traits. These dense maps are also very useful tools to assist sequence assembly in
whole-genome sequencing projects. For example, a genetic map based on 7015
SNP markers was used to assemble the common bean reference genome sequence
(see after). These 7015 SNP markers were developed based on next-generation se-
quencing data for 14 genotypes from the major gene pools and market classes of
common bean. In addition, by integrating genetic map data with genotyping data
generated from collections of genotypes, linkage disequilibrium (LD) patterns
across the genome can be investigated. This is a prerequisite for precise ‘genome-
wide association studies’ (GWAS) or association mapping. GWAS is an alternative
strategy to the classical biparental QTL mapping strategy to identify the genetic
basis of quantitative traits (Rafalski 2010). Since the diversity of markers and the
extent of LD may vary depending on the history of the collections, they should
be investigated prior to GWAS design (Shi et al. 2011; Galeano et al. 2012). Ulti-
mately, because of the low sequencing costs, high-density marker genotyping could
be replace by ‘genotyping by sequencing’ (GBS) based on a robust, cost-effective,
highly multiplexed approach (Elshire et al. 2011).

7.2 Sequencing the Genome: An Ancient Orphan Crop Joins

Modern Era

The genome of an Andean genotype from CIAT, G19833, was sequenced (Schmutz
et al. 2014) and recently released to the public (http://www.phytozome.org). The
total genome size is 521 Mb and represents 89 % of the 587-Mb genome (http://
data.kew.org/cvalues/). The assembled genome sequence was annotated using tran-
scriptome data and ab initio approaches, and 27,197 gene models and 4441 al-
ternately spliced transcripts identified a total of 31,632 protein-coding sequences.
In addition, it was determined that the transposable elements represent ~ 45 % of
genome, and ~ 40 % of the genome is retrotransposon. The vast majority of these
were inserted < 2 MA. A first draft of the entire common bean genome sequence of
a Mesoamerican genotype (BAT93) was also developed under the framework of the
PhasIbeAm consortium (Vlasovab et al. 2014).
The genome sequence has an immediate application by providing a reference
from which new markers can be developed. The synteny with other legume species
is also a relevant genetic tool. One example is the large collection of robust indel
markers discovered by low pass sequencing 14 diverse genotypes (Mafi Moghad-
dam et al. 2014) and mapping the reads to the reference genome to discover inser-
1  Common Bean 23

tion and deletion events. Scaffolds developed during the sequencing project were
used to discover a large number of single-nucleotide polymorphisms (SNPs). Care-
ful filtering of the SNPs leads to the development of Illumina Infinium SNP chip
(BARCBean6K3) containing ~ 6000 SNPs. The careful design provided a platform
that can be used for any market class and many markers that are suitable for bi-
parental QTL mapping as well as GWAS.

7.3 Current Challenge in the Post-Genomic Area for Breeding

Programmes

An important long-term challenge is the discovery of the gene(s) that control im-
portant production traits. This will need to be a cooperative worldwide effort that
involves breeders, geneticists and genomic and bioinformatics experts. Breeders
provide the essential skills of phenotyping and the identification and development
of genetic populations. Connecting phenotyping with the functional gene requires
the skills of pathologists, physiologists, and those with a deep knowledge of plant
anatomy. Those skilled with genomics and bioinformatics provide the expertise to
link the phenotypic and genotypic data with candidate genes. Once a candidate gene
is defined and the causative mutation is discovered, breeders will then have access
to best possible marker, one that is in the gene controlling the important phenotype.

7.4  Induced Mutagenesis as a Genetic Tool for Improvement

In common bean, both physical and chemical mutagens have been used to induce
mutations. Andersen and Down (1956) were the first to report an induced mutant in
this species using X-rays to modify growth habit, which resulted in the development
of the determinate navy bean cultivar Sanilac. Gamma radiation has also been used
to study and improve important traits affecting plant morphology (Frazier and Da-
vis 1966a; Nagata and Bassett 1984; Tulmann-Neto and Sabino 1994; Avinash and
More 2010), yield (Sarafi 1973), seed quality (Frazier and Davis 1966b; Hussein
and Disouki 1976; Wyatt and Dukes 1980; Allavena 1989), or resistance to pests
(Mohan et al. 1980) and diseases (Tulmann-Neto and Ando 1976; Zogorcheva and
Poriazov 1983; Allavena 1989).
Among chemical mutagens, EMS is most widely used in plants for the develop-
ment of large mutant populations. Porch et al. (2009) generated a common bean
population of 3000 M2 mutant lines using EMS, which was organized and classi-
fied based on mutated tissue, including root, stem, leaf, seed and whole plant traits.
EMS has also been used to generate mutants affected in plant architecture (Motto
et al. 1975), flower and seed coat colour (Moh 1971; Avinash and More 2010), seed
development (Silue et al. 2006), biological nitrogen fixation (Davis et al. 1988; Park
and Buttery 1989; Gautam et al. 1998) and phytic acid biosynthesis (Campion et al.
2009b). In addition, other chemical mutagens have been used for the induction of
24 A. M. De Ron et al.

common bean mutants, such as sodium azide (NaN3; Cary 1982; Jeng et al. 2010)
and N-ethyl-N-nitrosourea (ENU; Svetleva 2004).
Mutations can also be induced using molecular tags or DNA insertions. If the
tags insert into a gene coding or regulation region, they will disrupt the gene func-
tion. The most commonly used random insertion mutagens are the retrotransposon
Tos17, T-DNA, and the Ac/Ds and En/Spm transposon systems. Of these, T-DNA
insertional mutagenesis is the favoured approach due to its stability through genera-
tions of insertions and the low copy number per mutated genome, whereas muta-
tions promoted by transposable element are often unstable (Delseny et al. 2001).
As the sequence of the inserted element is known, the genomic region flanking
the insertion can be easily identified using standard cloning and polmerase chain
reaction (PCR)-based strategies, such as plasmid rescue (Behringer and Medford
1992), inverse PCR (IPCR; Ponce et al. 1998) and thermal asymmetric interlaced
PCR (TAIL PCR; Liu et al. 1995). Thereby, an insertional mutant collection consti-
tutes a useful tool for forward and reverse genetic approaches to identify new genes
and analyse their functions. Currently, there are several publicly available mutant
databases, mainly in the species Arabidopsis thaliana, Oryza sativa and Zea mays.
However, in species such as P. vulgaris, the lack of efficient transformation systems
is hindering the development of insertional mutagenesis resources.
Methods currently available for common bean transformation include indirect
gene transfer using Agrobacterium tumefaciens or A. rhizogenes and direct gene
transfer techniques, mainly particle bombardment or electroporation (Dillen et al.
1995; Kim and Minamikawa 1996; Aragão et al. 2002; Rech et al. 2008; Amugune
et al. 2011). Common bean, like other legumes, is generally considered recalcitrant
to Agrobacterium-mediated transformation due to poor regeneration in tissue cul-
ture (Svetleva et al. 2003; Colpaert et al. 2008; Arellano et al. 2009). However, high
transformation efficiency rates (75–90 % frequency) have been achieved by means
of A. rhizogenes-mediated root transformation (Estrada-Navarrete et al. 2006). Re-
cently, Aragão et al. (2013) has carried out the molecular characterization of the
first commercial transgenic common bean immune to BGMV. Molecular analyses
showed that the transgenes were structurally stable for eight self-pollinated genera-
tions and after backcrosses with a non-transgenic commercial variety. In addition,
the levels of small interfering RNA (siRNA) were analysed in seeds cooked for
10 min, demonstrating that transgenic beans are free of siRNA signals after cooking
and therefore suitable for human consumption.
Genetic transformation causes some public concern, especially in Europe. In
contrast, novel lines obtained by mutagens are much more acceptable to consum-
ers, breeders and governments. In this context, Targeting Induced Local Lesions in
Genome (TILLING) technology has been developed as an alternative to insertional
mutagenesis. TILLING is a non-transgenic method that uses gene-specific primers
for the identification of mutants of a gene of interest from a large mutagenesis popu-
lation (McCallum et al. 2000). TILLING has gained popularity as a reverse genetic
approach because it can produce an allelic series of mutants, including knockouts,
and it does not rely on the transformation method for gene discovery and verifi-
cation. Significant advances have been made in the development of a TILLING
platform in common bean because of the lack of insertional mutagenesis resources.
1  Common Bean 25

A TILLING consortium for tool development has been created, including the Uni-
versity of Geneva (Geneva, Switzerland), USDA/ARS/TARS (Mayagüez, Puerto
Rico) and CIAT (Cali, Colombia). To date, a population of 3000 M2 mutant lines
for TILLING has been generated by Porch et al. (2009) from the genotype BAT93, a
representative of the Mesoamerican gene pool. However, it is necessary to increase
this population, because a population of over 5000 mutant lines is required for ad-
equate genome coverage and an effective TILLING approach (Porch et al. 2009).
Moreover, the TILLING protocol for common bean has yet to be optimized. Once
the BAT93 TILLING project is completed, it will provide a source of characterized
genes for their application in molecular breeding of traits of interest.
Developing varieties with improved agronomic traits is a primary goal of the
common bean breeding programmes. In this context, induced mutation breeding has
become an effective method to increase common bean genetic variability available
to the plant breeder. Furthermore, renewed interest is being generated in induced
mutations since, as mentioned before, the sequencing of the common bean genome
is currently in its final stages, and its availability will significantly broaden oppor-
tunities for functional genomics research. Consequently, induced mutagenesis will
soon become a powerful tool for the isolation and functional characterization of
agronomically interesting genes, which can be used in common bean improvement.

8 Seed Production

8.1 Introduction

Successful crop production depends initially on the availability of high-quality

seed; therefore, production of high-quality seed has a major impact on growing
beans. Low-quality bean seed can cause a poor and an uneven stand resulting in
incidence of diseases, uneven maturity, harvesting problems and yield losses. High-
quality seed will reduce the incidence of seed-borne bacterial and fungal diseases
and has to guarantee the genetic purity of the genotype, and standards are estab-
lished by national seed laws and seed certification agencies to assure bean growers
that the seed they buy is accurately labelled with the correct variety.

8.2 Flowering and Pollination

Nowadays, almost all varieties of common bean grown at high latitudes are day-
neutral in their flowering response and plants flower as soon as they are physiologi-
cally ready. Bean flowers are self-pollinated as the anther sacs are borne directly
adjacent to the stigma and the pollen is released the day before the flower opens.
Normally, the stigma is self-pollinated before the flower opens and is accessible
to pollinating insects. However, a considerable amount of out crossing can occur
under certain conditions. High temperatures induce or increase the physiological
26 A. M. De Ron et al.

deterioration of seeds (Hampton et al. 2013). There is genetic variation for this con-
dition among bean varieties, and the pollen of one variety may lose viability while
another does not under similar conditions. If multiple varieties flower during a hot
period, it is possible that insects may move pollen from a fertile variety to another
cultivar lacking fertile pollen. Others factors that affect to outcrossing are the pres-
ence of barrier plants and isolation distance between plots.

8.3 Isolation Distance

Gene flow is a common phenomenon even in self-pollinated plant species. Faria

et al. (2010) evaluated the frequency of gene flow using transgenic bean cultivars
resistant to the herbicide glufosinate ammonium and their conventional counter-
parts as recipients of the transgene. The outcross occurred at a rate of 0.0074 %
observing that the frequency of gene flow was cultivar dependent and most of the
observed outcross was within 2.5 m from the edge of the pollen source. Ferreira
et al. (2007) observed that the highest frequency of natural hybrids, 0.136 %, oc-
curred at a distance of 0.5 m between the bean cultivars; the natural outcrossing
rate was practically zero beyond a distance of 3.25 m. Isolation requirements vary
depending on flower characteristic and biodiversity in the environment. For bean
commercial production, an isolation distance of 15 m is recommended with a taller
crop barrier (several rows of corn) to prevent pollinator movement. For stockseed
where genetic purity is essential, this distance should be increased to 50 m with a
tall barrier crop as well.

8.4 Genetic Maintenance

Population size depends on the variation within the population of beans to be grown
for seed. In general, commercial varieties are highly uniform and have little hetero-
zygosity; these varieties are mostly derived from a single elite plant. A bean variety
derived in this way has very little inherent genetic diversity, and therefore, 20 plants
saved for seed should be enough to preserve its genetic integrity.
Selection, or rouging, refers to removing unwanted off-types in the population
that is mainly practiced by growers. This selection is critical to maintain desir-
able variety characteristics. Rouging must be done throughout the growing season
inspecting the entire plant. During crop, it is needed to pay attention to earliness,
foliage colour, leaf shape, flower colour, growth form, trueness-to-type, vigour, and
disease and insect resistance. Rouging has to be done again on the seed. If there are
off-types at this stage to evaluate the purity of seed, crop is needed. It is critical to
select against disease in bean crops in order to produce quality seed, minimizing
risk of seed-transmitted diseases.
1  Common Bean 27

8.5 Seed-Transmitted Diseases

The most important diseases for bean seed production are the seed-borne diseases
that may transmit the pathogen via the seed to the next generation. Common beans
would have to be monitored for most important seed-transmitted diseases.
Viral Pathogens  BCMV is the most common and widespread virus of common
bean because it is transmitted by seed and by aphids. BCMNV is considered to be
endemic to Africa, and it has been spread throughout the world in infected seeds
(Davis et al. 2004).
Bacterial Pathogens  Halo blight (Pseudomonas syringae pv. phaseolicola) occurs
worldwide and can cause extensive losses under moderate temperatures and humid
moist conditions. Bacterial brown spot ( P. syringae pv. syringae) grow in wet
and cool conditions. Bacterial blight (Xanthomonas campestris pv. phaseoli) is
favoured by conditions of high moisture and humidity. Bacterial wilt (Curtobacte-
rium flaccumfaciens pv. flaccumfaciens) and fuscous blight (Xanthomonas fuscans)
are becoming a problem in bean seed production.
Fungal Pathogens ANT (Colletotrichum lindemuthianum) develops in regions
with wet periods, high relative humidity and moderate temperatures. Young plants
are infected from spores carried on seed or spores splashed from debris or nearby
infected plants. Fusarium (Fusarium oxysporum f. sp. phaseoli) can survive in soil
for long periods, and it also has been reported to be an external contaminant of seed.
Preventative measures for management of diseases of beans must include at least
a 3-year crop rotation, and all diseased plants have to be removed from the field to
prevent spread of the disease and also to avoid mechanical damage when the crop is
wet and clean equipment to prevent spread from one field to another.

8.6 Harvesting

Bean seed production is better done in the dry regions where pods can be left on the
plant to dry until harvest, without fear of disease. Timing of harvest is important in
order to produce high-quality bean seed that is mature, has an optimum germination
percentage and has high storage potential. Each variety has its own specific harvest
timing; the initial signal that the crop is ready to harvest is the relative maturity
of the pods and their yellow colour. Pods should generally be yellow coloured at
harvest in order to mature properly in the field, but the exact colour is specific of
the variety.

Acknowledgments  A. M. De Ron thanks the INIA Project RFP2013-00001 from the Span-
ish Government. R. Lozano and F. Yuste-Lisbona thank Junta de Andalucía grant to the PAIDI
Research Group AGR176 and Excellence Programme Project P10-AGR-06931 and Campus de
Excelencia Internacional Agroalimentario-CeiA3. P. A. Casquero thanks INIA project RTA 2011-
00076-C02-02. The authors thank Marcial (Talo) Pastor-Corrales for scientific assistance and
Howard F. Schwartz for providing pictures of bean diseases.
28 A. M. De Ron et al.

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1  Common Bean 33

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Chapter 2
Pea

Thomas D. Warkentin, Petr Smýkal, Clarice J. Coyne, Norman Weeden,

Claire Domoney, Deng-Jin Bing, Antonio Leonforte, Zong Xuxiao, Girish
Prasad Dixit, Lech Boros, Kevin E. McPhee, Rebecca J. McGee, Judith
Burstin and Thomas Henry Noel Ellis

1 Introduction

Pea ( Pisum sativum L.) is one of the first domesticated crops and is currently
grown in most temperate regions of the world. Pea belongs to the Leguminosae
family and as such is capable of fixing atmospheric nitrogen, thereby greatly
reducing the requirement for petrochemical-based inputs. World production of
dry pea ranged from 9.4–11.3 × 106 t to 6.0–6.6 × 106 ha between 2000 and 2012
(FAOSTAT 2013). These totals have been relatively steady over the past 50 years;
however, the key producing areas have shifted over that time. Eastern Europe was

T. D. Warkentin ()
Crop Development Centre, University of Saskatchewan, 51 Campus Drive,
Saskatoon, SK S7N 5A8, Canada
e-mail: [email protected]
P. Smýkal
Department of Botany, Palacky University in Olomouc, Slechtitelu 11,
Olomouc 783 71, Czech Republic
e-mail: [email protected]
C. J. Coyne
USDA, Washington State University, 59 Johnson Hall, Pullman, WA 99164-6402, USA
e-mail: [email protected]
N. Weeden
Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman,
MT, USA
e-mail: [email protected]
C. Domoney
Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, UK
e-mail: [email protected]
D.-J. Bing
Agriculture and Agri-Food Canada, 6000 C and E Trail, Lacombe, AB T4L 1W1, Canada
e-mail: [email protected]
© Springer Science+Business Media New York 2015 37
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_2
38 T. D. Warkentin et al.

the major producer from the 1960s to 1980s, then Western Europe from the 1980s
to 1990s, and since then North America, primarily Canada. China and India have
had relatively stable production of 2–3 × 106 t/year over the past 50 years. In terms
of world production of dry legume crops, dry pea trails only common bean which
had annual production of 17.6–23.3 × 106 t between 2000 and 2012, and the oilseed
legumes soya bean (161.3–265 × 106 t) and groundnut (33.1–42.1 × 106 t) during
the same period (FAOSTAT 2013). World production of vegetable pea ranged from
12.0–17.4 × 106 t to 1.6–2.2 × 106 ha between 2000 and 2012 (FAOSTAT 2013).
Vegetable pea production has been rising steadily over the past 50 years with China
and India being the major producers.
In order to expand world production of pea, breeders, agronomists, end users
and producers face several challenges. Grain yield gains must continue for pea to
remain an attractive option in crop rotations. This will require a concerted effort
from pea breeders internationally. In Western Europe, pea production has declined
in the past two decades as producers have focused on high-yielding winter wheat
and winter canola crops. This has led to a decline in pea-breeding activity. Pea

A. Leonforte
Nuseed Innovation Centre, 5 Ballinger Street, Horsham, VC 3400, Australia
e-mail: [email protected]
Z. Xuxiao
Chinese Academy of Agricultural Sciences (CAAS), Institute of Crop Science (ICS), No.12,
Zhong Guan Cun South Street, Haidian District, Beijing 100081, Beijing, China
e-mail: [email protected]
G. P. Dixit
Indian Institute of Pulses Research, Near Kanpur University,
Kanpur 208024, Uttar Pradesh, India
e-mail: [email protected]
L. Boros
Department of Seed Science and Technology, Institute of Plant Breeding
and Acclimatization—National Research Institute, Radzików 05-870, Poland
e-mail: [email protected]
K. E. McPhee
Department of Plant Sciences, North Dakota State University, 370G Loftsgard Hall,
P.O. Box 6050, Fargo, ND 58108, USA
e-mail: [email protected]
R. J. McGee
USDA-ARS, Grain Legume Genetics and Physiology Research Unit, Washington State
University, 305 Johnson Hall, PO Box 646434, Pullman, WA 99164, USA
e-mail: [email protected]
J. Burstin
INRA, UMR1347 Agroecology, Bat. Mendel, 17 rue de Sully, Dijon 21065, France
e-mail: [email protected]
T. H. N. Ellis
CGIAR Research Program on Grain Legumes, ICRISAT, Patancheru 502324, Telangana, India
e-mail: [email protected]
2 Pea 39

production has increased in North America and Australia over the past two decades
and similarly, pea-breeding efforts have increased.
In order to achieve yield gains in pea, many biotic and abiotic stresses must be
addressed through breeding. These stresses are specific to each region; however,
in general, fungal diseases are the key biotic stress in most pea-growing regions,
followed by various insects and viruses. Heat stress at flowering is the key abiotic
stress in many pea-growing regions, followed by early-season flooding. Address-
ing these stresses are key breeding objectives for pea breeders in their attempts to
increase and stabilize grain yields.
Greater international exchange of germplasm and increased use of diverse Pisum
accessions may aid in achieving new yield gains. Use of genomic tools should
enhance breeders’ ability to substantially enrich their breeding populations with
desired alleles, prior to the expensive exercise of yield testing in field trials.
Greater market diversification for pea will create more demand and expand
production. Dry pea has typically been used as dhal in Asian markets. A major new
use for dry pea is the Chinese vermicelli market which utilizes pea starch which is
effective because of its high amylose content. This market has expanded from zero
to more than 700,000 t/year over the past two decades (FAOSTAT 2013). Further
use of pea and pea fractions (protein, starch and fibre) in diverse food products
could promote expansion of the crop. Pea has good potential in new food applica-
tions due to its moderate protein concentration, slowly digestible starch and high
levels of soluble and insoluble fibre, all of which are attractive for addressing type
2 diabetes and obesity. In addition, pea has low allergenicity and to date is a non-
genetically modified organism (GMO), both factors making it attractive compared
to soya bean in some markets.

2  Origin and Systematics

2.1  Phylogeny and Taxonomy

Pea belongs to the Leguminosae plant family, the third largest flowering plant fam-
ily with 800 genera and more than 18,000 species (Lewis et al. 2005). The Papil-
ionoideae is the largest subfamily, with 476 genera and about 14,000 species, which
shared a common ancestor around 50 MA (Doyle et al. 1997; Lavin et al. 2005).
The largest group of papilionoids, Hologalegina, with nearly 4000 species in 75
genera, includes the large galegoid tribes (including Galegeae, Fabeae, Trifolieae),
united by the loss of one copy of the chloroplast inverted repeat. Tribe Fabeae Rchb.
(not Vicieae (Bronn) DC., nom. illeg.) currently consists of five genera: Lathyrus
(grass pea/sweet pea, about 160 species); Lens (lentils, 4 species); Pisum (peas, 3
species); Vicia (vetches, about 160–250 species) and the monotypic genus Vavilovia
formosa (Mikič et al. 2013; Smýkal et al. 2011; Schaefer et al. 2012). Tribe Fabeae
is considered one of the youngest groups in the legumes (Kupicha 1981; Steele and
Wojciechowski 2003), and Bayesian molecular clock and ancestral range analysis
40 T. D. Warkentin et al.

suggest a crown age of 23–16 MA, in the mid-Miocene (Lavin et al. 2005; Schaefer
et al. 2012). The centre of diversity and postulated area of origin of the Fabeae is in
the eastern Mediterranean (Kupicha 1981; Schaefer et al. 2012) with a minimum of
three dispersal events to the middle Atlantic islands and seven to the Americas. The
tribe is considered monophyletic, nested within the Trifolieae. The crown age of the
Pisum clade is estimated to 2.3–0.8 MA, while the divergence between Pisum and
Vavilovia dates back to 9.8–4.8 MA (Schaefer et al. 2012).
The genus Pisum L., originally described to be distinct from Lathyrus L.
(Linnaeus 1753), has recently been shown to be included in the Lathyrus/Vicia
complex (Schaefer et al. 2012). Interestingly, Lamarck (1778), who was certainly
aware of Linné’s description, designated pea as Lathyrus oleraceus. Depending on
how the Lathyrus/Vicia complex is treated, the genus Pisum may be incorporated into
a larger Lathyrus genus to achieve monophyly. Thus, the taxonomic nomenclature
used here will undoubtedly be revised. The classification of taxa within Pisum L.
based on morphology and karyology has changed over time from being considered
a genus with five species (Govorov 1937) to the currently widely accepted version
with two species, P. fulvum and P. sativum, recognized (Kupicha 1981; Davis 1970).
Numerous names have been proposed for wild representatives of P. sativum. In the
review of Yarnell (1962), P. humile (P. syriacum, P. sativum subsp. sativum var.
pumilio), P. elatius, P. abyssinicum and P. sativum were considered conspecific,
even though they often differ by inversions and translocations. In this chapter, we
will refer to the following taxonomic definitions of Pisum: P. sativum L. with subsp.
sativum (includes var. sativum and var. arvense), subsp. elatius (Bieb.) Aschers. and
Graebn (includes var. elatius, var. brevipedunculatum and var. pumilio), and subsp.
abyssinicum (A. Braun) Govorov and P. fulvum Sibth. and Sm.
P. abyssinicum, has been resurrected as a third species by some recent authors
(Maxted and Ambrose 2001; Vershinin et al. 2003; Jing et al. 2007), but for reasons
detailed below, this taxon will be maintained as a subspecies of P. sativum in this
chapter. Other ‘species’ such as P. jomardi, P. transcaucasicum and P. arvense have
also been included within P. sativum by most recent treatments (Jing et al. 2007;
Zaytseva et al. 2012). The most appropriate status for P. sativum subsp. abyssinicum
is still under debate (Maxted and Ambrose 2001; Zaytseva et al. 2012). This taxon
is native to Ethiopia and Yemen and has very low genetic diversity as demonstrated
by morphological, allozyme (Weeden and Wolko 2001) and DNA analyses (Pearce
et al. 2000; Vershinin et al. 2003; Jing et al. 2005, 2010). It possesses a distinct
phenotype (early flowering and strongly serrate leaflets) as well as unique alleles at
particular loci. Similar to most P. s. subsp elatius accessions, this taxon differs from
the standard P. sativum subsp. sativum karyotype by at least a reciprocal transloca-
tion (Ben-Ze’ev and Zohary 1973). Hence, it qualifies for species status on the basis
of phenotype and biological isolation. However, recent DNA sequence comparisons
have shown this taxon to fall within the humile/elatius/sativum cluster or between
it and P. fulvum, depending on the sequence being analysed (Jing et al. 2007, 2010;
Ellis 2011; Smýkal et al. 2011; Vershinin et al. 2003; Zaytseva et al. 2012). The
taxon has been used as a bridge between P. fulvum and P. sativum because it crosses
reasonably well with both. Many crosses have been attempted with abyssinicum
2 Pea 41

lines, and the most fertile crosses were to P. sativum subsp. sativum germplasm
rather than to subsp. elatius accessions, although the presence of the reciprocal
translocation definitely leads to reduced fertility in the F1 and F2 generations
(Weeden, personal communication). Thus, if the abyssinicum variation is to be giv-
en specific status, it appears appropriate for consistency sake to also raise at least
a portion of the elatius accessions to species status. From a practical viewpoint,
the current authors do not see much advantage to splitting the abyssinicum/elatius/
sativum germplasm into three or four species at the present time. Another taxon that
has recently been suggested to be included in Pisum (Maxted and Ambrose 2001),
V. formosa, we retain as a distinct genus (Smýkal et al. 2013; Mikič et al. 2013).
The centre of pea genetic diversity is the broad area of the Fertile Crescent
through Turkey, Syria, Iraq, Israel and Lebanon. It extends further east to Central
Asia (Iran, Afghanistan, Pakistan and Turkmenistan; Smýkal et al. 2011). Ethiopia
has been postulated as a secondary centre of diversity (van der Maesen 1998).
Vavilov (1950) considered Ethiopia together with the Mediterranean and Central
Asia as primary centres, and Near East as secondary. Pisum sativum subsp. elatius
and subsp. sativum are found naturally in Europe, northwestern Asia and extend
south to temperate Africa, while P. fulvum is restricted to the Middle East.

2.2  Origin and Domestication

Pea is one of the world’s oldest domesticated crops. Archaeological evidence dates
the existence of pea back to 10,000 BC in the Near East (Baldev 1988; Zohary and
Hopf 2000) and Central Asia (Riehl et al. 2013). Pea, among other grain legumes,
accompanied cereals and formed important dietary components of early civiliza-
tions in the Middle East and Mediterranean. In Europe, it has been cultivated since
the Stone and Bronze Ages and in India from 200 BC (De Candolle 2007). The Near
East and Mediterranean regions are also the area of origin and initial domestication.
Cultivation of pea spread from the Fertile Crescent to today’s Russia, and westwards
through the Danube valley into Europe and/or to ancient Greece and Rome which
further facilitated its spread to Northern and Western Europe. In parallel, pea was
moved eastward to Persia, India and China (Makasheva 1979; Chimwamurombe
and Khulbe 2011).
Phylogenetically, there are two wild populations variously described as subspe-
cies of P. sativum or as species, P. sativum subsp. elatius Bieb. and P. sativum subsp.
sativum (= P. humile Boiss and Noe (syn. P. syriacum (Berger) Lehm.; Ben-Ze’ev
and Zohary 1973; Smýkal et al. 2011). These two wild groups are morphologically,
ecologically and also genetically distinct (Ben-Ze’ev and Zohary 1973; Abbo et al.
2013). The domestication of cultivated pea from northern populations of ‘humile’
was proposed by Ben-Ze’ev and Zohary (1973), but the source could equally be the
‘northern elatius’ (Kosterin et al. 2010; Smýkal et al. 2011). Recently, P. humile was
included into so-called lost crops, for example, additional taxa that were at certain
points in time and in certain locations’ genuine crops, but were later abandoned
42 T. D. Warkentin et al.

(Abbo et al. 2013). It is notable that despite its wild-type seed dispersal mode and
wild-type seed dormancy, these southern P. humile are currently found only in sec-
ondary habitats and never invade adjacent less disturbed habitats, in contrast to
P. fulvum and P. elatius. Cytogenetic differences and analyses of genetic diversity
support the view that the majority of cultivated peas originated from a distinct gene
pool within var. humile (Zohary and Hopf 2000), although recent molecular studies
also highlight the likely genomic contribution from other wild forms and emphasize
the importance of introgression and recombination within the complex (Jing et al.
2010).
The domestication of pea has been experimentally tested, both in order to deter-
mine the genetic basis which led to the cultivated crop from the wild plant (Weeden
2007), as well as wild pea harvesting (Abbo et al. 2010). The so-called domesti-
cation syndrome in the case of pulses applies to increases in seed size, reduction
or elimination of pod shattering, and loss of germination inhibition, shoot basal
branching and seed toxins and antimetabolites (Smartt 1990; Zohary and Hopf
2000; Weeden 2007). Altogether, at least 11 loci involved in domestication traits
have been identified (Weeden 2007). In pea, explosive pod indehiscence and seed
dormancy (hard seededness) were probably the greatest barriers to domestication
(Smartt 1990). Pod dehiscence is primarily influenced by the Dpo gene (Lamprecht
1957a), although other genes also affect this trait (Weeden et al. 2002). The genetic
basis of hard seededness has yet to be fully elucidated, although it is clear that the a
mutation (lack of anthocyanin production) reduces testa thickness, thereby affecting
dormancy. Other traits selected during domestication and development of modern
cultivated forms, include several morphological characters that are determined by
one or a few genes. These genes include le (semidwarf growth habit), r (wrinkled
seed in garden types), af (conversion of leaflets to tendrils), and p and v (absence of
sclerenchymatic tissue in pods). Both a and r improved seed palatability, le and af
increased the efficiency of mechanical harvesting, and p and v lead to the develop-
ment of edible-podded types.
Monogenic inheritance is also known for several physiological traits that have
been altered during domestication. Wild Pisum in its native range displays a typical
winter habit in which plants germinate in autumn, overwinter in the vegetative state
and flower in response to increasing day length in spring (Weller et al. 2009; Abbo
et al. 2013). The obligate or near-obligate requirement for long days suits pea to a
winter cropping cycle and has been retained in some forage cultivars. However, most
of the cultivated pea accessions from higher latitudes have a quantitative long-day
response and are grown as a spring crop (Weller et al. 2009). Some pea varieties are
very early flowering and not photoperiod sensitive. The genes controlling flowering
in pea include Lf, Sn, Hr and E (Murfet 1973; Weller et al. 2009). The obligate
long-day (wild type) genotype is Lf, Sn, Hr, with E or e. The quantitative long-
day phenotype of many cultivars has the genotype Lf, Sn, hr. Day-neutral cultivars
are Lf, sn ( Hr is not strongly active in sn/sn plants), and the very early flowering
types are lf, sn. Hence, lf, sn and hr could all be considered ‘domestication’ alleles.
Domestication has also resulted in increased seed and pod size in pea, although
not as markedly as in other crops, with a correlated increase in leaf size and stem
2 Pea 43

strength (Swiecicki and Timmerman-Vaughan 2005; Weeden 2007). The genetic

basis of seed size appears to be quantitative (Timmerman-Vaughan et al. 1996),
and there are several genes known to influence pod and leaf size (Lamprecht 1953,
1954, 1957b, 1960, 1963). At present, no single or small set of these genes can be
identified as crucial for domestication.
Based on these morphological and genetic studies, P. humile/syriacum, P. elatius
and P. fulvum were identified as ‘wild’ germplasm in that they display traits such
as dehiscent pods and seed dormancy (thick testa), that are necessary for survival
in the wild and undesirable in a domesticated annual crop. In contrast, P. sativum
including var. arvense, transcaucasicum and asiaticum generally display indehis-
cent pods and little seed dormancy, and could be considered domesticated. P. ab-
yssinicum is early flowering, with indehiscent pods, moderately large seeds and
lacks seed dormancy. Based on this phenotype, it has been identified as partially
domesticated.
One interesting feature regarding the domestication of pea is that not all changes
have been unambiguous improvements. Rather many are trade-offs sacrificing cer-
tain adaptations for other advantages. For instance, incorporation of the a allele into
cultivars improved the taste of the seed but also made the plant more susceptible
to pathogens such as Pythium and Fusarium. Elimination of the Np gene increases
seed size but also increases susceptibility to bruchid attack (Berdnikov et al. 1992).
Incorporation of the ‘wrinkled seed’ (r) and ‘afila’ (af) alleles leads to a reduction
in yield in some environments. The sugar snap pea, with its combination of four or
five mutations ( a, r, p or v, n and sin) is notoriously susceptible to soil pathogens.
Once genes controlling main domestication traits are identified, along with the full
pea genome sequence, we might expect and look forward to comparative evolution-
ary studies across independently domesticated legumes.

2.3  Pea Genus Genetic Diversity

Based on morphology, Pisum sp. is one of the most diverse crop species known,
comparable to Zea mays, Cucurbita pepo and Brassica oleracea (Hancock 2012).
There are several user-defined classifications of cultivated pea diversity. Four sim-
ply inherited characters determine the main use types of peas within subsp. sativum:
the presence or absence of pod parchment, flower anthocyanin, leaflets occurrence
and whether the starch grains in the dry seed are simple or compound (Green 2008).
This classification is similar to that proposed by Lehmann (1954) except for the
afila type which was unknown at that time. Early data from electrophoretic pat-
terns of major seed proteins: albumin and globulin (Waines 1975), allozymes (Hoey
et al. 1996) and chloroplast DNA polymorphism (Palmer et al. 1985) separated
P. fulvum as a distinct species and P. sativum as an aggregate of ‘humile’, P. sativum
subsp. elatius and P. sativum, in agreement with the current view of the genus. Che-
mosystematic studies using flavonoids (Harborne 1971) showed that P. fulvum con-
tains quercetine 3-glucoside, primitive cultivars from Nepal and P. abyssinicum con-
tain kaempferol and quercetine 3-sophoroside, while modern pea cultivars contain
44 T. D. Warkentin et al.

kaempferol and quercetine 3-(cumaroyl-sophorotrioside). Petals of wild peas con-

tain delphinidin, petunidin and malvidin 3-rhamnoside-5-glucosides, while coloured
petals of cultivated garden pea contain, in addition, pelargonidin, cyanidin and peon-
idin 3-rhamnoside-5-glucosides (Harborne 1971). Unfortunately, the yellow colour
P. fulvum petals were not studied. Moreover, the Pisum genus contains the flavonoid
phytoalexin pisatin, which is shared with the genus Lathyrus but not found in Vicia
species (Bisby et al. 1994), which have wyerone instead. Serological reactions of
Pisum taxa by Kloz and Turkova (1963) indicated a close relationship of all studied
taxa, except of P. fulvum and P. abyssinicum. They were possibly the first to indicate
that P. abyssinicum might have originated from hybridization between P. sativum
subsp. elatius and P. fulvum. Hoey et al. (1996) using morphological, allozyme
and random amplified polymorphic DNA (RAPD) characteristics on a set of Ben-
Ze’ev and Zohary (1973) accessions showed a separation of P. fulvum and ‘southern
humile’, while cultivated peas were among P. sativum subsp. elatius accessions.
The position of ‘northern humile’ varied between a sister group to cultivated peas
and P. sativum subsp. elatius. More recently, studies of internal transcribed spacer
(ITS) sequence variation (Saar and Polans 2000) and histone H1 subtype five gene
(Zaytseva et al. 2012) have supported this. Recent phylogenetic studies based on
retrotransposon insertion markers support the model of P. sativum subsp. elatius
as a paraphyletic group, within which all P. sativum are nested (Nasiri et al. 2010;
Vershinin et al. 2003; Jing et al. 2005, 2010). Although pollination strategy is highly
relevant for genetic diversity, it has not been properly studied in wild pea. Pea is
considered a self-pollinating species; however, cross-pollination is likely to occur in
wild pea populations. The reported cross-pollination rate in cultivation ranges from
zero (White 1917) to 60 % (Harland 1948), depending on genotype and environ-
ment. The percentage reported for commercial cultivars is less than 1 % (Dostálová
et al. 2005). In addition to biological consequences, self-pollination reinforced
genetic barriers between wild and cultivated populations, facilitating fixation of the
desired genotype (Zohary and Hopf 2000).
Molecular analysis of pea diversity preserved in germplasm collections was done
using amplified fragment length polymorphism (AFLP; Ellis et al. 1998), its derived
retrotransposon insertion-based marker method, sequence-specific amplification
polymorphisms (SSAP; Pearce et al. 2000; Majeed et al. 2012; Vershinin et al.
2003) and gene sequences (Jing et al. 2007; Zaytseva et al. 2012). In all analyses, P.
fulvum and P. abyssinicum formed neighbouring but separate branches, a subset of
P. sativum subsp. elatius was positioned between P. fulvum and P. abyssinicum, and
further branches were found within cultivated pea. The most recent studies of P. ab-
yssinicum place it between P. fulvum and a subset of P. sativum subsp. elatius (Ellis
2011; Smýkal et al. 2011; Vershinin et al. 2003; Jing et al. 2010) and showed its
very low genetic diversity, which could be explained by passage through a genetic
bottleneck. Importantly, high conservation between retrotransposon sequence-spe-
cific amplification polymorphism (SSAP; Vershinin et al. 2003), retrotransposon
insertions (Jing et al. 2005) and gene-based derived (Jing et al. 2007) trees was
observed, in spite of the fact that they derive from different genomic compartments.
2 Pea 45

Another study on relationships among wild Pisum, using a combination of mito-

chondrial, chloroplast and nuclear genome markers (Kosterin and Bogdanova
2008; Kosterin et al. 2010), separated P. fulvum and P. abyssinicum accessions.
Interestingly, Afghan types (originating from Afghanistan, Iran, Pakistan) are not
nodulated by ordinary European and North American strains, but require specific
Rhizobium strains for symbiosis (Young and Matthews 1982). Afghan types were
clustered separately in diversity analysis based on retrotransposon insertions (Jing
et al. 2010). Genetic discontinuity in root-nodulating bacteria of cultivated pea was
shown in the trans-Himalayas between India and China (Rahi et al. 2012). The
example of coevolution was found in a south Turkey pea line, which was found to
form an effective symbiosis only with local Rhizobium strains but not with strains
from other parts of Turkey (Lie et al. 1987). These authors suggested that the ge-
netic uniformity of European R. leguminosarum strains is the result of selection and
domestication of Rhizobium strains originally derived from the gene centres of pea.
Several studies of pea germplasm using morphological descriptors and agronom-
ical traits and lately DNA markers have been published (see ‘Genetic resources and
utilization’, Sect. 4). These gave a consistent view. In spite of being a rather small
genus with two or three species, Pisum is very diverse and diversity is structured,
showing a range of degrees of relatedness that reflect taxonomic identifiers, eco-ge-
ography and breeding gene pools (Ellis 2011; Smýkal et al. 2011; Jing et al. 2012).
In summary, pea belongs to the early domesticated legume crops accompanying
cereals and formed an important dietary component of early civilizations. The Near
East and Mediterranean regions are both the area of origin and initial domestication.
In the process of domestication, two key traits have been modified, pod dehiscence
and seed dormancy. Additional traits include seed size, flowering-time control and
branching pattern. Pea belongs in the Fabeae tribe together with lentil, faba bean,
common vetch and grass pea. Recent phylogenetic analysis has shown the Pisum
L. genus to be positioned within the Lathyrus/Vicia complex to obtain monophyly.
Despite high morphological variation and an extensive geographical range, two true
species are recognized: P. sativum/elatius complex and the more distant P. fulvum,
consisting of the secondary gene pool. These can be intercrossed and fertile progeny
obtained, although there is some reduction in fertility due to chromosomal trans-
locations and nucleo-cytoplasmic conflict. Phylogenetically related V. formosa, a
perennial mountain species, consists of the tertiary gene pool.

3  Varietal Groups

Pea has a wide range of market classes and uses (See Fig. 2.1).
Field Pea  Also known as dry pea and combining pea. The mature seed phenotype
for field pea is round (genetically RR). Field pea includes yellow, green and red
cotyledon varieties typically used in the dehulled/split form in foods such as dhal.
New markets are emerging for pea flour in baked products, extruded snacks and
46 T. D. Warkentin et al.

Fig. 2.1   Diversity of seed

colour, shape and size in pea.
Among 15 genotypes shown,
testas have been removed
from one seed of every line
to show the cotyledon colour.
The phenotypes reflect
variation at several genetic
loci, including A, r, i and s

noodles. Starch fractions, typically from yellow cotyledon pea, are used widely in
China for production of vermicelli noodles. Protein and fibre fractions are also used
in the food industry.
Smaller market classes include:
• Dun (pigmented seed coat) which is also used in the dehulled/split form for
foods such as dhal
• Marrowfat (large seeds, blocky shape, green cotyledons, appealing flavour pro-
file) for snacks and mushy pea
• Maple (mottled seed coat) for bird seed mixtures
• Forage (high biomass) cut prior to dry seed maturity for ruminant feed
2 Pea 47

• Sprouts, that is, germinated seedlings used as a vegetable

• Feed (can include any of the above types) for use in the compound feed industry
typically for pigs and chickens

Vegetable Pea  Also known as garden pea and vining pea. The mature seed pheno-
type for vegetable pea is wrinkled (genetically rr). Vegetable pea includes:
• Freezer pea
• Snow pea
• Snap pea

4  Genetic Resources and Utilization

Total pea genetic resources are extensive with ex situ germplasm holdings of 73,931
accessions in over 28 plus national and international collections with duplicate sam-
ples of 9670 accessions preserved at the Svalbard Global Seed Vault at Spitsbergen,
Norway (Table 2.1).
The four largest active collections include the Australian Temperate Field Crop
Collection, Horsham, of 7432 accessions, N.I.Vavilov Research Institute of Plant
Industry (VIR), St. Petersburg, Russia, with 6790 accessions, the US Department
of Agriculture (USDA) 6827 accessions, and the French National Institute for Ag-
ricultural Research (INRA) of 8839 accessions held at Dijon, France. The Vavilov
Research Institute which originated in 1921 is the oldest and most famous of the
large pea germplasm collections and was started with Vavilov’s explorations. Very
representative and arguably the best studied is the John Innes (JI), Norwich Pisum
collection, containing 1200 P. sativum cultivars, 600 traditional landraces and 750
genetic stocks and reference lines together with wild Pisum samples. Australia has
the least duplicative and most diverse ex situ collection to date for Pisum. Also, a
large collection of 6105 is held by International Center for Agricultural Research in
the Dry Areas (ICARDA) which could be accessed easily until recent times. In ad-
dition, there are other exciting national collections of pea germplasm. An example
is in Israel, which hosts a collection of the crop’s wild relatives P. fulvum and P. sa-
tivum subsp. elatius var. pumilio collected in the Middle East. An estimated 20 % of
the world’s ex situ pea germplasm is duplicative (Smýkal et al. 2013), which would
leave 59,000 accessions as unique.
The highest traffic websites for ordering germplasm are the JI Centre (JIC; http://
www.jic.ac.uk/germplasm/) and the USDA (http://www.ars-grin.gov/npgs/). These
two sites have the highest turnover of international requests of readily available
Pisum accessions. For example, the USDA sends 3140 pea accessions per year
(5 year average) to researchers worldwide. The JIC Genetic Stocks (http://data.
jic.ac.uk/cgi-bin/pgene/) accessed through the ‘PGene Pisum Gene List’ contains
genes/traits recognized by the Pisum Genetics Association (http://hermes.bionet.
nsc.ru/pg/) and the associated gene stock JI line. Duplicate genetic stocks are
Table 2.1   List of selected pea germplasm collections preserving significant Pisum diversity (data from Smýkal et al. 2013)
48

Country Institute Website Cultivated Pisum sativum Total

Commercial Breeding Land races Mutant stock Wild Pisum sp.
varieties lines
Russia N.I. Vavilov Research Insti- http://www.vir.nw.ru 6653 – – – – 6790
tute of Plant Industry, St.
Petersburg
France INRA CRG Légumineuse à http://195.220.91.17/ 1315 496 174 4818 63 8839
grosses graines, Dijon legumbase/
Australia Australian Temperate Field http://www2.dpi.qld.gov.au 1170 1420 4514 87 205 7432
Crop Collection, Horsham
USA Plant Germplasm Introduc- http://www.ars-grin.gov 1504 64 3935 37 84 6827
tion and Testing Research
Station, Pullman
Syria International Center for http://www.icarda.cgiar.org 1185 1305 3240 0 228 6105
Agricultural Research in the
Dry Areas, Aleppo
Germany Leibniz Institute of Plant http://www.ipk-gatersleben. 3008 277 1553 71 47 5343
Genetics and Crop Plant de/
Research, Gaterleben
Italy Istituto del Germoplasma, http://www.igv.cnr.it 0 0 4558 0 0 4558
Bari
China Institute of Crop Science, http://icgr.caas.net.cn/cgris 532 45 3260 5 sets 0 3837
Chinese Academy of Agri-
cultural Sciences
India National Bureau of Plant http://www.nbpgr.ernet.in 210 1016 1207 8 0 3609
Genetic Resources, New
Delhi
UK John Innes Centre, Norwich http://www.jic.ac.uk 1071 61 600 585 368 3567
T. D. Warkentin et al.
Table 2.1  (continued)
Country Institute Website Cultivated Pisum sativum Total
2 Pea

Commercial Breeding Land races Mutant stock Wild Pisum sp.

varieties lines
Poland Plant Breeding and Accli- http://www.igr.poznan.pl 1254 638 21 297 75 2896
matization Institute Blonie,
Radzikow
Sweden NordGen, Nordic Genetic http://www.nordgen.org/ 472 1528 471 0 32 2849
Resource Centre, Alnarp sesto
Bulgaria Institute of Plant Introduc- http://www.genebank.hit.bg 750 500 196 150 3 2100
tion and Genetic Resources,
Sadovo
Brazil National Center for Vegeta- http://www.cnph.embrapa.br N.A. N.A. N.A. N.A. – 1958
ble Crops Research (CNPH)/
EMBRAPA
Spain Junta de Castilla y León. http://www.itacyl.es 649 328 543 103 32 1772
Instituto Tecnológico
Agrario de Castilla y León
Ethiopia http://www.ibc.gov.et/ N.A. N.A. N.A. N.A. – 1768
Ukraine Yurjev Institute of Plant http://www.bionet.nsc.ru N.A. N.A. N.A. N.A. – 1671
Breeding, Kharkov
Spain Institutot Nacional de http://www.inia.es 67 168 599 0 0 1648
Investigacion y Tecnologia
Agraria y Alimantaria
Czech Centre for Applied Research http://genbank.vurv.cz 1257 63 11 0 2 1414
Republic of Vegetables and Special
Crops, Olomouc
Czech AGRITEC, Research, http://genbank.vurv.cz 972 257 54 0 9 1326
Republic Breeding and Services Ltd.,
Sumperk
49
Table 2.1  (continued)
50

Country Institute Website Cultivated Pisum sativum Total

Commercial Breeding Land races Mutant stock Wild Pisum sp.
varieties lines
Hungary Institute for Agrobotany, http://www.rcat.hu 933 0 79 0 4 1205
Tapioszele
The Centre for Genetic http://www.cgn.wur.nl/pgr/ 504 86 345 0 0 1002
Nether- Resources, Wageningen
lands
Serbia IFVCNS, Novi Sad http://www.nsseme.com/en/ 665 127 74 42 55 991
Canada Plant Gene Resources of http://www.agr.gc.ca/ 449 76 55 0 11 616
Canada, Saskatchewan, pgrc-rpc
Canada
Israel Israel Plant Gene Bank, http://igb.agri.gov.il 0 0 0 0 333 343
ARO Volcani Center
Turkey Aegean Agricultural http://www.aari.gov.tr 0 0 236 0 0 236
Research Institute, Mene-
men/ IZMIR
Armenia Institute of Botany NAS RA, http://www.sci.am/ 4 0 0 0 0 19
Yerevan
Total 24,624 8455 25,725 6198 1551 73,931
N.A. not accounted
T. D. Warkentin et al.
2 Pea 51

maintained also at the Nord Genebank (Blixt and Williams 1982). The JIC Pisum
Genetic Stocks are partially duplicated at the USDA (Ambrose and Coyne 2009),
and at Polish (297) and Bulgarian (150 accessions) genebanks. Also maintained by
JIC is a fast neutron (FN) population started with 1400 seeds of JI 2822 subjected
to 20 Grays FN radiation (Domoney et al. 2013; Hofer et al. 2009). A very fruitful
targeting induced local lesions in genomes (TILLING) reference population from
the pea cultivar ‘Caméor’ and database (UTILLdb) resource is available for forward
and reverse genetics from INRA ( urgv.evry.inra.fr/UTILLdb; Dalmais et al. 2008).
The USDA G. A. Marx Pisum Genetic Stock collection was extracted from 80,000
lines Dr. Marx developed to study interactions between pea mutations (www.ars.
usda.gov/). Murfet and Reid (1993) developed and maintain developmental mutants
in Tasmania.
Only a small proportion (1876, approximately 2 %) of germplasm conserved
accessions, represent wild collected pea. Of these, there are 706 of P. fulvum, 624
P. s. subsp. elatius, 1562 P. s. subsp. sativum (syn. P. humile/syriacum) and 540
P. abyssinicum accessions (Smýkal et al. 2013). Wild Pisum species and subspe-
cies are expected to receive high interest as they are a fount of useful traits, for
example, resistance to pea seed weevil (Clement et al. 2009; Byrne et al. 2008;
Clement et al. 2002), rust (Barilli et al. 2010), a new resistance gene to powdery
mildew (Fondevilla et al. 2007b) and yield components (Mikič et al. 2013). Less
agronomically preferred germplasm (pigmented flower and pigmented seed coat)
were proven excellent sources of resistance to Aphanomyces root rot (Hamon et al.
2011) and Fusarium root rots (Weeden and Porter 2007; Grunwald et al. 2003).
Pisum sativum subsp. elatius and subsp. sativum are found in Europe, north-
western Asia and temperate Africa, while P. fulvum is found in the Middle East
(Abbo et al. 2008; Maxted and Ambrose 2001; Smýkal et al. 2011). A combined
gap analysis was conducted for six legume genera using more than 2000 unique
geo-referenced records. The resulting regression analysis illustrated that none of
the countries rich in Pisum species can be considered over-sampled, with Turkey,
Former Soviet Union (particularly the countries of the Caucasus), Syria and the
Balkans warranting further ex situ collection, as there is potential for finding
additional diversity (Maxted and Kell 2009). Passive in situ conservation of
legume species, including pea, occurs in several protected landscape ecosystems in
the Mediterranean and Near East regions, which are not aimed specifically to con-
serve wild crop relatives. Consequently, native legume populations are susceptible
to genetic erosion or even extinction (Maxted and Bennett 2001). Three reserves
were established within the Global Environment Facility project in Turkey (Kaya
et al. 1998). While no in situ reserves are formally established, wild pea populations
can still be found (Abbo et al. 2008, 2013; Mikič et al. 2012).
When available passport data on geographical origin are summarized, there is
a large bias (17 %) towards Western and Central European accessions, as these re-
gions represent modern pea-breeding activities. Substantially less well represented
are Mediterranean (2.5 %) and Balkan (2 %) regions, and the Caucasus (0.8 %) and
Central Asia (2 %) centres of pea crop domestication and diversity where higher
variation can be anticipated (Smýkal et al. 2013). There are still important gaps in
52 T. D. Warkentin et al.

the ex situ collections, particularly of wild and locally adapted materials (Maxted
and Kell 2009). An example of a significant gap was evident in a recent landrace
collection study of pea from China (Zong et al. 2009). Li et al. (2013) used eco-
geographical climatic characterization of 240 collection sites for 529 pea landra-
ces in China to identify locations with long-term abiotic stresses, especially during
the reproductive growth phase. This enabled 61 candidate accessions from these
stress sites to be prioritized for phenotypic validation to confirm tolerances to frost,
drought and heat. The Global Crop Diversity Trust (www.croptrust.org) has taken
on a full assessment of the gaps in Pisum germplasm held ex situ and in situ.
There are several international collection databases, which possess information
for pea, including: European Cooperative Program on Plant Genetic Resources
(ECPGR), Genetic Resources Information Network (GRIN) and System-wide
Information Network for Genetic Resources (SINGER) databases. Although these
databases provide information on around two million crop accessions, this infor-
mation is largely passport-based, and thus limited. The deposition and availability
of molecular, agronomic and morphological trait data is a very critical issue. In
the case of pea, this is largely handicapped by not being one of the CGIAR man-
date crops, and consequently with no coordinated international funding support.
Combining passport, morphological and genotypic data of many genebanks would
both improve germplasm management and enable search/query data exploration for
germplasm with multiple traits from a virtual world pea collection online (Furman
et al. 2006; Smýkal et al. 2008a).
A critical question remains, how can we efficiently utilize the positive alleles
found in the diversity of these Pisum germplasm resources? Climate change and
population pressures dictate the necessity of solving this challenge soon (Wheeler
and von Braun 2013). One proposal is to focus on reference sets for fine pheno-
typing and genotyping (Glaszmann et al. 2010). A recent meeting on crop wild
relatives proposed a bolder three-step approach to mine biodiversity for crop im-
provement, all highly relevant to pea (McCouch et al. 2013). The first step is to
sample sequence information (genotyping) of the genomes of all non-duplicate pea
accessions in the world’s gene banks. The second step is to analyse the phenotypes
of these pea accessions to evaluate their traits and overall agronomic performance.
The third step is to create an accessible bioinformatics infrastructure to catalogue
the diversity held in the world’s pea seed collections.
The following sections will summarize some of the progress to date in pea for
these three steps in achieving efficient utilization of germplasm resources. For the
analysis of pea diversity, simple sequence repeats (SSRs or microsatellites) have
been popular because of their high polymorphism and information content, codomi-
nance and reproducibility (Baranger et al. 2004; Loridon et al. 2005; Smýkal et al.
2008b; Zong et al. 2009; Kwon et al. 2012; Table 2.2). More recently, expressed
sequence tags (EST)-derived (eSSR) markers have become an important resource
for gene discovery and comparative mapping studies (De Caire et al. 2012; Mishra
et al. 2012). Alternately, retrotransposon repeats have been used to reveal diversity,
first applied in fingerprinting format of SSAP; Ellis et al. 1998; Vershinin et al.
2003) and developed into a high-throughput locus-specific genotyping technology
2 Pea 53

Table 2.2   Summary of the genotyping of some of the national pea germplasm ex situ collections
completed using DNA markers
Accessions Marker class Marker number Cluster Citation
(polymorphism) estimate
4538a RBIP, SSAP 27 3 Jing et al. (2012)
4429a SSR, RBIP 21, 25 6, 8 Smýkal et al. (2011)
3020a RBIP 45 7 Jing et al. (2010)
2120a SSR 21(115) 3 Zong et al. (2009)
373 SNP 384(356) – Deulvot et al. (2010)
310a SSR/RAPD 15/36(102) 3 Kwon et al. (2012)
299a Isozymes 18 – Swiecicki et al. (2000)
164 SSR, RBIP 10, 31 9 Smýkal et al. (2008b)
157 SSAP 129 4 Vershinin et al. (2003)
148a SSR/RAPD/ 66(121) 8 Baranger et al. (2004)
protein
122a RBIP 18(56) 4 Martin-Sanz et al. (2011)
86 RAPD, SSR 24(95), 25(54) – Tar’an et al. (2005)
71a SSAP 281 4 Ellis et al. (1998)
40 SRAP 7(162) 3 Esposito et al. (2007)
40 SSR 10(61) 2 Sarıkamış et al. (2010)
48a Gene 49 – Jing et al. (2007)
sequences
35 SSR 15(41) 2 Ahmad et al. (2012)
21 RAPD, AFLP 20(175), 2 Simioniuc et al. (2002)
11(462)
20 SSR 14 2 Ford et al. (2002)
RBIP  retrotransposon-based insertion polymorphism, SSAP sequence-specific amplification poly-
morphism, SSR simple sequence repeat, SNP single-nucleotide polymorphism, RAPD random
amplified polymorphic DNA and ISSRs, SRAP sequence-related amplified polymorphism, AFLP
amplified fragment length polymorphism
a
Contain core collection(s)

based on insertion/deletion of Ty1-copia PDR1 element and used for phylogeny and
genetic relationship studies in pea, providing a highly specific, reproducible and
easily-scorable method (Jing et al. 2007, 2010, 2012; Smýkal et al. 2008b, 2011).
Another class of Angela-family retrotransposon was identified and used for inter-
retrotransposon amplified polymorphism (IRAP) fingerprinting (Smýkal 2006;
Smýkal et al. 2009).
This knowledge has been used to characterize the distribution of genetic diver-
sity in Pisum (Baranger et al. 2004; Ellis et al. 1998; Jing et al. 2005, 2007, 2010,
2012; Pearce et al. 2000, Martin-Sanz et al. 2011; Majeed et al. 2012; Smýkal et al.
2008b; Sarıkamış et al. 2010; Tar’an et al. 2005; Vershinin et al. 2003; Zong et al.
2008, 2009) and these studies provide a consistent view. In spite of being a rather
small genus with two or three species, Pisum is very diverse and its diversity is
structured, showing a range of degrees of relatedness that reflect taxonomic identi-
fiers, eco-geography and breeding gene pools (Ellis 2011; Smýkal et al. 2011; Jing
54 T. D. Warkentin et al.

et al. 2012). Bayesian analysis of more than 4500 pea accessions from three large
collections (JIC, ATFC, CzNPC) analysed by retrotransposon insertion markers,
separated all wild peas ( P. fulvum, P. sativum subsp. elatius, and P. abyssinicum)
in one cluster, together with accessions of Afghan origin (Smýkal et al. 2011). An-
other cluster contained a large proportion of P. sativum subsp. sativum accessions of
Ethiopian origin (Smýkal et al. 2011). One hundred and forty accessions of Chinese
origin (Zong et al. 2009) were distributed more broadly into seven to eight clusters.
It was proposed that the distinct differentiation of the Chinese P. sativum genotypes
may in part reflect historic isolation of agriculture in eastern Asia from that in south-
ern Asia, Europe and northern Africa (Zong et al. 2009). Three relatively distinct
gene pools of Chinese pea landraces have been differentiated and formed under
natural and artificial selections (Li et al. 2013).
Recently, the analysis was complemented with addition of 1518 further Pisum
accessions selected from other major European collections leading to identification
of further diversity and formulation of a core collection (Jing et al. 2012). These
results showed that despite a wide diversity captured in historic cultivated germ-
plasm, relatively few genotypes with a high degree of relatedness have been used
as parents in modern pea-breeding programmes, leading to a narrow genetic base
of cultivated germplasm (Ellis 2011; Jing et al. 2010, 2012; Smýkal et al. 2011);
however, it is possible to broaden diversity using wild genotypes.
Although microsatellite and retrotransposon marker types are still used, their
potential is at its limits. With advances in model legume sequencing and genomic
knowledge, there is a switch to gene-based markers in pea (Aubert et al. 2006; Jing
et al. 2007; Bordat et al. 2011). This trend can be expected to further proliferate in
line with rapid advances in high-throughput single-nucleotide polymorphism (SNP)
generation and detection assays (Deulvot et al. 2010; Bordat et al. 2011). Recently,
a comprehensive transcriptome of pea was published (Franssen et al. 2011) and
several high-throughput pea transcriptome sequencing projects are underway and
should provide a complete set of pea genes. Much work remains to be done to
achieve the first step of genotyping all non-duplicate pea accessions as suggested
in the Feed the Future proposal. This is becoming increasingly feasible as the tech-
nologies for high-throughput sequencing continue to improve and costs are reduced
(Varshney et al. 2009).
It is a daunting task to phenotype the non-duplicate pea germplasm accessions for
the second step in the Feeding the Future proposal as it would involve tens of thou-
sands of accessions. Several large studies were published primarily for quantitative
disease reactions (Table 2.3). Germplasm data needs the same effort in curation as
the seed banks themselves, as well as an efficient method to share phenotypic data.
Generally, study authors are quite willing to share data, as indicated by Table 2.4.
The value of germplasm phenotyping is many fold. First is the value to all plant
scientists looking to germplasm to study specific traits and genes. Second is the
utilization by plant breeders for pea crop improvement. Once the variation is identi-
fied, breeders can test for heritability of a trait and move it into advanced breed-
ing/elite materials. If no useful germplasm is identified (e.g. pea cyst nematode
resistance), further screens can be conducted or alternative breeding methods pur-
sued (e.g. mutation, genetic modification).
2 Pea 55

Table 2.3   Replicated trials of agronomic and disease/pest reactions of sets of pea germplasm
Traits Accessions Citation
100 seed weighta 6827b Stout (personal communication)
Fusarium wilt, race 1a 3343b Muehlbauer, McPhee, McGee (personal
communication)
Fusarium root rota 3080b Grunwald et al. (2003)
Nematode resistancea 2253b Tedford and Inglis (1999)
Aphanomyces root rota 2195b Malvick and Percich (1999)
Mycosphaerella blighta 1993b Kraft et al. (1998)
Fusarium wilt, race 2a 1946b McPhee et al. (1999)
Proteina 1355b Jermyn and Slinkard (1977) and Coyne
Salinity, alkaline/acidity 780 et al. (2005b)
Leonforte et al. (2013)
Mycosphaerella blight 558 Zhang et al. (2006b)
Eco-geographical climatic 529 Li et al. (2013)
characterization
Sclerotinia white mould 497b Porter et al. (2009)
Mycosphaerella blight 581 Priliouk et al. (1999)
Seed mineral nutrientsa 458b Grusak et al. (2004)
Agronomic, biomass a
392b McPhee and Muehlbauer (2001)
Root traitsa 330b McPhee (2005)
Powdery mildew 317 Ali et al. (2007) and Azmat et al. (2012)
Pseudomonas syringae pv. pisi 242 Martín-Sanz et al. (2012)
Clover yellow vein virusa 215 Andrade et al. (2006)
Agronomic, mycosphaerella blight 169 Jha et al. (2013)
resistance, nutrition
Pseudomonas bacterial blight 169c Taylor et al. (1989)
Insects resistance (Melanagromyza 165 Mittal and Ujagir (2005)
phaseoli, Chromatomyia horticola,
Helicoverpa armigera)
Seed composition traits 157c Matthews and Ambrose (1994)
Agronomic, powdery mildew 153 Ali et al. (2007)
Agronomic 105 Amurrio et al. (1995)
Perenospora viciae 88 Davidson et al. (2004)
Fusarium wilt race 2 80 Bani et al. (2012)
Mycosphaerella blight 78 Fondevilla et al. (2005)
Agronomic 76 Amurrio et al. (1992)
Rust resistancea 52 Barilli et al. (2009)
Adaptive traits 37 Annicchiarico and Iannucci (2008)
Pea weevil resistancea 31 Clement et al. (2002)
a
Data available on Genetic Resources Information Network (GRIN)-Global www.ars-grin.gov/
npgs
b
USDA core collection included
c
John Innes Centre core collection

Eight core collections have been developed for pea by the national pea germ-
plasm programmes in Australia, China, Czech Republic, France, Poland, Spain,
the UK and the USA. The first pea core was constructed at JIC following Brown
(1989) recommendations and consists of 157 accessions with representatives from
56 T. D. Warkentin et al.

Table 2.4   Pea genes influencing plant and seed traits that are important in pea-breeding
programmes
Trait locus marker number of programmes
using MAS
Anthocyanin production A perfect 0
Enation virus resistance En 2
Powdery mildew resistance Er1 perfect 1
Fusarium wilt resistance (race 1) Fw several 0
Potyvirus resistance sbm1, sbm2 perfect 0
Absence of leaflets Af several 0
Winter hardinessa Hr perfect 0
Ascochyta blight resistanceb QTL several > 1
Frost tolerancec QTL several > 1
Photoperiod response Sn perfect 0
Photoperiod response Lf perfect 0
Aphanomyces toleranced QTL several > 1
Lodging resistancee QTL A001, A004 1
Seed shape R, Rb perfect 0
Seed compositionf rugosus perfect 3
Trypsin inhibitorc Tri perfect > 1
Pea Albumin 2 PA2 perfect 1
Protein contentg QTL several 0
Protein content Vc-2 perfect 0
a
Lejeune-Hénault et al. (2008) and
Weller et al. (2012)
b
Hamon et al. (2011)
c
Dumont et al. (2009)
d
Pilet-Nayel et al. (2002, 2005)
e
Zhang et al. 2006a
f
pgm mutants entered a number of
breeding programmes (UK, France,
Denmark) but markers were not used
for screening
g
Burstin et al. (2007)
MAS marker-assisted selection, QTL
quantitative trait loci

all the Pisum taxon phenotyped for nine seed quality characteristics (Matthews and
Ambrose 1994). Simon and Hannan (1995) developed the USDA core collection of
504 lines of P. sativum with a few P. sativum subsp. elatius based on geography and
flower colour.
The USDA core was refined to 310 accessions using 26 quantitative traits
(Coyne et al. 2005a). The Spanish core is a landrace collection based on passport
and quantitative data (Martin-Sanz et al. 2011). The Polish core of 266 accessions,
selected for diversity of type, taxon and described genes, was studied using iso-
zymes finding rare alleles in the landrace/primitive types (Swiecicki et al. 2000).
Baranger et al. (2004) reported on the INRA core collection of 43 Pisum accessions
selected based on protein and DNA marker diversity. With further use of molecular
data, Zong et al. (2009) created a core of 146 Chinese landraces using molecular
2 Pea 57

diversity data and then compared it to a core based on geography finding the mo-
lecular core more genetically diverse. Smýkal et al. (2011) proposed the application
of a model-based method for the formation of a molecular/eco-geographically di-
verse international pea core collection for a practical start to phenotyping the non-
duplicative pea germplasm. Core Hunter programme was developed and applied
to pea germplasm, resulting in a representative set from the entire Czech National
Pea Collection (CzNPC), Sumperk (1283 accessions), as well as six European com-
posed collections (4429 accessions; Jing et al. 2012; De Beukelaer et al. 2012).
International efforts are underway to phenotype and genotype various compositions
of fluid cores based on volunteer entries and not a strategic core per se.
A major shift to allele mining (Reeves et al. 2012) and association mapping
(Rafalski 2010) has occurred in pea germplasm collections. Rapidly, transcrip-
tomes for marker (EST-SSRs and SNPs) development (Lucau-Danila et al. 2012;
Kaur et al. 2012; Zhuang et al. 2012; McGee 2012a; Sanderson et al. 2011; Frans-
sen et al. 2011; Deulvot et al. 2010; Duarte et al. 2014; Sindhu et al. 2014) are
available, and we can imagine a complete gene list for pea in the near term. This
shift to gene-based germplasm exploitation has necessitated additional approach-
es to pea genetic resource conservation. Traditionally, USDA pea accessions
were maintained to preserve genetic diversity (e.g. landraces) within an acces-
sion. Pure lining accessions and creating a new pure-line accession has increased
the utility of germplasm collections (Nelson 2011). Consequently, precision in
phenotyping is achievable and high-throughput genotyping is feasible. Further,
pure lining is essential for trait associations for genome-wide association stud-
ies (GWAS) using genotyping-by-sequencing and resequencing approaches (Bra-
chi et al. 2011; Elshire et al. 2011). Genotyping-by-sequencing is very effective
at generating 10,000  plus  SNP genotypes in pea populations (Mazourek, personal
communication). Larger GWAS studies on a global scale are in progress (Warkentin,
personal communication). The choice of germplasm, extent of genome-wide linkage
disequilibrium (LD), and relatedness within the population determine the mapping
resolution, which together with marker density and statistical methods, are critical to
the success of association analysis. Estimates of the rate of LD decay in pea within
progressively more distantly related accessions tentatively suggest high LD among
cultivars (Jing et al. 2007), comparable to rice and maize. This estimate should be
considered preliminary, but would imply that a greater number of SNPs (hundred
thousands) might be required for effective genome-wide association mapping.
A project aimed at sequencing the pea genome started in 2013 (McGee 2012b).
Production of a draft pea genome will open opportunities for whole genome rese-
quencing of pea germplasm resources for high-throughput SNP and gene identifica-
tion similar to the case, for example, in rice (Xu et al. 2011) and chickpea (Varshney
et al. 2013).
Databases have become essential to curate the growing genomic and agronomic
information of pea and related legumes. Several of the pea transcriptomes are avail-
able online from Genbank. More useful are pea transcriptomes with user-friendly
tools such as GBrowse available online from Legume Information System (www.
comparative-legumes.org, Gonzales et al. 2005), KnowPulse (www.Knowpulse2.
usak.ca, Sanderson et al. 2011) and Cool Season Food Legume Genome Data-
58 T. D. Warkentin et al.

base (www.gabcsfl.org, Main et al. 2013). Further, databases are needed to host
­increasingly larger phenotype and genotype data sets, such as GRIN-Global and
Germinate 2.1 Pea (http://bioinf.scri.ac.uk/germinate_pea/app/index.pl; Lee et al.
2005). These databases use open source software so data can be extracted and
imported into various software packages for analyses. A recent example of combin-
ing passport data with habitats for ecogeographic analysis identified potential sites
for pea landraces with abiotic stress tolerances (Li et al. 2013).

5  Major Pea-Breeding Achievements

5.1 Canada

Over the past 20 years, steady progress has been made in improving the agronom-
ic and quality characteristics of field pea as evidenced in the cultivars released.
Yield gains in yellow cotyledon field pea cultivars, the most widely grown market
class in western Canada, has been approximately 2 % per year over the past 20
years when comparing the relative yields of prominent varieties including Carne-
val, Grande, Alfetta and Eclipse (released between 1993 and 1999) versus Crop
Development Centre (CDC) Golden, CDC Meadow, Agassiz and CDC Amarillo
(released between 2002 and 2012), based on the post-registration Saskatchewan
regional variety trials. Using the performance data of the second year entries in the
Western Canada Field Pea Cooperative Variety Registration Tests in 2001–2012,
the annual yield increase for yellow cotyledon pea entries arising from all breeding
programmes represented was 122 kg ha−1 which is approximately a 3 % annual gain.
Similar yield gains were achieved for green cotyledon entries over this time period.
The most important disease of field pea in western Canada is the Ascochyta
blight complex (referred to as black spot in Australia), of which Mycosphaerella
pinodes is the most important component. Twenty years ago, most cultivars were
rated as having ‘poor’ resistance to mycosphaerella blight, while now most cultivars
are rated as having ‘fair’ resistance, similar to that of the control cultivar. Radley.
Kraft et al. (1998), in an extensive evaluation of pea germplasm under conditions
of severe mycosphaerella blight, reported that no germplasm accessions had resis-
tance superior to Radley. Powdery mildew has been considered the second most
important disease of field pea in western Canada, as epidemics arise in most field
pea regions in most years, typically late in the season. Twenty years ago, none of the
pea cultivars recommended for western Canadian production was resistant to pow-
dery mildew, while today, nearly all cultivars are resistant. Resistance in all cases
is based on the single recessive gene er-1. Aphanomyces eutiches and various Fu-
sarium diseases are becoming important in some regions of western Canada and are
gaining increased attention from plant pathologists and breeders. Typically, these
soil borne pathogens are associated with the wettest regions of western Canada, and
those wet regions in which field pea has been grown extensively over the past 15
years or more.
2 Pea 59

After grain yield, typically the most desired trait of field pea growers is lodg-
ing resistance which facilitates harvest and reduces humidity in the canopy. Over
the past 30 years, a nearly complete shift has been made in western Canada from
‘leafy’ cultivars to ‘semileafless’ cultivars. Semileafless cultivars are those carrying
the afila gene that results in the replacement of leaflets by tendrils. Lodging rat-
ings began in official trials in 1993 through the use of a 1–9 scale (1 = no lodging,
9 = completely lodged). The advantage of the semileafless trait for improved lodg-
ing resistance became apparent as varieties with this leaf type typically had lodging
scores 2–3 points better than varieties with the leafy trait. The semileafless trait is a
prerequisite for improved lodging resistance but, in addition, increased stem stiff-
ness is required for good field resistance. Over the past 20 years, the typical lodg-
ing score of widely grown field pea cultivars has improved from scores of 5–6 to
scores of 3–4 at present. Thus, lodging resistance remains a key breeding objective.
Using the performance data of the second year entries in the Western Canada Field
Pea Cooperative Variety Registration Tests in 2001–2012, lodging has been signifi-
cantly reduced through breeding, with an annual reduction of 0.04 based on the 1–9
scale. It was interesting to note that, in general, high yield and lodging resistance
were positively associated.
As yield increased, days to maturity also increased at a rate of 0.37 day per year
(Western Canada Field Pea Cooperative Variety Registration Tests 2001–2012),
indicating the challenge of developing high-yielding but early-maturing varieties.
Under western Canadian conditions, field pea is typically the first crop harvested,
thus development of earlier maturing varieties is not critical, except for the benefits
of spreading out the harvest workload. The height (vine length) of breeding lines
has been increasing at a rate of approximately 2 cm/year (2001–2012). Plant height
was also positively related to yield; thus, it is likely that breeders have inadvertently
selected taller varieties while selecting for higher yield.
Retention of green cotyledon colour, or bleaching resistance, is a critical qual-
ity trait in green field peas. Colour must be stable after harvest and after at least
1 year of storage. Steady improvement has been achieved over the past 15 years
in green cotyledon bleaching resistance. Since 1994, green cotyledon varieties
in official registration trials have been rated for bleaching resistance using a 1–5
scale, where 1 = no bleaching, 5 = completely bleached, and a key check cultivar
arbitrarily assigned a score of 3 (or 2.5) in each trial. Most cultivars recommend-
ed for production in western Canada are now rated as having ‘good’ resistance to
bleaching, while 15 years ago, most were rated as having only ‘fair’ resistance.
Marrowfat cultivars with large seed size (350–400 mg), fair–good green cotyle-
don bleaching resistance, powdery mildew resistance and yield 90 % of that of the
yellow check cultivars are now available. Marrowfat cultivars with good lodging
resistance are still lacking. Marrowfat peas are typically used in snack foods in Asia
and as ‘mushy peas’ in the UK. Maple pea cultivars with powdery mildew resis-
tance and yield 90 % of that of the yellow check cultivars are now available. Maple
peas are typically used in seed mixtures for domestic and wild birds. One recently
released dun pea cultivar (CDC Dakota) with powdery mildew resistance and yield
greater than the yellow checks is now under pedigreed seed m ­ ultiplication. Dun peas
60 T. D. Warkentin et al.

are typically dehulled and split then used in dhal applications, similar to regular yel-
low cotyledon peas. Forage pea cultivars which produce large biomass, small seed
size, good lodging resistance, powdery mildew resistance and grain yield 90 % of
that of the yellow check cultivars are now available. Forage peas are typically grown
in mixtures with annual cereals such as oat, barley or triticale, harvested when the
cereal reaches the soft dough stage and used as forage or silage for ruminants.

5.2  The USA

Pea-breeding achievements in the USA have been similar to those described above for
Canada. Pea breeding has a longer history in the USA, particularly for the vegetable
pea types. The US programmes have made excellent progress in green seed bleaching
resistance, as well as in improving resistance to root rot pathogens and viruses.

5.3 Australia

Field pea production across the Mediterranean-type climates of Southern Australia

was initially based on introduced and improved landrace plant types (i.e. dun
types) that characteristically grew vigorously over winter and were tall (i.e. long
internodes). The development of the modern semidwarf, semileafless varieties for
Australia followed a breeding effort over more than 20 years to pyramid genes
for adaptation and plant type. This process primarily involved complex crossing
and recurrent selection programmes between introduced spring-type semileafless/
semidwarf germplasm and Australian-adapted conventional cultivars. Specifical-
ly, the newly developed semidwarf germplasm was taller, more active in winter
growth and showed reproductive development that commenced in mid-spring to
avoid major frost risk (Leonforte and Brouwer 1999). A critical development for
Australia was the trait pyramiding of lodging resistance (Leonforte et al. 2006)
with reduced pod parchment (i.e. sugar pod trait) to reduce pod shattering at har-
vest. The unique combination of these traits led to the release of the first broadly
adapted semidwarf cultivar Kaspa which has since rapidly dominated production
across Southern Australia. The focus of recent breeding has been to optimize the
phenology of the Kaspa plant type, which is late flowering and determinate for
shorter season climates, particularly in Western Australia (WA). This has led to
recent releases such as Pulse Breeding Australia (PBA) Gunyah and PBA Twilight
that are early flowering, and display more stable yield than Kaspa in short season-
climates. In Australia, the average rate of genetic gain for yield is estimated to be
around 1–1.3 % per annum from field pea breeding over the past 20 years.
Field pea, like other pulses, is comparatively sensitive to a number of abiotic stress
factors, particularly involving soil nutrition such as salinity and ­alkaline-induced bo-
ron toxicity, reproductive frost damage, heat stress (Dita et al. 2006) and specific
herbicide actions. In Mediterranean-type climates, the main limitations to field pea
2 Pea 61

production occur during reproductive development, as developing flowers, pods and

seeds are highly sensitive to abortion and damage from increasingly high and low
temperature extremes and water-limiting stress. A low-cost approach of long-term
and strategic selection for grain yield in high-risk environments has, however, proven
to be highly successful for genetically improving grain yield reliability of the pea
crop for Mediterranean climates per se (McMurray et al. 2011). The component traits
that contribute to higher water-limiting tolerance relate mostly to phenology and bio-
mass production prior to flowering (Sadras et al. 2012). Higher stress tolerance has
been identified in landrace accessions for toxicity to boron (Bagheri et al. 1994),
salinity (Leonforte et al. 2013) and for iron deficiency (Kabir et al. 2012), and new
seedling-screening techniques to improve tolerance levels are now being routinely
used in Australia (Leonforte et al. 2009). There is also preliminary evidence for use-
ful variation for heat stress tolerance during flowering and podding (Petkova et al.
2009), and some evidence for higher tolerance to reproductive frost damage based on
the yield response of the Australian variety ‘Sturt’ (Hawthorne 2007) and pod dam-
age (Shafiq et al. 2012).
Field pea is affected by many fungal and viral diseases, bacterial blight and
pests, particularly in winter-sown cropping regions where there is a long vegeta-
tive growth phase. Black spot disease (Ascochyta blight), described earlier in this
chapter, is a major limitation to field pea production in Southern Australia, and its
management was recently reviewed by Khan et al. (2013). There is no robust source
of resistance to this disease although the development of an erect plant type during
vegetative growth has most likely improved overall resistance of the crop (Le May
et al. 2005). Making large numbers of intercrosses between partially resistant lines
and recurrent selection should help to develop more resistance (Fondevilla et al.
2007a). One Greek and one Ethiopian line have been used in a study involving
two cycles of recurrent selection by Beeck et al. (2008), which showed significant
simultaneous improvements in black spot resistance as well as stem strength.
Downy mildew caused by Peronospora viciae (Berk.) Caspary and powdery
mildew caused by Erysiphe pisi DC. cause disease periodically in pea in Australia.
Downy mildew is prevalent in cooler, wetter growing regions (i.e. southwestern
Australia) and more typically affects seedling growth, but rarely causes systemic
whole plant infection. Major genes for resistance have been identified and effective
screening established (Davidson et al. 2004). However, rapid pathogen specializa-
tion is a widespread problem in many regions including Australia (Davidson et al.
2011). Powdery mildew can be a serious disease of field pea but more so in warmer
and more humid Mediterranean climates at the start of flowering (i.e. South Austra-
lia, southern New South Wales). There are two major genes for resistance, er1 and
er2, conferring high and stable resistance to this disease (Katoch et al. 2010) which
have been used extensively to develop resistant varieties globally. A third major
gene (er3) conferring resistance has also been identified from P. fulvum Sibth. and
Sm. (Fondevilla et al. 2008) but has not been exploited in breeding. Other more
regionally important foliar fungal diseases include pea rust ( Uromyces pisi (Pers.)
Schrot. ; Barilli et al. 2009) and septoria blotch ( Septoria pisi West). Both can cause
severe damage in wet seasons; however, only moderate resistance has been found
62 T. D. Warkentin et al.

to rust (Rai et al. 2011) and low resistance to septoria blotch (Leonforte et al. 2004).
Bacterial blight caused by Pseudomonas syringae pv. pisi Sackett and pv. syringae
van Hall are other localized but potentially devastating diseases that can occur in
cool temperate and Mediterranean-type climates. Breeding has mostly focused on
pyramiding available major genes for race-specific resistance to pv. pisi (i.e. from
seven races; Hollaway and Bretag 1995, Elvira-Recuenco et al. 2003).
Many aphid-transmitted viruses produce a range of disease symptoms individu-
ally or in combination in field pea. These include cucumber mosaic virus (CMV),
pea early browning virus (PEBV), pea enation mosaic virus (PEMV), luteo viruses-
pea leaf roll virus (PLRV) or bean leaf roll virus (BLRV), poty viruses-bean yellow
mosaic virus (BYMV) and pea seed-borne mosaic virus (PSbMV), alfalfa mosaic
virus (AMV), pea streak virus (PeSV) and red clover vein mosaic virus (RCVMV).
Selection for major gene resistance to PSbMV and poty viruses is now incorporated
into breeding strategies (van Leur et al. 2007).
Root rot diseases are less widespread in Mediterranean regions. They are gener-
ally caused by a combination of several common soil fungal pathogens: Aphanomy-
ces root rot ( A. euteiches Drechs.), Pythium tip blight (Pythium ultimum), Fusarium
root rot ( Fusarium solani f. sp. pisi (Jones) Snyder and Hansen), Rhizoctonia root
rot ( Rhizoctonia solani Khun) and Fusarium wilt ( Fusarium oxysporum Schlecht.).
Robust resistance is only found for Fusarium wilt; efforts are focused on improving
resistance to Aphanomyces. Only partial resistance controlled by several quantitative
trait loci (QTL) is available for this disease (Pilet-Nayel et al. 2002, 2005). In pea,
useful pest resistance has only been identified for pea weevil ( Bruchus pisorum L.)
which is a widespread global problem. Resistance genes are in the secondary gene
pool ( P. fulvum; Clement et al. 2002). Transfer of genes for resistance from P. ful-
vum has now been completed by introgression from P. fulvum into cultivated field
pea through backcrossing to produce several advanced pea weevil resistant lines
(Clement et al. 2009; Aryamanesh et al. 2012).

5.4 Europe

A major achievement in modern pea breeding in Europe was the commercial

deployment of the afila (semileafless) trait in the 1970s. The first successful cul-
tivar was Solara from Cebeco Zaden (now Limagrain). Solara became a parent
in numerous crosses by breeders throughout Europe over the next decade. Field
pea-breeding activities expanded in Europe in the 1980s and 1990s as EU policy
provided subsidies for the local production of high-protein crops to address the
massive deficit in protein in the continent. Pea breeding in Europe is conducted
primarily in the private sector with the public sector focused on upstream research.
The companies having the most market share in the pea industry in Western Europe
in the 1980s and 1990s were Cebeco Zaden (The Netherlands), Florimond Deprez
(France), Serasem (France), Svalof Weibull (Sweden), Danisco Seeds (Denmark),
DLF Trifolium (Denmark) and Sharpes (UK). In addition, many other small com-
panies were actively involved. However, with changes in EU policy that were less
2 Pea 63

favourable to protein crops, production of field pea and faba bean in Europe has
declined over the past 15 years. This led to consolidation and reduction in pea-
breeding activity. Currently, Limagrain, that consolidated the former Cebeco Zaden,
DLF Trifolium and Sharpes programmes, is the largest pea-breeding programme
in Europe, followed by Florimond Desprez and RAGT, that grouped breeding pro-
grammes from Serasem and GAE Recherche. In France, breeding programmes con-
ducted during the past 20 years have mainly targeted yield, lodging and disease
resistance. Post-registration trials conducted in 2012 and 2013 compared historical
short semileafless varieties (Solara registered in 1987, Baccara registered in 1992)
with the most recent cultivars (e.g., Arvalis registered in 2013). A yield increase
of 1.1 t ha−1 has been achieved to reach 6.6 t ha−1, accompanied by a much better
lodging resistance (56 cm height at harvest for Kayanne, as compared to Solara,
26 cm, or Baccara, 22 cm).
Similar trends occurred in Eastern Europe in that pea-breeding expansion was
led by the release of semileafless, semidwarf varieties such as Sum in Poland in the
1980s. Pea yields increased by 50 % over the period 1977–1995 in Poland primarily
due to the cultivation of these new varieties (Swiecicki et al. 1997). Also in Poland,
Prusinski (2007) estimated annual pea yield increases at 42–45 kg ha−1 from 1989
to 2006, and Boros estimated annual pea yield increases at 46 kg ha−1 for edible
peas and 44 kg ha−1 for fodder peas for the period 2001–2010. Currently grown
pea varieties have yield potential of 5–6 t ha−1. The major pea-breeding companies
centred in Eastern Europe are Poznań Plant Breeders Ltd., Plant Breeding Smolice
Ltd., Danko Plant Breeders Ltd., and Selgen.
To supplement the benefits of cultivars with improved lodging resistance, use of
fungicides for management of Ascochyta blight is most common in Europe, followed
by North America and Australia. Breeding for powdery mildew resistance is com-
mon in Eastern European breeding programmes as the disease can cause yield losses
of 10–65 % by significantly reducing seed weight (Ondrej et al. 2003). Improved
resistance to downy mildew and Fusarium wilt races 1 and 2 are also important
breeding achievements in Eastern Europe. In warm seasons, aphid dissemination of
viral diseases is more prevalent. Their importance varies across regions depending
on alternative hosts, seed transmission and aphid control. Screening methods were
developed for virus diseases PEMV and PSbMV (Hochman and Dostalova 2010).

5.5 India

Field pea breeders have mainly concentrated on leaf type (afila), dwarf plant type
and powdery mildew resistance over the past three decades. Recently, varieties have
been developed which combine resistance to powdery mildew with dwarf stature and
semileafless trait. Each of these characters has played a significant role in reverting
the negative trend of area under the crop. The semileafless trait allows for penetra-
tion of sunlight to the lower canopy, and mechanical support to prevent lodging and
reduce bird damage. The dwarfing gene has enhanced productivity through improved
response to fertilizers, irrigation and dense plant population. Recently, several short
64 T. D. Warkentin et al.

duration varieties like Adarsh, Vikas and Prakash have been developed which escape
terminal stress and have the yield potential of 2.5 t ha−1. During the 1980s, a severe
incidence of rust occurred in pea in northern and eastern regions of India and screen-
ing work initiated for resistance to this disease. In the past two decades, efforts have
been made to identify slow rusting lines like FC1. A few rust tolerant varieties like
HUDP 15, Prakash, Swati, Aman and Pant P 42 are now available.

5.6 China

Thirty years ago, local cultivars of field pea dominated the main dry pea production
areas in rain-fed hilly fields of China. Since then, 45 cultivars were bred and re-
leased by pea breeders from nine public research institutions. Of these, 34 were for
dry pea production, and 11 were for various types of vegetable production. Among
the 34 dry pea cultivars, 26 have normal leaf type and 8 have semileafless type.
Among the vegetable cultivars, six varieties are snow pea, three are snap pea, two
are tendril-less for leaf vegetable production. All cultivars were bred through single
plant or pure-line selection from segregating populations derived from crosses be-
tween cultivated genotypes or from natural mutations of local cultivars. Interspecies
crosses and induced mutation methods were not applied.
While the majority of this chapter is focused on developments in dry pea breed-
ing, many similar improvements in yield and disease resistance have been achieved
in vegetable pea breeding over the past three decades. In North America and
Europe, the majority of the vegetable pea-breeding activities have been conducted
in a few private companies, with the leading contributors being General Mills, Inc.
(Le Sueur, Minnesota), Syngenta (Nampa, Idaho), Crites Moscow (Moscow, Ida-
ho), Pure-line Seeds (Moscow, Idaho), Brotherton Seed Company (Moses Lake,
Washington) and Seminis (Filer, Idaho). These companies have released many
varieties with key improvements including increased yield, resistance to soil-borne
and foliar fungal pathogens, resistance to aphid-vectored virus diseases and resis-
tance to abiotic stresses. In addition, they have commercialized snap pea and petit
pois types, improved synchrony of harvest, and improved flavour and colour.

6  Specific Goals in Current Pea-Breeding Programmes

The objectives of the Canadian programmes (University of Saskatchewan at

Saskatoon, Saskatchewan and Agriculture and Agri-Food Canada at Lacombe,
Alberta) include the development of early maturing, high-yielding field pea culti-
vars with resistance to powdery mildew and improved resistance to mycosphaerella
blight and lodging, with superior quality for export and domestic markets. Emphasis
is placed on the development of cultivars for human consumption markets, with
approximately 60 % of the activity focussed on the yellow cotyledon market class,
30 % of the activity on the green cotyledon market class and 10 % on speciality field
pea markets including marrowfat, dun, maple and forage.
2 Pea 65

In the USA, there are two public pea-breeding programmes, that is, the US-
DA-Agricultural Research Service (ARS) programme is located at Washington
State University in Pullman, Washington, and the North Dakota State University
programme is based in Fargo, North Dakota. A private seed company, ProGene,
LLC, is based in Othello, Washington. All three programmes breed peas for the
target environments of the Pacific Northwest and Northern Plains regions of the
country. Typically, the more arid regions favour green cotyledon varieties and yel-
low cotyledon varieties are grown in the more humid regions of the northern plains.
Testing and evaluation of advanced lines is accomplished through a network of
regional variety trials and state-wide variety trials conducted by agronomists at state
universities and experimental stations. The primary breeding objectives of the pro-
grammes are similar to those of the western Canadian programmes. In addition,
there is considerable breeding effort focused on the development of autumn-sown,
food quality cultivars. Disease resistance breeding that is important in the USA
includes resistance to PSbMV, BLRV, PEMV, Aphanomyces root rot, Fusarium root
rot, Fusarium wilt (races 1 and 2) and powdery mildew. Recently, efforts have also
been directed to screening for tolerance to low pH soils and aluminium toxicity.
In Australia, the national field pea-breeding programme is centred at Horsham,
Victoria, with testing sites in key locations across the continent. The major objec-
tives of the programme are similar to those described for western Canada, with
specific emphasis on the dun seed type based on the cultivar Kaspa (light tan-red
seed coat), resistance to bacterial blight, downy mildew, BLRV and PSbMV, toler-
ance to subsoil boron and NaCl toxicity, and reproductive frost damage.
In India, breeding efforts are focused on yellow cotyledon pea with non-
pigmented seed coats, with smaller programmes on vegetable pea. Current breeding
objectives include increasing harvest index, development of short duration (100–
110 days) varieties, resistance to pea weevil, tolerance to terminal drought through
early maturity and frost tolerance.
In China, semileafless leaf type is one of the specific breeding goals for dry pea
production. Vegetable pea varieties are being developed with normal leaf type and
short duration fitted to a two crop season, such as pea–maize, in areas of China at
40°N and above. Vegetable varieties of pea are special breeding goals for production
around cities in southern China.
In Western Europe, high yield, lodging resistance and improved resistance to
Aphanomyces root rot are the key objectives for spring field pea. Over the past four
decades, the target market has primarily been the compound feed industry, and thus
less emphasis is placed on seed visual quality than is the case in North America, but
the seed protein content is critical for registration. The recent emergence of more
frequent drought and heat waves during the reproductive period has led to consider-
ation of tolerance to these traits in breeding programmes. In France, some breeding
companies and the French national agronomic research institute INRA have under-
taken to develop autumn sown field pea varieties, as a strategy to increase yield
potential and stability through a longer crop cycle, higher biomass production and
earlier maturity to avoid late-cycle drought and heat stress (Hanocq et al. 2009).
The development of winter field pea varieties has added more breeding targets to
those listed above for spring peas, particularly the improvement of winter ­hardiness,
66 T. D. Warkentin et al.

the fitting of flowering time to avoid flower initiation during frost at the end of
winter, and also seed filling during drought and heat at the beginning of summer,
and Ascochyta blight resistance. Following recent research, results on the control
of flowering time and frost tolerance in pea (Lejeune-Hénaut et al. 2008), a new
pea type registration has been launched: The winter peas responsive to photoperiod.
In Eastern Europe, breeding objectives for field pea include development of high-
yielding varieties with improved resistance to lodging, multiple disease resistances
( E. pisi, Fusarium subsp., Ascochyta, Uromyces), virus resistances (PEMV, PS-
bMV), resistance to abiotic stress and improved grain quality. For green cotyledon
peas, desirable traits include optimal size, shape and colour, high content of resis-
tance starch, and high content of vitamins and carotenoids (luteins, β-carotene).

7  Breeding Methods and Specific Techniques

At the University of Saskatchewan, approximately 300 new crosses are generated

each year during the course of three crossing cycles (summer, fall and winter). The
F2-derived family method is used. Visual selection for seed quality traits is con-
ducted in each generation, including size, shape, dimpling, cotyledon and seed coat
colour and seed coat integrity. Selection for powdery mildew resistance is conducted
in the F1 generation of complex crosses, or the F2 generation of single crosses based
on natural epidemics late in the season. Approximately, 10,000 F2-derived F3 lines
are evaluated in single replicate micro-plots. Approximately 15–25 % of these lines
are advanced each generation, based on selection for lodging resistance, maturity,
mycosphaerella blight resistance and seed quality, with the number of evaluation
sites increasing through the generations. The Agriculture and Agri-Food Canada
pea-breeding programme uses pedigree selection in combination with single-seed
descent. The two-year Field Pea Co-operative (Co-op) Registration Test is grown
at 10–12 locations in western Canada. Data summaries from the most promising
varieties arising from the Co-op Test are presented to the Prairie Recommending
Committee for Pulse and Special Crops. This group of experts votes on whether or
not to recommend to the Canadian Food Inspection Agency that individual varieties
be registered in Canada. Successful varieties are evaluated in provincial regional
trials in western Canada to further evaluate performance in specific pea-growing
regions. Data arising from these trials are summarized and published in provincial
seed guides for use by seed growers, farmers and agronomists.
The USDA-ARS programme employs a pedigree breeding method. Approxi-
mately, 300 crosses are made each year. F1’s are grown at the WSU Spillman
Agronomy Farm (SAF). F2’s are typically sent to a counter-season nursery and se-
lected for simply inherited traits. The F3 through F6 generations are grown at SAF
in paired rows and evaluated within and between families. Single plants are selected
in F3–F5, and selected bulks are taken in F6. The selected F7’s are evaluated for
agronomic performance and produce sufficient seed for the preliminary yield trials
(PYT) which are grown at one location. Breeding lines advanced from the PYT are
2 Pea 67

entered into advanced yield trials (AYT) that are typically grown at four locations
for several years. Lines selected from the AYT are entered into the appropriate state-
wide and regional yield trials.
The field pea-breeding programme at Horsham, Australia, includes 450–550
crosses per year followed by pedigree selection. Individual plant or pod based
selections (3000–5000) will be taken from F2 to F4 segregating populations in the
field. Single-seed descent, recurrent and mass selection and summer seed multipli-
cation are strategically employed. The introgression or maintenance of key major
genes (e.g. A, I, b, af, le, er 1, sbm-1, rpv, p, v) is achieved through backcross-
ing, pedigree systems of selection and effective phenotyping (e.g. downy mildew,
boron toxicity, PSbMV). For more complex traits (e.g. blackspot, bacterial blight,
BLRV), segregating populations are maintained as bulk lines to increase frequency
combinations of minor genes (e.g. improved lodging resistance) that will additively
contribute to improved variation. Mass selection is used for eliminating genes in
relation to poor grain quality traits (e.g. shape, size, dimples and colour). Progeny
lines (i.e. 3000–5000) are initially tested in short paired rows. Preliminary testing
occurs in short row plots at four to five regional locations with concurrent seed
multiplication and yield testing in Victoria. Fixed line selections are grown over
several years to permit observations of performance (e.g. grain yield) under differ-
ent environmental conditions and enable the selection of lines that are more broadly
adapted over years and environments.
Pea-breeding techniques in Europe are generally similar to those described
above for North America and Australia. Promising lines are submitted by the breed-
ing company to official testing for assessment of variety value for cultivation and
use (VCU) and testing for distinctness, uniformity and stability (DUS). Positive
results of the VCU and DUS assessments are a precondition to variety entry into
a national list. DUS testing is required for granting of Plant Breeders’ Rights and
is conducted under The International Union for the Protection of New Varieties of
Plants (UPOV) guidelines. The VCU process must be conducted in individual coun-
tries, while the DUS process need only be conducted once. Cultivars on the national
list of one European country are eligible for production and marketing in the entire
European Union territory after their admission to the Common Catalogue.
Several national and international organizations are actively engaged in
field pea improvement in India, with the largest at the Indian Institute of Pulses
Research (Kanpur), followed by GB Pant University of Agriculture and Technol-
ogy (Pantnagar), CCS Haryana Agricultural University (Hisar), Punjab Agricultural
University (Ludhiana) and Banaras Hindu University (Varanasi). In general, these
field pea-breeding programmes include 250–300 crosses per year followed by
pedigree selection. Single plant-based selections (2000–3000) are usually taken
from F2 to F4 segregating populations in the field based on improved grain yield,
dwarf plant type and powdery mildew resistance. The introgression of major genes
is achieved through backcrossing, pedigree selection and phenotyping. Fixed line
selections are grown over 3 years to permit observations of performance under
different environmental conditions and enable the selection of lines that are more
broadly adapted over years and environments.
68 T. D. Warkentin et al.

Recently, EMS mutation methods have been introduced in pea variety im-
provement in China, by establishment of M2 to M3 populations of several green
pea and dry pea varieties. This has led to the release of varieties including Zhong
Wan No. 6.

8 Integration of New Biotechnologies in Breeding

Programmes

Over the past three decades DNA markers, molecular techniques and genomic tools
have been developed for pea (Smýkal et al. 2012), and it is of interest to ask how
many of these are being used in public or private breeding programmes.

8.1  Gene Identification and Novel Allele Discovery

In pea, considerable effort has been directed towards gene identification. The genetic
diversity within available germplasm collections is reasonably well characterized,
having been analysed for variation in a wide range of molecular marker types (see
‘Genetic Resources and Utilization’ Sect. 4). Many polygenic traits important for
pea breeding (flowering time, seed size, lodging susceptibility, resistance to many
pests and pathogens—see sections above) have been subjected to genetic analyses,
and at least specific regions of the genome influencing the respective trait have
been determined. In a number of cases, particularly flowering time, plant height
and branching and nodule formation, the actual genes have been identified and
their interrelationships characterized. With the availability of many convenient SSR
and SNP markers in pea, breeders are now routinely investigating traits important
for specific habitats (boron susceptibility, bleaching susceptibility) or crop types
(winter hardiness, amylose level). Mutation screening populations, and more
recently TILLING populations, have been developed, greatly facilitating the rec-
ognition and confirmation of those genes conditioning specific phenotypes. For
many crops, TILLING may be regarded as a useful and more practical alternative
to GM technology for knocking out expression of target genes. TILLING in pea
has been extremely successful (http://www-urgv.versailles.inra.fr/tilling/pea.htm,
Dalmais et al. 2008), and a wide array of genes and alleles has been generated for
the research community. TILLING targets have first been designed to prove or dis-
prove the role of candidate genes in the processes involved, for example, internode
length (le) and tendril-less (tl) mutations, where natural mutants have been available
previously (Dalmais et al. 2008; Hofer et al. 2009).
Once the function of a gene is validated, reverse genetic methods allow the
identification of novel alleles in mutant and/or genetic resource collections. TILL-
ING or EcoTILLING screens can identify genotypes carrying different alleles of
the genes of interest for their testing and subsequent use as donor progenitors in
2 Pea 69

b­ reeding programmes (e.g., Weller et al. 2012). It may be expected that novel mu-
tants affecting flowering time, seed composition and yield are in the pipeline and
will be in demand by breeders. Equally, the development of high-throughput and
rapid screening methodologies for the detection of deletion mutants for seed qual-
ity targets (Domoney et al. 2013) may be expected to expand the potential for the
identification and introduction of novelty by breeders.

8.2  Marker-Assisted Selection

In addition to the identification of genes responsible for or influencing important

traits, the mapping of these genes on the pea genome and the understanding of the
interactions of genes involved in the expression of quantitative traits have allowed
pea breeders to organize their breeding activities and objectives into programmes
that are more efficient and predictable. Genotypes with specific flowering times,
pathogen responses, seed or pod qualities (see e.g., Fig. 2.2), or other traits can be
selected before crosses are designed. Similarly, the steps necessary to obtain and the
likelihood of recovering the desired genotypes can be determined with much greater
precision. Linkage drag effects can be estimated, encouraging breeders to screen for
specific recombinants before investing considerable efforts on genotypes with gene
combinations difficult to break.
Marker-assisted selection (MAS) permits screening for genes before the charac-
ter is expressed in the plant, by allowing the identification of heterozygous geno-
types that carry the desired recessive allele, but will not express the trait, and by
enabling the screening for individual genes controlling a polygenic trait so that
those plants with more of the desirable alleles can be identified and used in future
crosses. These more indirect uses of biotechnology have had a significant effect on
the efficiency of pea-breeding programmes throughout the world.
Several markers have been associated to traits of interest in the past decade.
These characters for which the genetic basis is at least partially known should be
amenable to MAS. In cases where the gene(s) responsible for the trait have been
identified, DNA sequence polymorphism can be used to generate a ‘perfect’ marker
that correlates 100 % with the presence or absence of the desired allele. This is
particularly the case for mono- or oligogenic traits. For more complex traits, mark-
ers associated with QTL have been mapped. Table 2.4 lists a number of genes that
are critical in pea breeding and for which ‘perfect’ markers or genetically linked
markers have been identified.
Despite the availability of these useful markers, relatively few pea-breeding pro-
grammes are using MAS routinely. There are several reasons for this: In some cases
(e.g. presence of anthocyanins, absence of leaflets and seed shape), the trait is easily
scored directly, heterozygotes can be conveniently identified in the next generation,
and often both parents were homozygous for the desired allele. Thus, these traits
might not be expected to be prime candidates for MAS, despite the saving in space
and time that can result from early selections based on markers particularly for
70 T. D. Warkentin et al.

Fig. 2.2   A plant of JI 1194,

a parent of one mapping
population at JIC, Norwich

seed traits. Other genes that require phenotypic screening under specific conditions
(e.g. presence of the pathogen, or under controlled photoperiods) are more suitable
for MAS. However, for these traits with often complex heredity, the marker–trait
linkage has to be tight enough for efficient MAS. Additionally, allele diversity is
generally low in cultivated breeding pools. When broader parentage is used, there
are greater incentives to screen for the retention of genes and markers representing
the cultivated background, while introgressing the trait and region of the genome
that contains a novel gene of interest.
There are however several instances where a MAS approach has been adopt-
ed thus far. For instance, MAS for enation mosaic virus is used in New Zealand
because direct testing with the pathogen is not possible (there is no pea enation virus
in, or allowed into, that country), and resistance to enation virus is highly desired
in varieties released. MAS is also being used to introgress the pea seed albumin 2
mutation (Vigeolas et al. 2008) into a cultivated background for improved animal
feed. The development of perfect markers for the locus (Tri) controlling the activ-
ity of trypsin inhibitors in pea seeds offered a facile alternative to cumbersome
biochemical assays (Page et al. 2002) that is relevant to some winter pea-breeding
programmes. Due to close linkage between Tri and Vc-2 (a locus encoding a class of
2 Pea 71

vicilin, a major seed storage protein), the marker for the latter (Chinoy et al. 2011)
could be used to distinguish lines carrying particular combinations of alleles at the
Tri locus and a pseudogene at Vc-2, where the latter allele may be associated with a
reduced seed protein concentration. In France, MAS for pyramiding QTL for frost
tolerance and A. euteiches disease resistance in elite genetic backgrounds is under-
way at INRA (Baranger et al. 2010).
The number of markers useful for selection has been relatively modest until re-
cently (300 SSR markers were mapped in Loridon et al. 2005). Now high-though-
put, cost-effective and easy to score marker technologies have been developed for
pea in different programmes (e.g., Deulvot et al. 2010; Leonforte et al. 2013). These
may enhance MAS in pea in the near future. This approach may be favoured by
the generation of service genotyping platforms for these markers that will allow
breeders who are not always familiar with and/or using marker technology to out-
source this activity. Furthermore, as breeding programmes avail more of the wider
pea germplasm and the ever-increasing diversity of traits offered by this resource,
alongside the greater emphasis currently on translational research, it is likely that
greater reliance will be placed on the use of markers. Finally, the availability of
large SNP marker collections and the pea genome sequence (McGee 2012b) should
allow genomic selection programmes to enhance breeding for complex traits.

8.3  Other Biotechnologies

As is the case for many pulses, pea is not particularly amenable to tissue culture.
Although it can be done, growing plants from single cells or explants remains slow.
Thus, recent advances are not groundbreaking but show promise. Regeneration pro-
tocols are available (Sanchez and Mosquera 2006; Rajput and Singh 2010), and
Esposito et al. (2012) have developed a method for increasing perhaps tenfold the
number of F1 plants produced from a cross. Both anther culture and double haploid
production have been explored (Lulsdorf et al. 2011) but the approach has yet to be
applied in breeding programmes.
The production of genetically modified pea plants and seeds expressing a foreign
protein that conferred resistance to a major pest was a very early success story of
legume crop transformation. In many parts of the world where pea is cultivated,
the pea bruchid beetle (B. pisorum) prevails. Expression of the α-amylase inhibitor
(α-AI) from French bean ( Phaseolus vulgaris L.) in transgenic pea proved the util-
ity of this inhibitor in protecting plants in the field and stored seeds from attack by
the pea bruchid (Schroeder et al. 1995; Morton et al. 2000). However, the commer-
cial development of these lines was abandoned when transgenic seeds appeared to
invoke a T-cell response in mice which was not evident when either the non-trans-
genic pea or French bean seeds were tested. This observation and subsequent stud-
ies led to the conclusion that differential glycosylation of the α-amylase inhibitor
expressed in the non-host plant had triggered a pre-immune response (Anonymous
2006); other minor changes to seed proteins were also documented for the transgen-
ic seeds, likely reflecting pleiotropic effects on post-translational processing (Islam
72 T. D. Warkentin et al.

et al. 2009). The negative publicity that this research received is regrettable and, as
noted by others (Anonymous 2006), has not served the best interests of scientists,
breeders or consumers. In fact, the rigour applied to the testing of the modified
seeds far surpassed any analysis that is performed on material from standard breed-
ing programmes, including those involving wild relatives. It is difficult to assess to
what extent the lack of acceptability of genetically modified foods in some parts
of the world contributed to the negative publicity of this research and to the aban-
donment of other more applied research projects. Related research has produced
additional transgenic pea lines, but none with such obvious agronomic benefit as
the α-AI lines; pea lines having moderate reductions in seed trypsin inhibitor activ-
ity (Welham and Domoney 2000) and others with modified nodulation (Schneider
et al. 1999) and resistance to PSbMV (Jones et al. 1998) contributed to fundamental
research projects. These and others generated in the pursuit of academic research
projects (Weigelt et al. 2009) have led to a fuller understanding of biological pro-
cesses that may be investigated more thoroughly using mutagenesis. The latter pro-
grammes are more likely to lead to modified germplasm that will be accepted more
readily into breeding programmes in the current climate. Transformation systems
for pea, although not very efficient, are unlikely to represent a barrier and are also
improving (Clemow et al. 2011). The exploitation of pea seeds as a vehicle for the
production of valuable proteins, including pharmaceuticals, has also been demon-
strated clearly (Mikschofsky and Broer 2012; Mikschofsky et al. 2009).
We may expect, with the current rate of development of new genomic tools (e.g.,
gene-editing techniques) coupled with high-throughput germplasm screens, the
powers of association mapping and advanced phenotyping, plus advances in ge-
nome sequencing, that breeding programmes will move to a new level in the next
5–10 years. Programmes in which pyramiding of genes is currently very challeng-
ing (for instance, breeding for snap peas where at least three recessive genes must
be combined to obtain the appropriate pod character, added to other desired disease
resistance and flowering time traits) will become much more rapid and accessible.

9  Seed Production and Variety Commercialization

In the University of Saskatchewan programme, pre-breeder seed development

is initiated concurrent with F6 yield testing. Single plants are selected from the
best-performing F6 lines, then grown as F6-derived microplots concurrent with
first-year Cooperative Registration Testing and as F6-derived long plots concur-
rent with second-year Cooperative Registration Testing. Breeders’ seed of new
varieties is typically bulked after long plot evaluation. Commercialization occurs
through an agreement with Saskatchewan Pulse Growers (SPG), the organization
which represents pulse growers. Variety release occurs on a royalty-free basis in
exchange for SPG support of breeding. Breeders’ seed is distributed by SPG to se-
lect-status seed growers who multiply the seed and ultimately sell certified-status
seed to farmers.
2 Pea 73

In the Agriculture and Agri-Food Canada (AAFC) programme, selected F9 lines

are grown in strips in the year prior to registration testing in the home location
for seed multiplication and further purification, and for further observation and
selection. Any off-type plants are removed, resulting in uniform and pure breeding
lines. The commercialization of the AAFC’s field pea varieties is based on a tender-
ing mechanism. The breeder prepares the performance information on candidate
varieties and provides it to the Office of Intellectual Property of Canada (OIPC).
OIPC posts the information to the public as a proposal of commercialization. All
proposals are reviewed by a committee, and a successful company is granted the
right for commercialization of a specific variety. After the commercialization rights
to a variety has been granted to a seed company, seed production is carried out at
the AAFC Seed Unit, Indian Head, SK. Then the seed company multiplies the seed
for future sales.
In the USDA-ARS breeding programme, lines identified for potential release
are grown in strips 1–2 years before variety release is anticipated. Approximately
200 single plants are selected that conform to variety type. Seed from each plant is
grown for 2 years in paired rows and selected for ‘trueness to type’. After the second
year, the remaining progeny are bulked and transferred to either the Washington
State Crop Improvement Association or another licencing entity. That organization
is responsible for the production of foundation and registered seed and marketing.
The NDSU breeding programme utilizes a similar approach.
In Australia, lines identified for commercial release are included in both
advanced breeding trials and the national variety testing (NVT) programme to max-
imize evaluation regionally. Multiplication of breeders’ seed commences once lines
are promoted to NVT testing. Commercialization occurs via an open tender process
for either individual lines or on the basis of a short-term licence (5 years) for a va-
riety pipeline. Pulse Breeding Australia (PBA) is an unincorporated joint venture
between Australian state governments and GRDC, established in 2005 to manage
the breeding and variety release strategy. PBA works closely with the seed licensee
to develop a variety release strategy that maximizes uptake of new genetic advance-
ments by industry. The seed licensee is responsible for commercial seed production
and marketing and seed or endpoint royalty collection on behalf of stakeholders.
In European programmes, pre-breeder seed production typically begins once
lines have entered multilocation yield trials, and it is carried out by individual breed-
ing companies. Breeders’ seed multiplication is accelerated after a variety succeeds
in VCU and DUS evaluations.
The Indian Council of Agricultural Research (ICAR) organizes seed production
of varieties of different crops through ICAR Institutes, State Agricultural Universi-
ties and organizations like the National Seed Corporation, State Farm Corporation
of India. In China, public and private pea seed production systems are operating in
northern China.

Acknowledgments  Appreciation is expressed to Devini DeSilva for critically reviewing the

references section of this chapter.
74 T. D. Warkentin et al.

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Chapter 3
Chickpea

Teresa Millán, Eva Madrid, José I. Cubero, Moez Amri, Patricia Castro
and Josefa Rubio

1 Introduction

Chickpea ( Cicer arietinum L.) is a unique cultivated species in the genus Cicer. It is
an annual, self-pollinated crop adapted to post-rainy season either in spring sowing
or summer-dominant rainfall regions (Berger and Turner 2007). It has been a food
crop since ancient times in the Mediterranean basin from where it was dispersed to
the Indian subcontinent becoming a basic constituent of Asian diets.
Nowadays, it is grown all over the five continents in around 50 countries, with
90 % of its cultivated area (around 13 × 106 ha) in developing countries. India ranks
first in the world in respect of cultivated area (68.5 %) followed by Pakistan (8.7 %)

T. Millán ()
Department of Genetics, Campus de Rabanales, Edif C5 2ª planta, Córdoba, Spain
e-mail: [email protected]
E. Madrid
Institute for Sustainable Agriculture, CSIC, Avda. Menendez Pidal sn,
Apdo 4084, Córdoba, Spain
e-mail: [email protected]
J. I. Cubero
Departmento de Genetica, Universidad de Córdoba, Campus Rabanales, Córdoba, Spain
e-mail: [email protected]
M. Amri
Regional Field Crop Research Center of Beja (CRRGC), PB 350, Beja 9000, Tunisia
e-mail: [email protected]
P. Castro
Genetic Improvement of Fruits and Vegetables Lab, Rm. 214, Bldg. 010A, BARC West,
10300 Baltimore Avevue, Beltsville, MD 20705-2350, USA
e-mail: [email protected]
J. Rubio
Area de Mejora y Biotecnologia, IFAPA Centro “Alameda del Obispo”, Avda. Menendez Pidal
s/n Apdo: 3092, Córdoba, Spain
e-mail: [email protected]
© Springer Science+Business Media New York 2015 85
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_3
86 T. Millán et al.

Fig. 3.1   Evolution of world chickpea production

as well as in total production (68 %) and followed by Australia (5.9 %), that is one
of the major exporting countries. This crop is mainly grown under rainfed condi-
tions with a world average yield of 931 kg ha−1 in 2012 but reaching a maximum of
6044 kg ha−1 in Israel where this crop is grown under irrigated conditions. Chickpea
production has increased over the past 30 years from 6.1 to around 13 × 106 t. It
has been particularly stunning that the increase of this crop in Africa and Oceania,
where the total production was at minimum, doubled (Fig. 3.1). This increase can
be explained by (i) the development of new high-yielding varieties tolerant/resis-
tant to main diseases, pests and abiotic stresses and (ii) successful integrated crop
management practices. The number of importing countries has increased from 65 in
1991 to 164 in 2011, suggesting the highest global demand for this crop in the past
20 years (Faostat 2014).
Chickpea cropping systems have been recently reviewed by Berrada et al. (2007).
In the Mediterranean basin, springtime (February–mid-April) is the traditional sow-
ing date, but with the new winter chickpea varieties, sowing can be advanced to
January, whereas in the Indian subcontinent it is sown from mid-September to No-
vember after the rainy season. Chickpea has been mainly cultivated in rotation after
multiple base crops such as wheat, teff, oat, rice, pearl millet, sorghum, maize or
cotton, although intercropping has also been put into practice in subsistence farming
areas. Production stability and yields have been limited by various constraints such
as susceptibility to major worldwide-distributed diseases, Ascochyta blight and Fu-
sarium wilt or terminal drought.
This crop, as a legume, improves soil fertility by fixing atmospheric nitrogen,
meeting up to 80 % of its nitrogen requirement from symbiotic nitrogen fixation
(Gaur et al. 2012). Chickpea-specific mesorhizobia are present in soils where
chickpea has been traditionally grown but seed inoculation is required in the new
chickpea-cultivated lands or in marginal soils. Better N2 fixation can be achieved
3 Chickpea 87

by selecting rhizobial strains of superior N2-fixing capacity but also depends on

chickpea cultivars (Kantar et al. 2007).
Chickpea is a high-quality and cheap source of protein for people in developing
countries (particularly in South Asia), who are largely vegetarian. Additionally, it is
rich in nutritionally important unsaturated fatty acids and vitamins such as ribofla-
vin, niacin, thiamin, folate and the vitamin A precursor β-carotene. Different studies
have provided some evidence to support the potential beneficial effects of chickpea
components in lowering the incidence of various cancers, high-density lipoprotein
(HDL) cholesterol, type-2 diabetes and heart diseases (Roy et al. 2010; Jukanti et al.
2012).
C. arietinum has a relatively low DNA content estimated to be 738.09 Mb
(Varshney et al. 2013) distributed in eight pairs of chromosomes. Genomic tools in
chickpea have been progressing very rapidly in the past years. Nowadays, reference
genetic maps are available including cross-genome markers from the model species
Medicago truncatula, which made it possible to assign chickpea linkage groups
(LG) to Medicago chromosomes. (Millán et al. 2010; Nayak et al. 2010; Gujaria
et al. 2011; Thudi et al. 2011). Furthermore, five out of the eight chickpea chromo-
somes could be isolated by flow-cytometry and specific sequence-tagged microsat-
ellite site (STMS) markers amplified on sorted chromosomes and allowed to assign
four linkage groups to particular chromosomes (Vláčilová et al. 2002; Zatloukalová
et al. 2011). All this previous information, together with the recent publication of
the first draft of the whole-chickpea genome sequence by two different groups (Jain
Rajesh et al. 2013; Varshney et al. 2013), provides powerful tools to be used for
searching genes involved in agronomic traits. The relatively simple genome of this
species together with its importance to world agriculture and available genetic and
genomic tools make this crop as a possible “model” for cool-season food-legume
genomics.

2  Systematic and Origin

The genus Cicer belongs to the family Leguminosae, subfamily Papilionoideae and
the monogeneric tribe Cicereae Alef., including 35 perennials, 8 annual and 1 un-
specified wild species. It is a component of the Galegoid group (or cool season
legumes) together with tribes Viceae, Trifoliae and Loteae (Zhu et al. 2005). The
tribe Cicerae has been subclassified in sections Cicer (= Monocicer), Chamaecicer,
Polycicer and Acanthocicer based on morphological traits and geographical dis-
tribution (van der Maesen et al. 2007). The single cultivated species C. arietinum
belongs to the section Cicer.
Based on their crossability with chickpea, the wild Cicer annual species have
been classified into gene pools reflecting their distance from the cultivated species
as proposed by Harlan and de Wet (1971). According to this definition, the primary
gene pool consists of C. arietinum, the wild annual progenitor, Cicer reticulatum
Ladz. and the closely related Cicer echinospermum P. H. Davis. The secondary gene
88 T. Millán et al.

pool is composed by Cicer bijugum K. H. Rech, Cicer pinnatifidum Jaup and Spp.
and Cicer judaicum Boiss. Sterility is associated with the first-generation hybrids
for those species. Interspecific or wide hybridization has been identified as a poten-
tial means of increasing the genetic variation and introduction of resistance genes in
cultivated species from wild species (Singh et al. 2008).
Chickpea was one of the first domesticated grain legumes together with other
crops such as wheat, barley, rye, pea, lentil, flax and vetch in a reduced area of
southeast of Turkey (Ladizinsky and Adler 1976; Abbo et al. 2003). The domestica-
tion seems to have occurred from the wild ancestor C. reticulatum (sin. C. arietinum
subsp. reticulatum) with a monophyletic origin as revealed by the low genetic varia-
tion of the cultigen (C. arietinum subsp. arietinum) (Moreno and Cubero 1978).
Only in the early Bronze period (fifth millennium BC) can chickpea be considered
as an established crop in the Near East. A few seeds dated ca. 6000 BC have been
found in Bulgaria, and two millennia later, in Greece. Thus, it seems that chickpea
belonged to the first agricultural complex reaching Europe through the Black Sea
(Zohary et al. 2012). Afterward, chickpea was likely taken to India by the Aryan
tribes in the second millennium BC who probably brought the crop from Iranian
tribes, as suggested by De Candolle on linguistic evidence. It was also spread over
the Mediterranean basin and through the Nile River and was expanded to the east
of Africa. Chickpea was taken by Spanish colonizers to the New World in 1492. It
crossed the Atlantic Ocean in the first Columbus travel of discovery. Chickpea was
always a humble crop that almost never appeared on the royal tables, being on the
contrary a useful food for both humans and animals and a good companion of man
around the globe.

3  Varietal Groups

C. arietinum is divided into two main cultivar groups for breeding purposes ‘desi’
and ‘kabuli’ types. This distinction is made mainly on the basis of a small number
of morphological characters, principally the seed shape and colour. White flower,
thin seed coat, large seed (200–680 mg) and more of them with a “ram’s head”
shape and cream-coloured seeds, smooth seed surface, lack of anthocyanin pig-
mentation and semi-spreading growth habit are present in the kabuli chickpea. On
the other hand, pink flower, thick seed coat, small seed (100–200 mg) angular, dark
seeds, anthocyanin pigmentation of stem, rough seed surface and either semi erect
or semi-spreading growth habit are characteristics of desi ones (Pundir et al. 1985).
This classification overlaps, to a certain extent, with the macrosperma and micro-
sperma races proposed by Moreno and Cubero (1978) studying quantitative as well
as qualitative traits. Additionally, a third type designated as pea-shaped character-
ized by medium to small seed size and cream-coloured seeds has been proposed
(Upadhyaya et al. 2008a).
Desi types, mostly grown in India, Pakistan and East Africa, cover around 85 %
of chickpea cultivated area. In these countries, seeds are usually dehulled and split
3 Chickpea 89

before cooking. Kabuli chickpeas are grown mainly in the Mediterranean basin,
the Near East, Central Asia and America where whole seeds are used for human
consumption after soaking and boiling. It is believed that the kabuli chickpea was
introduced into India through Kabul, Afghanistan (therefore named kabuli), in the
mid- to late-seventeenth century (Singh 1987). Kabuli probably evolved from the
desi type in the Mediterranean basin and oligogenic traits like flower colour, seed
coat thickness and seed size seem to have played an important role in its evolution
(Moreno and Cubero 1978; Gil and Cubero 1993).
Significant differences in agronomic traits have been observed between these
two groups. Ascochyta blight resistance, cold tolerance and erect growth habit are
more frequently found in kabuli types, whereas Fusarium wilt resistance, heat and
drought tolerance and early flowering are prevalent in desi types (Singh 1987).
They also differ in quality components such as seed coat thickness, crude fibre
content, mineral and trace element composition, polyphenolic content and in vitro
digestibility (Jambunathan and Singh 1981; Gil et al. 1996). Kabuli types were re-
ported to be nutritionally superior to desi types in terms of cooking time, biological
value and sensory properties (Singh et al. 1991) and receive higher market price
than desi types. The price premium in kabuli types generally increases as the seed
size increases.
The distribution of genetic diversity in kabuli seems to be much narrower than
in the desi predominant chickpea type. Both types are also nearly uniform in cyto-
plasm, indicating no evolution of hybridization barriers (Moreno and Cubero 1978).
The desi and kabuli groups tend to have maintained distinct morphological types
and may have different gene blocks for important yield components appearing as
two separate groups when they are clustered based on molecular marker analysis
(Iruela et al. 2002).
These differences have been employed in chickpea-breeding programmes using
desi × kabuli (and vice versa) crosses to obtain new disease-resistant cultivars with
higher yields, large seed size and vigour of desi types (Gaur et al. 2007).

4  Genetic Resources and Utilization

The genetic resources provide basic material for selection and improvement through
breeding, leading to ensure food security needs of the world’s rapidly rising popula-
tion. They comprise diversity of genetic material contained in traditional varieties,
modern cultivars, crop wild relatives and other wild species (Farshadfar and Far-
shadfar 2008; Upadhyaya et al. 2008b). The collections represent both insurance
against genetic erosion and as sources of resistance/tolerance to diseases and pests,
climatic and other environmental stresses.
The two major chickpea germplasm collections are maintained at the Consulta-
tive Group on International Agricultural Research (CGIAR) centres, the Interna-
tional Crops Research Institute for the Semi-Arid Tropics, http://www.icrisat.org
(ICRISAT) and the International Center for Agricultural Research in Dryland Areas,
90 T. Millán et al.

http://www.icarda.org (ICARDA) with more than 20,000 and 13,000 accessions,

respectively. ICRISAT mainly focusses on desi types while ICARDA maintains
mostly kabuli types. Both centres, ICRISAT and ICARDA, maintain wild Cicer
sp. Other important chickpea collections are conserved by the National Bureau of
Plant Genetic Resources, India (NBPGR) with around 14,000 accessions, Centre
for Legumes in Mediterranean Agriculture Australia (CLIMA) with approximately
8000 accessions and the United States Department of Agriculture, http://www.ars-
grin.gov (USDA) with about 6000 accessions (Rubio et al. 2009; Upadhyaya et al.
2011). Other collections have been described by Gaur et al. (2012).
In spite of the large number of germplasm accessions available at the gene banks,
there has been a very limited use of these accessions in chickpea breeding; thus, in
India, only ten lines contributed to the 35 % of genetic base in chickpea (Upadhyaya
et al. 2008b). The main reason for the modest utilization of germplasm is the lack of
information on a large number of accessions. Thus, core and mini core collections
(about 10 and 1 % of the total accessions, respectively) have been suggested as an
opportunity for the utilization of genetic diversity in crop improvement (Upadhyaya
and Ortiz 2001). In chickpea, core and mini core subsets, representative of the entire
chickpea collections, have been obtained. Upadhyaya et al. (2001) developed in
ICRISAT a chickpea core collection consisting of 1956 accessions, which repre-
sented the global chickpea germplasm collection. ICRISAT developed a reference
set consisting of 300 accessions representing diversity from the entire spectrum of a
composite collection made between ICRISAT and ICARDA (Upadhyaya and Ortiz
2001). Mini core collections have been useful to identify accessions with good ag-
ronomic traits and resistance to different diseases (Pande et al. 2006a) as well as in
applied breeding for the development of broad-based elite breeding lines/cultivars
with superior yield (Upadhyaya et al. 2008b).
The Generation Challenge Program (GCP; www.generationcp.org) is contribut-
ing to intensify the molecular characterization of core and mini core collection to
identify genetically diverse parents for mapping and utilization in breeding pro-
grammes (Gaur et al. 2012).

5 Major Breeding Achievements and Specific Goals

in Current Breeding

Breeding efforts have contributed substantially to improve chickpea yield potential

but the lack of stable production still continues to be a major concern for the adop-
tion of this crop by farmers. The major constraints limiting chickpea production in-
clude various abiotic and biotic stresses, particularly important are fungal diseases
(Ascochyta blight and Fusarium wilt), pests (pod borer) and drought or cold stress.
Parasitic plants could also be a big problem in such particular environments such as
Mediterranean conditions (Gaur et al. 2007; Chen et al. 2011) (Fig. 3.2).
3 Chickpea 91

Fig. 3.2   Chickpea screening for resistance/tolerance to different diseases and parasitic plants in
specific clinic fields in Tunisia (a) Ascochyta blight (b) Fusarium wilt (c) Broomrape ( Orobanche
foetida) (d) Dodder ( Cuscuta spp.)

5.1  Biotic Stresses

Ascochyta blight, caused by the Ascomycota fungus Ascochyta rabiei (Pass.) Labr.
(teleomorph Didymella rabiei (Kovatsch) Arx.), is one of the most serious diseases
of chickpea worldwide, which affects all aerial parts of the plant. It reduces chick-
pea seed yield significantly reaching 100 % of losses when favourable conditions
such as cool (5–15 °C) and wet weather occurred (Pande et al. 2005). The use of
varieties with improved levels of resistance is considered the most economical so-
lution for long-term disease management. So that, efforts to develop and commer-
cialized blight chickpea resistant cultivars have been made (Gaur et al. 2007; Taran
et al. 2013). Currently, the development of blight-resistant lines has made possible
the introduction of winter sowing in the Mediterranean region with the prospect
of increasing chickpea production that could be doubled (Singh and Reddy 1996).
However, reaching high levels of resistance to blight is complex because many
genes with minor to moderate effects control this trait and only partial resistance is
available (Pande et al. 2005; Taran et al. 2007; Bhardwaj et al. 2010).
The pathogen survives between crop seasons either on infected plant debris or
in contaminated seeds. A wide pathogenic variation has been described and those
are grouped in two or three pathotype systems (Udupa et al. 1998; Chen et al. 2004,
92 T. Millán et al.

2011). Breeders try to search for different sources of resistance and pyramid differ-
ent resistance genes into the same cultivar to improve both the resistance level and
the durability of this resistance. Different sources of resistance/tolerance have been
employed to achieve this goal, and as a result, a significant number of cultivars with
improved Ascochyta blight resistance have been released. This information was
summarized in Pande et al. (2010) and Millan et al. (2013). Additional sources of
resistance have also been identified among wild Cicer species, including C. judia-
cum, C. pinnatifidum, C. echinospermum and C. reticulatum (Pande et al. 2006b)
Fusarium wilt, caused by the vascular pathogen Fusarium oxysporum Schlech-
tend: Fr. f. sp. ciceris, is, along with Ascochyta blight, the most important fungal
disease in chickpea. Fusarium wilt epidemics can be devastating to individual crops
and cause up to 100 % loss under favourable conditions. Persistence of the patho-
gen in soil for many years even in the absence of the host renders its control dif-
ficult. Consequently, the use of resistant cultivars is the most economic, effective
and ecofriendly method of controlling such pathogen. However, the effectiveness
of resistant cultivars is limited by the existence of different races of pathogens. To
date, eight physiological races (0, 1A, 1B/C, 2, 3, 4, 5 and 6) have been reported
from India, Spain, Tunisia and the USA (Haware and Nene 1982; Jimenez-Gasco
et al. 2004). Resistance to wilt in chickpea has been reported to be race-specific and
controlled by major resistance genes (Gaur et al. 2007; Sharma and Muehlbauer
2007; Castro et al. 2012a).
The screening of international germplasm has led to the identification of sources
of resistance to wilt in both cultivated (desi and kabuli types) and wild chickpea
germplasm (Millan et al. 2013). However, the resistance sources in kabuli types
are limited compared to the desi types. Haware et al. (1992) evaluated a world
collection with 13,500 germplasm accessions for race 1, identifying 160 resistant
accessions but only ten of them were kabuli type. The desi type accession WR315 is
extensively recognized as possessing resistance to all known wilt races. It has been
widely used in inheritance and mapping studies and is considered to be a very inter-
esting source of wilt resistance for chickpea-breeding programmes (Haware 1998).
Two kabuli accessions (ICC 14194 and ICC 17109) with complete wilt resistance
and extra large seeds, a key quality determinant in the market, were detected by
Gaur et al. (2006). Significant progress has been made in Fusarium research and
cultivars including resistance to multiple races are now available (Malhotra et al.
2007; Singh et al. 2009).
Botrytis grey mould (BGM) caused by Botrytis cinerea Pers. ex. Fr. is the second
most important foliar disease of chickpea after Ascochyta. BGM causes complete
crop loss in several South Asian countries (Pande et al. 2006c). The limited reports
available on genetics of BGM resistance in chickpea suggest that a few major genes
control resistance in the host to BGM (Anuradha et al. 2011). There is no adequate
level of genetic resistance to BGM in the cultivated genotypes. However, high lev-
els of resistance have been found in the wild Cicer species, including C. judaicum,
C. bijugum, C. echinospermum and C. pinnnatifidum (Pande et al. 2006c). Thus,
several wide and intraspecific hybridizations have been carried out to transfer the
identified disease resistance genes in wild types and land races to commonly ad-
opted and widely grown chickpea cultivars.
3 Chickpea 93

Other fungal diseases considered of local importance could also affect chickpea
productions. Rust ( Uromyces ciceris arietini) has been reported to be a problem
in central Mexico and Italy (Ragazzi 1982; Díaz-Franco and Pérez-García 1995).
The resistance is controlled by a single gene ( Uca1/uca1) (Madrid et al. 2008) and
moderate levels of incomplete and partial resistance are available (Rubiales et al.
2001). Phytophthora root rot (caused by Phytophthora medicaginis) affects cool
season plantings (Chen et al. 2011). Until now, five genotypes (FLIP 97-132C, FLIP
97-85C, FLIP 98-53C, ILC-5263 and NCS 9905) evaluated under controlled con-
ditions in Pakistan exhibited highly resistant response to the disease (Akram et al.
2008). C. echinospermum appears to be the most promising source of resistance in
wild species after field and controlled condition evaluations in Australia (Knights
et al. 2008).
Parasitic plants as broomrape ( Orobanche crenata and Orobanche foetida) may
cause serious losses in chickpea productions in winter sowing under Mediterranean
conditions (Rubiales et al. 1999; Roman et al. 2007). Sources of resistance to O.
crenata in Spain (Rubiales et al. 1999) and to O. foetida in Tunisia (Amri personal
communication) have been identified. Despite the low broomrape infestation levels
observed in chickpea compared to other grain legume species, rapeseed or wild
species recently, more aggressive and virulent new Orobanche populations are aris-
ing (Amri et al. 2009). Field dodder (Cuscuta spp.) is another parasite that was
reported damaging chickpea production in many regions in the world (Goldwasser
et al. 2012a; Chen et al. 2014). In highly infested fields, this parasite can cause up to
100 % loss in grain production (Singh et al. 2007). Sources of resistance for Cuscuta
campestris (field dodder) (ICCV 95333 and Hazera 4) exhibiting high resistance
were identified in Israel (Goldwasser et al. 2012b).
In addition, efforts have been made to identify sources of resistance to both pests
pod borer ( Helicoverpa armigera Hübner) and leaf miner ( Liriomyza cicerina Ron-
dani) in the cultivated and wild species at ICRISAT and ICARDA (Gaur et al. 2007).

5.2  Abiotic Stresses

Terminal drought is globally the most serious abiotic stress to chickpea productivity
and the most important factor for instability of yield in major production countries
as Asia and Africa, where chickpea is mainly grown as a rainfed crop on residual
moisture. Cultivars may escape (early maturity) or tolerate terminal drought in-
creasing the efficiency of water use. Promising accessions (ICC 4958, ICC 1882,
ACC 316 and ACC 317) and varieties with a vigorous and deeper root system to
improve drought tolerance have been developed (Saxena et al. 1993; Gaur et al.
2008; Cancy and Toker 2009). In addition, transgenic plants have been developed
at ICRISAT having either a dehydration responsive element or a gene that increases
proline accumulation in the plant (Gaur et al. 2007).
Salt stress imposes a significant limitation of productivity related to the adverse
effects on the dry weights of both shoots and roots and also on nodulation and
nitrogen fixation (Manchanda and Garg 2008). Limited efforts to identify salin-
ity tolerance within chickpea indicated low genotypic variation and few varieties
94 T. Millán et al.

with tolerance to moderate levels of salinity (ECe ranging from 4 to 6 dS/m) have
been developed. Karnal Chana 1 (CSG 8962) and Genesis 836 (ICCV 96836) were
developed in India and Australia, respectively (Maliro et al. 2004). Recently, ICRI-
SAT identified several lines that gave a higher yield than the salinity tolerant culti-
var Karnal Chana 1 (Krishnamurthy et al. 2011; Gaur et al. 2012).
Finally, both freezing (< – 1.5 °C) and chilling (between – 1.5 and 15 °C) are
known to affect chickpea at various development stages from germination to matu-
rity (Croser et al. 2003) and should be considered in chickpea breeding for winter
sowing in Mediterranean environments. Two chilling tolerant cultivars (‘Sonali’
and ‘Rupali’) have been released in Australia (Clarke et al. 2005) and should be
included in winter sowing breeding programmes to avoid problems in pod filling in
fresh spring. Also, ICARDA and ICRISAT breeding programmes developed cold-
tolerant cultivars adapted to winter sowing (Gaur et al. 2007).

5.3  Phenological Characters

Flowering time is influenced by photoperiod and temperature and is a major task

to improve crop adaptation. Early flowering associated with early maturity is pre-
ferred to escape from terminal drought, high temperature or frost at the end of the
season (Gaur et al. 2007). A moderate and positive genetic correlation between days
to flowering and seed weight was reported by Hovav et al. (2003), suggesting that
it is difficult to breed early-flowering cultivars without compromising seed weight.
However, ICRISAT developed two extra large and early-maturing kabuli types from
Mexican origin, which suggests that it is possible to breed early varieties with extra
large seeds (Gaur et al. 2006). The first landmark variety was ICCV 2, which ma-
tures in about 85 days, and it is perhaps the world’s earliest maturing variety of ka-
buli chickpea. Two super-early desi chickpea lines, ICCV 96029 and ICCV 96030,
which mature in 75–80 days in southern India were developed by Kumar and Rao
(1996). Further advancements have been made in breeding for super-earliness and
several short-duration high-yielding varieties of chickpea, both in desi and kabuli
types (Gaur et al. 2012).

6  Breeding Methods

Productivity, yield stability in different environment conditions and resistance/

tolerance to main damaging diseases are the major goals in chickpea-breeding
programmes. These constraints become more pronounced especially with the cli-
mate change affecting both the development of the crop and its enemies (develop-
ment of new pathogens/pests and changing in the aggressiveness and virulence of
others). The development of chickpea crop in a sustainable agricultural system is
facing several challenges: (i) being more efficient in the development of new vari-
eties resistant/tolerant to main biotic and abiotic stresses and (ii) strict adoption of
these new developed varieties by farmers that could result in an improved produc-
tivity, reducing yield fluctuations.
3 Chickpea 95

Fig. 3.3   Combined bulk and pedigree method in chickpea-breeding for tolerance/resistance to
both Ascochyta blight and Fusarium wilt diseases (chickpea-breeding programme—Tunisia).
Seeds coming from the same selected plant are sown in both blight and wilt clinic fields during the
same cropping season. Crosses are mainly performed at ICARDA

Any breeding programme can be achieved through three main steps or compo-
nents: (i) the genetic variation which is the base of the breeding programme, (ii)
strict and rigorous selection within that variation and (iii) evaluation and confirma-
tion of the selected lines (Salimath et al. 2007). Chickpea can be considered a strict
self-pollinated crop. Hence, inbreeding to fix genes and develop pure-lines cultivars
are the main breeding objectives. Mass or pure-line selection from landraces was
the simplest method initially employed. Later, crossing programmes and subse-
quent different modification of pedigree methods and backcrossing programmes for
qualitative traits have been developed (Gaur et al. 2012). Most chickpea-breeding
programmes have been confined to intraspecific hybridization that includes desi ×
desi, kabuli × kabuli or desi × kabuli crosses (Gaur et al. 2007). As mentioned in
Sect. 3 of this chapter, desi and kabuli types have different genetic backgrounds
differing in disease resistance, tolerance to abiotic stresses and quality components.
Depending on the objective of the breeding programme, single, three-way or mul-
tiple crosses are used. Single crosses have been the most widely adopted. Figure 3.3
shows an example of the methodology followed in the national chickpea-breeding
96 T. Millán et al.

programme in Tunisia at the National Institute of Agronomy Researches/Regional

Field Crop Research Center of Beja (INRAT/CRRGC) while the crosses are mainly
performed at ICARDA. Hybridization also allowed the obtention of recombinant
inbred lines (RILs) by single-seed descent (SSD) methods (Johnson and Bernard
1962) where plant materials combining interesting traits have been selected (Rubio
et al. 2006). Three-way or multiple cross approaches provide additional opportuni-
ties to study gene interactions with the aim of combining different traits from differ-
ent parents in a new cultivar. Hybridization allows the exploitation of transgressive
inheritance of some characters such as yield or seed size.
Singh (1987) summarized the effectiveness and specificity of various breed-
ing methods in respect to the desired specific traits and reported that: (i) pedigree
method could be used for resistance to biotic stresses; (ii) bulk-pedigree method
for drought tolerance and winter hardiness; (iii) modified bulk method for abiotic
stresses, seed size, earliness and plant type; (iv) backcross method for inter specific
hybridization and (v) limited backcross for desi × kabuli introgression and resis-
tance breeding. Pedigree methods are not frequently used due to the cumbersome
data collection and the fact that this approach limits the breeding programme to
only a few crosses. The combination of bulk and pedigree methods is widely used
among many chickpea-breeding programmes (Gaur et al. 2012). The backcrossing
is in general applicable to incorporate one or few oligogenic characters into a well-
adapted elite variety. Thus, tolerance to Ascochyta blight and double podding were
successfully introgressed into CDC Xena, CDC Leader and FLIP98-135C using
markers linked to the quantitative trait loci (QTLs) for both traits (Taran et al. 2013).
Introgression from the wild related species into the cultigens has been also em-
ployed. Beneficial traits such as cold tolerance and a high degree of resistance to
wilt, root rot, and Botrytis grey mould have been introgressed from C. reticulatum
and C. echinospermum into cultivated chickpea (Singh et al. 2005; Ramgopal et al.
2012). Similarly, the C. reticulatum accession ILWC 119 was included in a cross-
ing programme giving two cyst nematode resistant chickpea germplasm lines ILC
10765 and ILC 10766 (Malhotra et al. 2002).

7 Integration of New Biotechnologies in Breeding

Programmes

The integration of genomic technologies in chickpea breeding will greatly improve

the efficiency and time required of breeding programmes in the development of bet-
ter cultivars (Gaur et al. 2012; Castro et al. 2013).

7.1  Genetic Maps

Molecular markers are considered valuable tools for crop improvement due to
their usefulness in characterizing and manipulating genetic loci responsible for
­monogenic and polygenic traits. Markers have made it possible for the ­development
3 Chickpea 97

of genetic maps, the necessary framework for any marker-assisted selection (MAS)
programme. Availability of well-saturated genetic linkage maps is a prerequisite
for tagging traits with molecular markers, thus enabling their use in MAS and posi-
tional cloning of genes of interest.
In chickpea, the first published maps were developed analyzing isozymes in F2
populations derived from interspecific crosses (Gaur and Slinkard 1990; Kazan
et al. 1993). Since then, tremendous advances in DNA marker technology and map
development have been achieved, allowing for the identification of markers close to
genes or genomic regions with agronomical importance. Even though maps are still
incomplete, the chances of finding new polymorphic markers have been consider-
ably increased, essentially due to the development of STMS markers (Hüttel et al.
1999; Winter et al. 1999; Sethy et al. 2003, 2006; Lichtenzveig et al. 2005; Choud-
hary et al. 2006; Nayak et al. 2010). The use of STMS markers in different popula-
tions gave the possibility of exchanging information between maps. These markers
have also made it possible to build a consensus chickpea map (Millán et al. 2010)
following the nomenclature previously proposed by Winter et al. (2000), which has
been considered as the map of reference. Nayak et al. (2010) completed the map
published by Winter et al. (2000) adding 175 new markers and provided anchor
points for comparing chickpea linkage groups with linkage groups in the model spe-
cies M. truncatula. More recently, the development of next-generation sequencing
technologies allowed the obtaining of the first chickpea transcriptome (Hiremath
et al. 2011). Large-scale molecular markers were obtained using the transcriptome
information and comprehensive genetic maps were developed (Gujaria et al. 2011;
Thudi et al. 2011; Hiremath et al. 2012; Jhanwar et al. 2012). High-density ge-
netic maps of gene-based markers represent a powerful resource to enhance genome
analysis, thus providing an important opportunity to directly tag genes related to
agronomical traits. Due to the low levels of genetic diversity present within the
gene pool of cultivated chickpea, the high-density genetic maps have been devel-
oped in the interspecific cross ICC 4958 × PI 489777 (Table 3.1), considered as
Table 3.1   Second generation genetic maps developed in chickpea recombinant inbred lines (RIL)
populations
Referencea Newly developed No. of loci Coverage (cM) Average inter-
markers markers distance
(cM)
Gujaria et al. (2011)a SSR, GMMs, CISR 300 766.56 2.55
Thudi et al. (2011)a BES-SSR, DarT 1291 845.56 0.65
Choudhary et al. EST–SSR, ITPs, SNPs 406 1497.7 3.68
(2012)a
Gaur et al. (2012)a SNPs, SSR 368 1808.7 1.7
Hiremath et al. CKAMs, TOGs-SNPs 1328 788.6 0.59
2012)a
Stephens et al. SSRs, SNPs 401/417 658.7/752 1.74/2.16
(2014)b
a
RIL derived from Cicer arietinum (ICC 4958) × C. reticulatum (PI 489777)
b
RIL derived from Lasseter × ICC3996/ S95362 × Howzat
GMMs genic molecular markers, SSR simple sequence repeat, EST expressed sequence tag,
SNP single-nucleotide polymorphism, CKAMs chickpea KASPar assay markers, TOGs tentative
orthologous genes, ITPs intron targeted primers, BES bacterial artificial chromosome (BAC)-end
sequences
98 T. Millán et al.

the ­reference mapping population. As far as we know, this population was only
phenotyped for wilt (races 4 and 5, Winter et al. 2000). So, the high-resolution map
developed in this cross could not be used for tagging the genomic regions associated
to other agronomic traits, such as Ascochyta blight, which is the most important
constraint in chickpea. Recently, the construction of two intraspecific genetic maps
has been reported (Stephens et al. 2014) using the RIL populations Lasseter × ICC
3996 and S95362 × Howzat, both segregating for blight resistance. In addition to
the reference interspecific map, it may be very useful performing a high-resolution
mapping in new crosses segregating for more characters valuables for breeding ap-
plications.
Moreover, the availability of the draft genome in desi and kabuli chickpea has
opened the possibility of anchoring genetic maps and positioning QTL on the physi-
cal one (Jain Rajesh et al. 2013; Varshney et al. 2013; Madrid et al. 2014). The
identification of markers with complete association with QTLs will boost the de-
velopment of “perfect” markers in pulses (Kumar et al. 2011). Such markers are
extremely useful for guiding the introgression of multiple genes, because they in-
crease selection efficiency and avoid recombination events between markers and
QTLs (Hospital 2009).
Major efforts of breeding programmes are concentrated in the development of
resistant lines to the main fungi affecting crops (Ascochyta blight and Fusarium
wilt). The majority of authors consider the resistance to blight as a quantitative
trait and several QTLs have been identified in the chickpea genetic map (Millan
et al. 2013). QTLs for resistance to blight have been located and validated on link-
age groups LG4 (QTLAR1 and QTLAR2), LG2 (QTLAR3), LG3 (QTLAR4) and LG8
(QTLAR5) of the chickpea map employing different mapping populations (Millan
et al. 2013). Another QTL was also detected in LG6 using the cross ICCV 96029 ×
CDC Frontier (Anbessa et al. 2009).
Resistance to Fusarium wilt in chickpea has been described to be race specific
and controlled by major resistance genes, the majority of which are recessive in
nature. Resistance genes to races 0, 1, 2, 3, 4 and 5 ( foc-02, foc-1, foc-2, foc-3, foc-4
and foc-5) have been found to form a cluster located on LG2 of the chickpea map
(Sharma and Muehlbauer 2007; Cobos et al. 2009; Gowda et al. 2009; Halila et al.
2010). However, one of the two resistance genes for race 0 ( foc-01) was found in
LG5 (Cobos et al. 2005) confirming that the resistance is controlled by two inde-
pendent genes ( foc-01 and foc-02), as Rubio et al. (2003) reported before by classical
genetic studies.
Markers associated with other diseases as rust and BGM have been localized. A
gene that controls resistance to chickpea rust ( Uca1/uca1) has been located in LG7
tightly flanked by two STMS markers (Madrid et al. 2008). On the other hand, three
genomic areas controlling resistance to BGM have been identified by Anuradha
et al. (2011). QTL1, sited in LG6, explained 12.8 % of the total phenotypic variation
while QTL2 and QTL3 explained 9.5 and 48 %, respectively, both of them located
in LG8 (names of the groups are referred to chickpea consensus map).
In addition, several important characteristics such as quality components and
agronomic traits have also been mapped and flanking markers were identified.
3 Chickpea 99

­ igmentation of the flower (pink/white = P/p = B/b), brown/yellow seed testa (T3/

P
t3), purple/green epicotyl (Gst/gst) and seed coat thickness (Tt/tt) are some of the
phenotypic traits with simple inheritance mapped in the chickpea genetic map and
located in LG4 (Tekeoglu et al. 2002; Cobos et al. 2005). Erect/prostrate plant
growth habit (Hg/hg) is another trait with simple inheritance which has been re-
ported to be located in LG3 (Winter et al. 2000; Cobos et al. 2009). Double pod is
a mutation controlled by a single recessive gene designated as s or sfl (Muehlbauer
and Singh 1987) linked to TA80 (Rajesh et al. 2002) in LG6 (Cho et al. 2002; Cobos
et al. 2005). Regarding yield, different QTLs have been identified in LGs 2, 4, 5 and
7 by different authors (Cobos et al. 2007; Gowda et al. 2011). Early flowering is
another trait related to yield improvement. It seems to have a positive effect on yield
under the Mediterranean environment (Siddique et al. 2003; Rubio et al. 2004).
QTLs controlling this trait have been detected in LGs 1, 2, 3, 4 and 6. The QTLs
with high limit of detection (LOD) values in LG3 have special interest for breeding
applications as they were validated in different environments and in both intra- and
interspecific populations (Cho et al. 2002; Cobos et al. 2009). A recent study pub-
lished studying drought tolerance identified 45 robust main-effect QTLs (M-QTLs)
explaining up to 58.20 % phenotypic variation and 973 epistatic QTLs (E-QTLs)
explaining up to 92.19 % phenotypic variation for several target traits. Neverthe-
less, there is a QTL-hotspot in LG4 explaining about 58.20 % phenotypic variation
containing seven markers (ICCM0249, NCPGR127, TAA170, NCPGR21, TR11,
GA24 and STMS11) that could be further used in MAS (Varshney et al. 2014b)

7.2  Marker-Assisted Breeding

Molecular markers closely linked to a particular agronomic trait facilitate the de-
tection of favourable alleles in breeding programmes. MAS is particularly useful
in the case of breeding for disease resistance in order to avoid complex and time-
consuming evaluations as well as for pyramiding different resistance genes in the
same genotype. However, the efficacy of MAS relies on the saturation of genomic
areas of interest with robust, highly polymorphic, easy to interpret and cost-effec-
tive markers (Collard and Mackill 2008).
Despite the effort carried out during the past years to saturate genetic linkage
maps and identified markers tightly linked to traits of interest in chickpea, the adop-
tion of MAS in chickpea breeding has not been widely employed. Most cultivars
of chickpea are the results of conventional plant-breeding programmes, where trait
evaluation and phenotypic selection under field or greenhouse conditions are the
routine procedure. With the advent of molecular markers and genetic maps, there
has been an increased interest in the use of marker technology to facilitate chickpea
crop improvement. As previously mentioned, molecular markers have been used for
identification and mapping of genes and QTLs for agriculturally important traits in
chickpea. However, the extent to which markers have to be employed in chickpea-
breeding programmes has not been clearly determined. To date, only few studies
100 T. Millán et al.

reported the employment of MAS in conventional chickpea-breeding programmes

(Taran et al. 2013; Varshney et al. 2014a). In addition to the employment of mo-
lecular markers in breeding programmes, they can be used to develop new genomic
resources to be used in genomics studies or to introduce new genetic combinations
in the programmes. For example, Castro et al. (2010) employed the most associated
marker with the Foc5 gene located in LG2 (TA59) to assist the selection of resistant
or susceptible genotypes in order to develop near isogenic lines (NILs).
Regarding Ascochyta blight, the sequence characterized amplified region
(SCAR) markers SCY17590 and SCAE19336, tightly linked to QTLAR2, were suc-
cessfully employed to tag a source of Ascochyta blight resistance in a collection
of chickpea genotypes (Imtiaz et al. 2008). Recently, a new codominant molecular
marker (CaETR) was developed by Madrid et al. (2013) based on allelic sequence
length polymorphism in an ethylene receptor-like gene located in the QTLAR1
(Madrid et al. 2012). It was probed for the usefulness of the markers SCY17590
and CaETR to discriminate between resistant and susceptible chickpea genotypes.
Those markers have been used in other studies in order to check its efficiency in
MAS of blight-resistant genotypes in different breeding programmes (Bouhadida
et al. 2013; Castro et al. 2013). These two markers contribute efficiently in the
selection of new chickpea varieties with better combinations of alleles to ensure
resistance to Ascochyta blight.
Recently, the use of molecular markers in a backcrossing breeding programme
(MABC) was reported. Markers were applied to introgress blight resistance (LG4b
and LG8) and double podding (LG6) into adapted chickpea cultivars (Taran et al.
2013). In addition, MABC has been used to introgress resistance to Fusarium wilt
race 1 and Ascochyta blight in C214, an elite cultivar of chickpea (Varshney et al.
2014a). This approach permits the selection of plants with more than one set of QTL
for resistance to blight and double podding without phenotyping.
In spite of these efforts of using molecular markers in breeding programmes, it
is imperative to characterize molecular markers useful for MAS targeting the most
important agronomic traits, such as Fusarium, Ascochyta, yield, growth habit, etc.
On the other hand, the possibilities for genetic improvement and selection ap-
proaches are limited in chickpea when stress tolerance is present in sexually incom-
patible gene pools. To solve this problem, transgenic chickpeas have been developed.
Transgenic chickpeas expressing either the cry1Ac/b or the cry2Aa gene and the
bean-amylase inhibitor gene are resistant to Helicoverpa and bruchids, respectively,
but these chickpeas have yet to be commercialized. Unfortunately, attempts to gener-
ate transgenic chickpeas with increased tolerance to drought and salinity or with in-
creased methionine content have been less successful (Acharjee and Sarmah 2013).

7.3  Functional Genomics

The aim of functional genomics is to discover the biological function of genes and
to determine how sets of genes and their expressed products interact in a ­particular
3 Chickpea 101

phenotype. Understanding the functional characteristics and expression of a

­particular trait may also involve techniques such as expressed sequence tag (EST)
library construction, mutant identification, RNA interference (RNAi) experiments
and overexpression studies.
Preliminary investigations have been carried out in chickpea to determine im-
portant functional genes involved in traits such as abiotic tolerances (Mantri et al.
2007), seed quality (Gremigni et al. 2004) and biotic disease resistances (Jaiswal
et al. 2004; Coram and Pang 2005a, b). Until now, transformation studies on chick-
pea are preliminary (Indurker et al. 2010; Acharjee and Sarmah 2013; Kanakala
et al. 2013), and no RNAi essays have been performed. The most detailed functional
studies have been made using an enriched library of EST sequences, microarray
experiments or Supersage studies (Coram and Pang 2005a, b, 2006; Molina et al.
2008, 2011). Nevertheless, these studies did not establish correlation between the
genes identified and the genomics regions genetically mapped related with agro-
nomic traits. With the advent of the complete genome sequence, the identification
of genes directly located on these regions will be within reach in an easy way.
One of the most common techniques to perform functional genomic studies is
the real-time quantitative polymerase chain reaction (qPCR) technology. It has
emerged as the most accurate and sensitive method for gene expression analyses
(Derveaux et al. 2010). To perform these studies it is necessary to validate internal
control genes first. In chickpea, different combinations of reference genes were
given depending on the stress under study (Castro et al. 2012b) enabling an accurate
and reliable normalization of qPCR results.

8  Seed Production

In the past years, new successful chickpea varieties have been originated over the
world mainly by international or national research institutions or growers associa-
tions. The profit margin from chickpea seeds is low and, generally, does not attract
private sector investment because chickpea is highly self-pollinated and many farm-
ers use their own seeds stored on farm (Van Gastel et al. 2007). This is the com-
mon situation for small farmers in developing countries, where food legumes are
very important in family nutrition, but, generally, they do not have access to seeds
from improved food legume varieties. In contrast, developed countries such as the
USA, Canada or Australia, mainly exporters, require high-quality seeds to be able
to provide homogeneous raw material to be processed by the industry. Typically,
seed quality parameters in chickpea were focused on seed size, shape and seed
coat colour, but nowadays the demand of new varieties suitable for pre cooked or
processed chickpea seeds is increasing. Chickpea seed production has been widely
reviewed by Van Gastel et al. (2007), who described seed classes following the Or-
ganization for Economic Co-operation and Development (OECD) nomenclature. In
general, new varieties obtained in chickpeas are pure lines, but still a small amount
of heterozygosity could be present in the breeder or foundation seed. Around 500
102 T. Millán et al.

selected plants from each variety should be harvested and threshed separately to
­initiate variety seed production. The next step should be sowing seeds from each
plant in a single progeny row in order to discard rows with off-type plants.
Careful crop management practices such as sowing in uniform fields should be
applied. In addition, requirements for previous cropping in the seed field should
specify the crops that should not be grown for a limited time preceding the produc-
tion of the seed crop. In chickpea, the land selected to produce seeds should be free
of any other chickpea variety for at least 2 years for pre-basic and basic seeds. For
certified seeds, only 1 year between two crops of different varieties is required (Van
Gastel et al. 2007). A minimum isolation distance of 1–2 m between two fields is
considered to be enough. However, slightly longer isolation distances are recom-
mended for pre-basic seed and 3 m for basic and certified seeds. It is also suggested
touse a relatively high plant population density to improve the competitive ability
of chickpea plants to weed (Van Gastel et al. 2007).
Seed storage conditions are other important factors to take into account. Reduced
moisture and low temperature increase the longevity of the seed. Storing seeds at
less than 13 % moisture, however, has adverse effects on viability (Siddique and
Krishnamurthy 2014). Seed standards (physical purity, percentage of germination,
pest and diseases) have not exactly the same parameters in each country. Harmoniz-
ing seed certification procedures to develop a flexible or internationally acceptable
seed certification scheme should be desirable for the benefit of the national seed
industries (Van Gastel et al. 2007).
Possibly, chickpea seed producers associations will play in the future a major
role in enhancing adoption of improved chickpea cultivars in developing countries
as occurred in Ethiopia, the largest producer, consumer and exporter of chickpea in
Africa. In this country, 90 % of the seed demand is being met by the farmers orga-
nized as seed growers (Fikre 2014).

9  Conclusions and Future Prospects

Chickpea crop has a promising future. It is already a basic food in many Asian
countries and it is recognized as a source of biologically active compounds (Roy
et al. 2010). It is also a crop with low inputs adapted to low water requirements.
However, chickpea belongs to the category of “low value seed crop” because it is
highly self-pollinated; so in most cultivated areas, farmers continue to grow old
varieties and landraces using their own sowing seeds (Gaur et al. 2010). Research
on chickpea crop has been done with successful results in international and national
institutes but it is necessary to solve the transference of knowledge to private sector
and to solve commercialization of the new varieties.
In spite of the progress made in the last years in the developing of genetic maps
and the identification/location of different genes/QTLs related with the main agro-
nomic traits affecting chickpea, the molecular basis of these traits remains unknown.
Isolation and validation of genes underlying the genes/QTL for the traits of interest
is an essential step to determine gene function. Development of a genome-wide
3 Chickpea 103

physical map or local physical map around the gene/QTL region and then sequenc-
ing those are the next steps in this direction (Gaur et al. 2012). The availability of
the reference genome for desi and kabuli types is facilitating this approach (Madrid
et al. 2014) and will allow the development of diagnostic markers enhancing the
adoption of molecular breeding for increasing chickpea productivity.

Acknowledgments  Authors would like to acknowledge research funding support from the Span-
ish Ministry of Science and Innovation (MICINN, project RTA2010-00059), co financed with
European Regional Development Fund (FEDER). E. Madrid is a researcher funded by the ‘Juan
de la Cierva’ programme of the Ministry of Science and Innovation.

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Chapter 4
Lentil

Thomas R. Stefaniak and Kevin E. McPhee

1 Introduction

Lentil ( Lens culinaris Medik.) is one of the first, if not the very first, domesticated
grain crop. If the beginning of agriculture define humanities transition from wan-
dering hunter-gatherers to members of a civilization, then lentil shares credit with
only a few other species for making this transition possible. Archeologists found
carbonized remains of lentil seed along with einkorn, emmer, and barley; suggest-
ing that rotation with cereals was a feature of agricultural systems from the very
beginning of early agriculture. The domestication of lentil and the development of
agriculture made it possible for humans to become sedentary and turn from hunt and
gathering to agricultural production.
Lentil plants are indeterminate semiprostrate members of the Leguminosae
or Fabaceae family. Legume etymology is probably from the Latin word legere,
which means to gather. Lentil leaflets are pinnately compound with one to eight
pairs of leaflets. The older leaves terminate with a prehensile tendril. Lentil plants
can have one to many primary branches depending on genotype and population
density. Lentil flowers can be white, pink, and purple to pale blue in color. The fruit
of the lentil plant is a pod, which is a defining feature of all legumes. The lentil pod
generally contains two round convex lens-shaped seeds. Indeed, the English word
lens, comes from the Latin word for this plant: lens, lentis, and lentil. Lentil roots
form a slender taproot system that can range from shallow with many branching
to deep with little branching depending on genotype and growing conditions. As

T. R. Stefaniak ()
North Central Research Extension Center, North Dakota State University,
5400 Highway 83 S, Minot, ND 58701, USA
e-mail: [email protected]
K. E. McPhee
Department of Plant Sciences, North Dakota State University,
370G Loftsgard Hall, P.O. Box 6050, Fargo, ND 58108, USA
e-mail: [email protected]
© Springer Science+Business Media New York 2015 111
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_4
112 T. R. Stefaniak and K. E. McPhee

is the case with all ­legumes, the roots form a symbiosis with rhizobacteria that fix
atmospheric nitrogen into the soil. This trait allows the plant to grow on nitrogen-
poor soils.
Lentil seed is an excellent source of energy, dietary protein, fiber, and micronu-
trients. Lentil is similar to the other pulses in total energy content and lower than
soybean (Urbano et al. 2007). However, this difference with soybean is due to the
substantially higher fat content of soybean (Gebhardt and Thomas 2002). Protein
content in lentil is the highest among food legumes consumed without industrial
processing (Sharma 2009). Protein in lentil can range from 21.4 to 25.5 % (Rasheed
et al. 2010). The necessity of combining lentil or other legumes with cereals in a
vegetarian diet to promote good human health was recognized thousands of years
ago. Cereals lack adequate amounts of the essential amino acid lysine. The amino
acid profile of lentil has favorable leucine/isoleucine and leucine/lysine ratios rank-
ing lentil highly as a vegetable protein source (Fernandez et al. 1996; Nestares et al.
1996). The protein profile as well as the high vitamin and mineral content of lentil
complements that of cereals in a vegetarian diet (Urbano et al. 2007).
Lentil grain is harvested after the crop has reached physiological maturity result-
ing in hard unpalatable seed. Eating uncooked or undercooked mature lentil seeds
in sufficient quantities will cause illness in monogastric animals including humans,
as is the case with all the other major legume crop species. This is because lentil
contains anti-nutritional constituents including phytic acid, trypsin inhibitors, and
various tannins among others. These constituents are inactivated with cooking or by
germinating the seeds. One of the traits highly favored in lentil is the rapidity with
which it can be cooked relative to other whole grain legumes.
Due to a combination of socioeconomic status, cultural/religious mores, and
climate, the region of the globe that consumes the most lentils is south central to
southwest Asia. Diets in this region are largely vegetarian. Whole lentil seed is usu-
ally prepared in soups, stews, or porridge-like dishes. Its popularity is also due to
the fact that lentil softens with cooking even without presoaking. Cooked lentil is
also puréed and served in porridge like stew such as dahl, which is a typical dish
of India. Many other Asian and North African countries have their own version of
dahl such as Turkey’s Kirmizi Mercimek Corbasi (Yadav et al. 2007). In Ethiopia
lentil and wheat flour are moistened and baked into a type of bread called Sambusa
(Yadav et al. 2007). In northern India, lentil flour is sometimes baked with wheat in
the traditional bread naan. The Egyptians boil lentils in water, drain, and serve alone
or tossed with a variety of sauces. In North Africa, lentils are combined in soups
with chickpeas and white dry bean. More recently, lentils have become popular and
served as sprouts in salads especially in Western vegetarian diets.
Lentil production in the developing world is grown mostly for subsistence and
not for export. The organization Consultative Group on International Agricultural
Research (CGIAR) reported that about 70 % of lentils produced are consumed in
the country in which they were grown (CGIAR 2014). Many of the varieties are
landraces that are potentially quite ancient and do not respond well to external in-
puts. India traditionally produces the greatest amount of lentils; however, in 2005
Canada surpassed India and accounted for 12–30 % of global production (NDSU
4 Lentil 113

extension 2006). India, Turkey, Ethiopia, China, Syria, Iran, the USA, Canada, and
Bangladesh are the top lentil-producing countries (CGIAR 2014).
Many of the most populated regions of the developing world are dependent on
lentil production and consumption primarily for two reasons. First, lentil is well
suited to production systems in these areas because the crop has evolved with rela-
tively little human influence to be productive on marginal land. Like all legumes,
lentil fixes atmospheric nitrogen into a form that makes it available to the crop.
This makes the application of costly nitrogen fertilizer unnecessary. Second, just
as lentil well compliments cereals in a human diet, they also do so in a crop rota-
tion by reducing populations of insects and pathogens, and by leaving nitrogen in
the soil.
The largest lentil-importing nations between 2000 and 2005 were in the develop-
ing world and include India, Bangladesh, Egypt, Colombia, Algeria, and Sri Lanka
(Skyrpetz 2006). India is both a major importer and producer of lentil. Dependence
on imported lentil in the developing world is due to a combination of a high human
population to arable land ratio, and the low yield potential of the landraces of lentil
grown in these areas.
By far, the largest lentil exporter in the world is Canada. The United Nations
Food and Agriculture Organization (FAO) reported that 31.7 % of annual produc-
tion is exported and that the top five exporters were Canada, India, Turkey, Austra-
lia, and the USA between 2001 and 2006 (FAO 2008). Indeed, collectively these top
five exporters account for over 80 % of global export. North America and Australia
are able to lead lentil exports due to a combination of a low human population
to arable land ratio, and the higher yield potential of the cultivars of lentil which
have improved response to inputs that are grown in these areas. Additionally, len-
til ­consumption is much lower in North America and Australia when measured as
availability for consumption.
While it is true that producers in developing regions do not have access to the
level of input resources that their counterparts in wealthier nations do, much effort
has and is being expended on improving varieties and cultural practices. The need
for these improvements is critical as can be seen in the fact that yields in countries
such as India, yield has remained stagnant for much of the past four decades while
increases in cereal yields have been steady (McNeil et al. 2007). The International
Center for Agricultural Research in Dry Areas (ICARDA), which is headquartered
in Lebanon, is an organization that is engaged in extensive efforts to improve lentil
production. Efforts in the area of breeding and genomics include projects to assem-
ble and genotype a reference collection for association mapping to improve stress
tolerances, as well as biotechnological approaches (ICARDA 2013).
In the past two decades, lentil production has increased substantially in d­ eveloped
countries, especially in Canada and the USA. Concurrently, research efforts have
been ramped up to increase productivity. The need for this is further indicated in the
fact that the increase in lentil harvested has been largely due to increased acreage,
and not increasing yields over the past 20 years (McNeil et al. 2007). In C ­ anada,
considerable resources are devoted to lentil improvement. The primary player here
is the Crop Development Center (CDC) at the University of S ­ askatchewan. In
114 T. R. Stefaniak and K. E. McPhee

a­ ddition to developing superior lines for increased productivity, the CDC is heavily
involved in improving nutritional traits in their breeding programs.
In the USA, the vast majority of lentil production is in the northern Midwest
(Montana and North Dakota) over to the Palouse region of the Pacific Northwest.
Consequently, the primary universities involved in breeding and production re-
search are North Dakota State University (NDSU), the Montana State University
(MSU), the University of Idaho, and Washington State University (WSU). Research
in Canada and the USA has different priorities that address the use of greater inputs
available to producers in these regions. Because lentil breeding in North America is
relatively young compared to crops such as wheat and maize, considerable opportu-
nities exist to better adapt lentil to North America. Specifically, positive responses
to improved fertility, better water management, and chemical control of pests are all
goals of Canadian and American breeding programs.

2  Origin and Systematics

2.1  Prehistorical Data

The domestication of lentil ( L. culinaris Medik.) is as old as agriculture and

indeed human civilization itself. This domestication event was possible because
the two ingredients necessary for humanities transition from hunter-gatherer, to
farmer came together in time and space. Those two ingredients were a favorable
growing season/region, and plant species amenable to cultivation. In the Fer-
tile Crescent ­region, reliable sources of moisture, good soil fertility, and species
with potential for domestication were all available to the Neolithic tribes of the
region.
The oldest evidence of the consumption of lentil traces back to 10,000 BC in
Greece where carbonized remains have been found (Sandhu and Singh 2007). It is
uncertain whether these seeds were gathered or harvested from cultivated plots as
this predates when archeologists believe humans began farming around 8000 BC
(Brown et al. 2009). Additionally, these lentil seeds were small and indistinguish-
able from seed from wild Lens species (Sandhu and Sing 2007).
In Israel lentil seed dating to 6800 BC were discovered that appeared to have
been stored in large quantities, indicating that seed stock was being preserved for
the purpose of future cultivation (Sandhu and Sing 2007). Further archeologi-
cal evidence that lentil was being cultivated and traded around this time comes
from carbonized lentil seeds from the relatively distant locations of Tell Ramad
in Syria (6250–5950 BC), Beidha in Jordan, Hacilar in Turkey (5800–5000 BC),
and Sabz in Iran (5500–5000 BC) (Van Zeist and Bottema 1971; Helbeck 1959,
1963, 1970) These discoveries represent the oldest evidence that humans had
begun farming.
4 Lentil 115

2.2  Historical Data and Literature

As is the case in modern times, legumes were regarded differently than cereals in
the ancient world. Then as now, legumes are described mostly in terms of their
nutritional value and not their delectability. Pliny describes the growing of lentils
from seed and its varieties. He mentions its medicinal properties and a variety of
ways of boiling or otherwise cooking lentils for various remedies (Wright 2001).
They are seen as a poor man’s source of protein when he cannot afford meat. This
is seen in the fact that mention of legumes, including lentil, are absent from many
of the accounts by ancient historians (Flint-Hamilton 1999). Indeed, it was not
­recommended that farmers produce a surplus of legumes as they are of little value
for trade. However, though legumes generally receive little attention in ancient lit-
erature, the one that is probably mentioned to the greatest degree is lentil.
An early mention of lentil from the fifth century BC comes from the comic play-
wright Aristophanes (Flint-Hamilton 1999). He wrote in Plutus that Chremulos has
no further need of lentils because he has found wealth (Flint-Hamilton 1999). Later
in the second century, Athenaeus, in Deipnosophistae, uses the description of a meal
of lentil and a foul-smelling perch to indicate the unsophistication of the hosts.
In the Judean/Christian tradition lentil is also mentioned. In Genesis (34), where
Esau sells his birthright for a bowl of lentils. The passage states, “Then Jacob gave
Esau bread and pottage of lentils; and he did eat and drink, and rose up, and went
his way: thus Esau despised his birthright.” In Ezekial 4:9, it is written, “And you,
take wheat and barley, beans and lentils, millet and emmer, and put them into a
single vessel and make your bread from them.” 2 Samuel 17:28–29 says, “Brought
beds, basins, and earthen vessels, wheat, barley, flour, parched grain, beans and
lentils, honey and curds and sheep and cheese from the herd, for David and the
people with him to eat, for they said, the people are hungry and weary and thirsty
in the wilderness.”

3  Origin and Domestication

Lentil is believed to have been domesticated in the Fertile Crescent region of the
Middle East in what is present day Iraq. Indeed, archeological evidence confirms
the presence of lentil as far back as 8500–6000 BC in the Turkey/Syria/Iraq region
(Yadav et al. 2007). The first agricultural revolution transformed human societies
in this region from hunter-gatherers to agriculturalists. As is true in modern times,
people generally have preferred to eat animals over plants. Animal husbandry was
probably the impetus to the raising of crops. This is because the raising of livestock
in this hard-scrabble environment was relatively inefficient. Even in modern times,
food from animals requires more energy input than production of food from plants.
Lentil was among the first crops because not only could it be used as animal fodder
but it also could be a good source of protein when meat was scarce.
116 T. R. Stefaniak and K. E. McPhee

As previously discussed, the nutritive aspects of lentil were important in influ-

encing Neolithic man to begin its cultivation. Lentils were among the grain legumes
consumed as an alternate protein source to animals that were not in great supply.
Lentil was also identified as a crop that can improve the health of the soil. Though
they did not know why, Greek and Roman farmers noticed that it could be grown in
fields depleted by the more valued cereal grains. Additionally, ancient agronomists
observed that the seed from a desirable plant tended to produce a plant that was very
similar to the plant from whence the seed was collected. The ability of lentil to fix
atmospheric nitrogen and its self-pollinating habit made it an attractive wild spe-
cies for domestication by ancient agronomists. For all these ­reasons, from the very
beginning of the agricultural revolution, lentil was an important part of cropping
systems, along with wheat, barley, pea, flax, emmer wheat, and einkorn.
Because lentil is self-pollinated, domestication first took the form of selecting
desirable plants and retaining their seed for the next growing season. In its most
basic terms, a species is domesticated when its seed is easily established, and its
harvested tissues can be collected efficiently. As is the case with most if not all crop
species, seed dormancy was the primary phenotype to be improved. Along with
­dormancy, the removal of pod dehiscence was required. In lentil domestication,
seed size was also important as it relates to ease of harvest and yield. Indeed, a ma-
jor phenotypic difference between the cultigen L. culinaris and its wild relatives is
seed size. The other traits that most obviously separate cultivated lentil from its wild
relatives include greater leaf area, leaf number, plant height, number of f­lowers,
larger pods, longer rachis, and shorter peduncle.

3.1  The Spread of Lentil

Lentil cultivation spread with the spread of agriculture starting in Southwest Asia
and fanning out from there to Greece, Central and Western Europe via the Danube,
south through Africa along the Nile, and eastward to India (Harlan 1992). This
spread was rapid as can be evidenced from the fact that archeologists have found
the remains of lentil as far west as the eastern Iberian Peninsula by about 5450 BC
(Cubero et al. 2009).
Due to an environment not conducive to the preservation of botanical remains,
it is unclear when lentil reached the Nile delta, though it was no doubt early in
its ­domestication, because of this regions close proximity to the center of domes-
tication for lentil. However, further up the Nile in the tomb of the twelfth dynasty
­(2400–2200 BC) remains of lentils have been recovered (Erskine et al. 2009).
Lentil reached the Indian subcontinent around 2000 BC (Cubero et al. 2009).
Tradesmen likely following what would later become the silk road brought lentil
to what now is Uzbekistan, Kyrgyzstan, Pakistan, Afghanistan, and into India it-
self as part of an Indo-European invasion. Evidence suggests that this introduction
represented very little genetic diversity as seen in the lack of variability in the l­ ocal
landraces still in use based on molecular evidence. This is surprising when it is
considered that India quickly became, and remains the largest lentil-growing region
in the world.
4 Lentil 117

4 Taxonomy

4.1  The Genus Lens

Cultivated lentil (L. culinaris) belongs to the genus Lens and early researchers used
comparisons of morphology and “crossability” to connect or separate putatively
distinct individuals. Molecular genetic techniques made possible the clustering of
individuals based on actual DNA sequences and many early classifications were
modified. A detailed description of the changes in Lens classification over the years
is beyond the scope of this chapter. In summary, species included in the genus
went from four in 1979; L. culinaris, L. orientalis, L. nigrican, and L. ervoides
­(Ladizinsky 1979), to two in 1984; L. culinaris (subspecies culinaris, orientalis,
and odemensis), and L. nigricans (subspecies nigricans and ervoides), back to four
in 2000; L. culinaris (subspecies culinaris, orientalis, odemensis, and tomentosis),
L. nigricans, L. ervoides, and L. lamottei. The most recent classification scheme
also established in 2000 contains six species and is as follows: L. culinaris (subspe-
cies culinaris and orientalis), L. odemensis, L. tomentosis, L. nigricans, L. ervoides,
and L. lamottei (Cubero et al. 2009).

4.2  Biological Species

When new specimens are identified, breeders are interested in whether they can be
crossed and give rise to viable progeny and molecular characteristics are secondary.
Experiments attempting to establish reproductive barriers between putative species
and subspecies can further complicate classification. However, for breeding prog-
ress, because the cultigen of lentil is L. culinaris ssp. culinaris; what can be crossed
to it represents the most readily available reservoir of genetic variability for breed-
ing improvement.
Cultivated lentil was initially subdivided into two subspecies macrosperma
(seed diameter 6–9 mm) and microsperma (seed diameter 2–6 mm) based on seed
size by Barulina in 1930 (Sandhu and Singh 2007). Indeed, most if not all breed-
ing progress has been made by utilizing alleles from accessions that fit into these
classifications. The macrospermas have yellow cotyledons and little or no pigment
in their flowers. The microspermas have red, orange, or yellow cotyledons and
pigmented flowers.
More recently, Ladizinsky (1979) reported that the cultigen L. culinaris ssp. cu-
linaris and L. orientalis share a common karyotype and cross freely with one an-
other. This is why L. orientalis is now placed firmly as a subspecies of L. culinaris.
L. nigricans has a slightly different karyotype and different accessions within that
­species cross with varying degree of success with L. culinaris ssp. culinaris (Balyan
et al. 2002). As a result, those L. nigricans accessions that do cross with L. culinaris
were reclassified as L. culinaris ssp. odemensis (Ladizinsky et al. 1984). Hybrid-
ization experiments tended to verify or reassign accessions from subspecies of a
­species to separate species and vice versa.
118 T. R. Stefaniak and K. E. McPhee

In conclusion, hybridization experiments have supported the most recent classifica-

tion scheme of six species within the Lens genus (Cubero et al. 2009). In terms of gene
pool levels, L. orientalis clearly belongs in the primary gene pool of L. culinaris and is
one of its subspecies. L. odemensis, because viable progeny can be obtained through
embryo rescue, is in the secondary gene pool of L. culinaris and remains a separate
species. L. tomentosis, L. nigricans, L. ervoides, and L. lamottei belong to secondary
or tertiary gene pools due to the requirement of embryo rescue to obtain hybrids.

5  Varietal Groups

Lentil is composed of several varietal groups or market classes based on testa color
and cotyledon color. The color classes red and green each have within them three
relative sizes. The seed sizes of red lentils place them in the smaller seeded micro-
sperma botanical category and are graded in the USA as extra small, small, and
medium. The sizes of green lentils originating from macrosperma types are graded
as small, medium, and large. The cultivar CDC Lemay is an example of the spe-
cialty small-seeded type French Green. Large-seeded types range in millimeters of
diameter from 6 to 9, medium from 5 to 6, and small from 3 to 5, with extra small
being up to 3. Much of the production of large green lentils is in North America,
and principal importers of large green types include Colombia and Western Europe
(Saskatchewan Ministry of Agriculture 2013). Pardina or Spanish brown types have
a brown speckled testa with yellow cotyledons (Fig. 4.1). Red lentils have tradition-
ally been produced and consumed in India and the Middle East. More recently, the
USA, Canada, and Australia have become major producers of red lentils (Muehl-
bauer et al. 2009).

Fig. 4.1   ‘Armuñesa’ (left) and ‘verdina’ (small green, right) lentil landraces. (Courtesy of the gene
bank CRF-INIA, Alcalá de Henares, Spain)
4 Lentil 119

6  Genetic Resources

Preserving and exploiting genetic diversity is of paramount concern to lentil breed-

ers since genetic variation is required for breeding progress. Lentil is a relatively
small-seeded grain species so ex situ storage of seed does not require unreasonably
large storage facilities. The ICARDA, headquartered in Beirut, Lebanon, is well
positioned geographically and culturally to be the primary institute for research on
lentil improvement and; therefore, maintains the world collection of over 10,000
accessions of Lens. Included in these are almost 9000 accessions of cultivated
Lens from 70 different countries representing four major geographic regions, 1373
ICARDA breeding lines, and 583 wild Lens taxa from 24 different countries (Fur-
man et al. 2009). Most of these were obtained on collection missions conducted
by ICARDA personnel. A large proportion of these are from Southwest Asia and
North Africa, because as is the case with most species the greatest genetic variation
can be found in or near the center of origin and domestication. Interestingly, het-
erogeneities within families descended from individuals from some landraces have
resulted in significant phenotypic variation (Erskine and Choudhary 1986). These
results suggest that useful genetic variation can be obtained from within particular
landraces.
Four other institutes house substantial collections. They are the Australian Tem-
perate Field Crops Collection (ATFCC) in Victoria, Australia, with 5250 acces-
sions (Redden et al. 2007), the United States Department of Agriculture (USDA) in
Pullman, USA, with 3011 accessions (USDA 2013); the N.I. Vavilov All-Russian
Research Institute of Plant Husbandry (VIR) in St. Petesbourg, Russia, with 2687
accessions (VIR 2013), and the National Bureau of Plant Genetic Resources in New
Delhi, India with 2212 accessions (Dwivedi et al. 2006). The genetic resources
available to lentil researchers are substantial and would be unwieldy to utilize for
breeding or genomic research in their entirety.
In order to make efficient use of the worldwide collections for improvement core
collections have been assembled. These core collections are a subsample of an en-
tire collection and represent the overall genetic, as well as agroclimatological diver-
sity of lentil. Assembly of these collections have relied on efforts to cross-reference
accession identifications with phenotypic and passport data. Maintaining passport
data is simply an exercise in faithfully recording locations where accessions were
obtained. However, those data are of little use without accurate characterization of
phenotypes.
Comparison among accessions is most easily accomplished by assembling
a panel including individuals from differing regions, growing them in a uniform
environment, and recording their responses. Erskine et al. (1985) measured nine
quantitative traits in 615 accessions from 13 lentil-growing regions. They found
that seed size, pod height, and time to flowering were useful in grouping accessions
into regional groups.
Studies of the magnitude of variability within regional collections have been
conducted and are indicative of patterns of diversity. These studies can be regard-
ed as representative of regions included in the larger collection held by ICARDA.
120 T. R. Stefaniak and K. E. McPhee

Sindhu and Mirsha (1982) evaluated 30 lines from centers in the All India Coordi-
nated Pulse Improvement Program. They predicted substantial genetic advance for
eight agronomic traits including grain yield. Ramgiry et al. (1989) evaluated the
same agronomic traits in 21 Indian accessions and reported similarly high genetic
variances. Lakhani et al. (1986) reported substantial heritabilities for germination
traits using 100 Indian genotypes. They concluded that sufficient genetic variation
existed in their sample to improve germination rates. Contradictory results were
reported in an evaluation of 78 genotypes collected from around Bangladesh; where
very little phenotypic variability was measured for five of six agronomic traits (Sar-
war et al. 1982). Baidya et al. (1988) reported highly significant differences be-
tween 96 genotypes collected in Bangladesh, for days to flowering, plant height,
seed weight per plot, and plant dry weight. It should be noted that in all the above
experiments the phenotypic variances were substantially greater than the genotypic
variances suggesting that environmental conditions could greatly hamper selection
for the respective traits in the field.
In order to characterize their usefulness, genotypes in the ICARDA collection
have been screened for particular tolerances such as drought (Sarker et al. 2005; Er-
skine et al. 1985), chilling (Erskine et al. 1981), and responses to regional pathogen
populations (Erskine and Witcombe 1984). A seminal report published by ICARDA
called the Lentil Germplasm Catalog is a summary of phenotypic data. Much of
the overall genetic diversity is accounted for in the landraces and wild relatives
collected near and within lentil’s region origin. Additionally, breeding lines and
germplasm that were derived in disparate climatic regions were included in order
to ensure alleles for tolerance to a wide array of stresses were sampled. Genetic and
statistical analyses that included hierarchical cluster analysis of agronomic traits
and two step cluster analyses of agroclimatological data linked to geographical lo-
cations of collection were used to ensure diversity (Furman 2006).

7  Major Breeding Achievements

7.1  Yield Potential

The first concept that should be made clear in the beginning of any discussion of
plant breeding is that the most important trait is yield for agronomic crops. Many
treatments on plant breeding discuss tolerance to disease, extreme weather, insect
predation, etc. Improvement of these traits is for the purpose of maintaining or
increasing yield. This is true even in the case of quality traits, that is, breeders
are trying to improve iron content in lentil. If lines are developed that have higher
iron content with no grain yield penalty, then harvesting more iron per unit of crop
area has been achieved. Lentil production has increased dramatically over the past
40 years (Fig. 4.2a, 4.2b, 4.2c). This increase is partly due to increased acreage
and partly due to increased yield (Fig. 4.2a, 4.2b). Conventional wisdom holds that
 c
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and assumed to be below 1000. (Adapted from http://faostat.fao.org/site/567/DesktopDefault.

2012 in three of the top producing developed countries. *Data for Australia are not available
121
122 T. R. Stefaniak and K. E. McPhee

about 50 % of yield increases are due to breeding efforts and 50 % are due to im-
proved management practices.
Production has increased in some countries of the developing world and remained
flat or even decreased in others over the past 40 years (Fig. 4.3). Yields have been
flat or very erratic in developing world where domestic consumption is heavily reli-
ant on local production. In this time period, India has at times been the top exporter
and increased production has been solely a function of increased production area.
Clearly, much opportunity exists for breeding improvement of Indian cultivars.

7.2  Genetic Variability Bottleneck

The history of lentil breeding surprisingly short given its ancient domestication.
Before modern breeding techniques were developed, lentil production in South Asia
was severely constrained by the unavailability of genotypes that possessed appro-
priate flowering and maturity traits. At this time, cultivars with little variability
were available to farmers and breeders, which limited potential genetic improve-
ment for a whole host of stress traits. Cultivars from this region are designated
pilosae and have superficial characteristics that differentiate them from nearly all
other lentils in other regions (Barulina 1930). When lentil was first brought to In-
dia by the Sanskrit-speaking race, it was virtually unknown to the local inhabit-
ants ­(Cubero 1981). Attempts to grow this crop in a new and different environment
placed substantial selection pressure, primarily for flowering time and maturity on
the introduced landraces creating a genetic “bottleneck.” This bottleneck was the
impetus for major breeding efforts for the India subcontinent.
In the 1980s, ICARDA researchers began addressing the bottleneck by introduc-
ing several cultivars from Western Asia into India and Pakistan. However, it was
observed that when these Western cultivars were only flowering, the indigenous
genotypes were nearing maturity (Ceccarelli et al. 1994). Therefore, attempts at
breaking the bottleneck by the simplest means, that is, introduction, has only been
possible with Western lines having similar phenology. Linkage between maturity
alleles and other important agronomic traits have hampered efforts to broaden ge-
netic diversity in lentil. It is also important to note that tolerance to most if not all
stresses is inherited quantitatively. Maturity is also a polygenic trait. This makes
introgression of appropriate maturity alleles into germplasm with an array of stress
tolerances challenging.
Effort by researchers at ICARDA beginning in 1981 to minimize the bottleneck
effect focused on hybridizing later maturing West Asian lines with indigenous lines
in order to introgress favorable maturity alleles into exotic germplasm (Erskine
et al. 1998). These efforts represent major achievements for lentil breeders of the
subcontinent because they allow for selections to be made in local nurseries with-
out the need to manipulate photoperiod. High-yielding early-maturing cultivars that
have been released include Barimasur-2 (Sarker et al. 1999a), and Barimasur-4 in
Bangladesh (Sarker et al. 1999b).
 4 Lentil 123

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2012 in three of the top producing developing countries, Bangladesh, India, and Turkey. (Adapted
from http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor)

Development of Barimasur-4 provides an illustrative example of the widening

of the bottleneck in South Asian lentil. The cross was made at ICARDA in Aleppo,
Syria using a locally adapted landrace ILL 5888 for the female parent and an exotic
disease-resistant line FLIP 84–112 L (ILL 5782) for the male parent. A segregating
F3 population was sent to Bangladesh where single plant selections were made.
Lines from the single plants were advanced to the F6 using a family selection strat-
124 T. R. Stefaniak and K. E. McPhee

egy, and advanced testing commenced. Barimasur-4 was eventually released and
described as being an early-maturing high-yielding cultivar with good resistance to
rust ( Uromyces viciae-fabae (Pers.) de Bary, Pucciniaceae, Uredinales) and stem-
phylium blight (caused by Stemphylium botryosum Wallr.; Dematiaceae, Hyphales).
Thusly, exotic disease resistance was assembled into a package with an adapted
landrace. Substantial achievements have been made in breeding for tolerance to
other diseases as well.

7.3  Biotic Stresses

Before resistance was available, Ascochyta blight caused by Ascochyta lentis and
Ascochyta fabae were among the most serious disease threats to lentil production
globally. Fortunately, breeding for tolerance has been one of the greatest successes
for lentil breeders. Germplasm has been released that have been shown to be As-
cochyta blight resistant including ILL 5588 (Erskine et al. 1996), “CDC Vantage”
(Vandenberg et al. 2002a), and “CDC Plato” (Vandenberg et al. 2005) among many
others. New species and pathotypes continue to emerge making breeding progress
challenging. Ascochyta blight can affect not only the growing plant but also se-
verely reduce seed quality. Ascochyta-infected seed can transmit the disease to the
next growing season. The germplasm ILL 5588, previously mentioned, also has
resistance to lentil vascular wilt (caused by Fusarium oxysporum Schlechtend.:Fr. f.
sp. lentis (Vasudeva and Srinivasan) W. L. Gordon; Erskine et al. 1996). This germ-
plasm was used to develop the Ascochyta-resistant cultivar “Northfield” (Ali 1995).
Numerous other cultivars and germplasm have been released that have resistance to
Ascochyta (Materne and McNeil 2007).
In South and West Asia as well as parts of North Africa, rust is the most im-
portant foliar disease of lentil (Erskine et al. 1994). In some epidemics complete
crop losses have been observed. Genetic variation has been identified in several
sources (Nene et al. 1975; Reddy and Khare 1984; Mishra et al. 2005; Singh
and Sandhu 1988). Inheritance of resistance has been determined to be from a
single dominant gene (Sinha and Yadav 1989). Released varieties resistant to rust
include the above-mentioned ‘Barimasur-2’ (Sarker et al. 1999a), ‘Barimasur-4’
in Bangladesh (Sarker et al. 1999b), and ‘Chakkouf’ (Idrissi et al. 2012) among
others.
Vascular wilt caused by F. oxysporum f. sp. lentis Vasudeva and Srinivasan is a
serious constraint to lentil productivity. Tolerance to vascular wilt has been identi-
fied in wild species of Lens and introgressed into L. culinaris. Bayaa et al. (1995)
identified resistant alleles in L. montbretii (Fisch et Mey) Davis et Plitm. This source
of resistance was used to develop the germplasm ILL 5588 which was released and
is available to the public from ICARDA (Erskine et al. 1996).
Stem rot caused by Botrytis cinerea infects lentil crops in many countries. Resis-
tance to botrytis was discovered in the former Soviet Union (Khare 1981). As with
Ascochyta this pathogen can also affect the seed and be transmitted to the next crop
via infected seed. The first cultivar with resistance was released in Pakistan and
4 Lentil 125

called ‘Masoor-93’ (Tufail et al. 1995). The Botrytis species B. fabae also causes a
stem rot, complicating resistance breeding (Materne and McNeil 2007). In Australia,
genotypes were identified that had good resistance to both B. fabae and B. cinerea.

7.4  Abiotic Stresses

Increasing tolerances to extreme temperatures is a strategy that can improve pro-

ductivity by allowing a crop to avoid stress during critical stages of development.
In India, the lentil crop is planted after the rains of the monsoon in October to De-
cember at the beginning of the dry season. Therefore, it must have cold tolerance
sufficient to establish a good stand in the winter and then appropriate phenology
to complete its life cycle utilizing the moisture stored in the soil. Screening for
cold tolerance is a challenge primarily because environmental conditions for field
evaluations are not reliable. Ideally, the stressful temperatures would occur at a con-
sistent phonological stage and at a constant rate of decline. Also, the lack of snow
cover should be consistent. Screening for cold tolerance must be done in multiple
locations and years due to the inconsistent environmental conditions (Erskine et al.
1981). Genetic variation for cold tolerance has been identified in 238 accessions
through screening of a world collection of 3592 lines (Erskine et al. 1981).
Drought during the critical seed-fill stage of development can be avoided by
sowing lentil in the winter. Substantial yield increases have been realized in re-
search conducted in these countries by increasing cold tolerance so that the crop
matures before the hot/dry months of the year (Muehlbauer and McPhee 2007). To
accomplish this yield gain, phenology of the crop must also be altered so that pods
are set before excessive heat causes pod abortion, and grain fill is completed before
drought conditions occur. This strategy shows much promise, although it has yet to
be implemented on a large scale. Data from yield trials corroborate this potential
and cultivars as well as germplasm have been released. WA8449085, WA8449090,
and WA8449041 are germplasm that are adapted to this strategy (Spaeth and Muehl-
bauer 1991). Two of these WA8649090 and WA8649041 (Spaeth and Muehlbauer
1991) were used as parents to develop the winter-hardy cultivar, Morton (Muehl-
bauer and McPhee 2007). In yield trials, Morton yielded 73 % more than the highest
spring-sown variety (Muehlbauer and McPhee 2007). Additionally, Morton yielded
63 % better than the winter-hardy check.
Improvements in tolerance to inhospitable soils have been substantial in lentil.
In Australia, lines have been identified that are extremely tolerant to boron toxicity,
and it is projected that if superior alleles from these lines can be introgressed into
adapted cultivars, yield could increase by 91 % (Hobson et al. 2006). Saline soils
also constrain lentil production. Fortunately, significant genotypic difference to salt
stress have been identified (Katerji et al. 2001; Ashraf and Waheed 1990).
126 T. R. Stefaniak and K. E. McPhee

8  Specific Goals in Current Breeding

8.1 Cooperations

One of the most innovative goals of plant breeding in lentil-producing regions of the
developing world is to establish participatory plant breeding (PPB) and increasing
popularity of participatory variety selection (PVS) networks. In PVS, local growers
are recruited to test cultivars and provide feedback regarding their own wants and
needs, and in PPB actually participate in conducting trials and making selections.
These participations are particularly useful in the developing world because local
producers tend to be isolated geographically from researchers. In India, where hun-
dreds of languages and dialects are spoken, producers can be isolated linguistically.
Consequently, lentil producers in these countries are slow to adopt newer improved
cultivars. Additional reasons for this are insufficient capital, and cultural biases.
The four phases of PVS are: (1) identification of farmer preferences, (2) search
for material to fill those needs, (3) testing of that material in local producer’s
fields or nearby research station, and (4) dissemination of information and culti-
vars to ­individuals in wider areas of the region (Solanki et al. 2007). Phase 1 is
­accomplished by making available an array of genotypes from local landraces to
prereleased experimental lines to directly to the growers and they are queried for
their impressions and results. Sometimes these demonstrations actually take place
at a research station and the growers are brought to them for discussion. Phase 2
involves a search for available germplasm that more closely meets the grower’s
preferences. Phase 3 allows farmers to grow and compare the selected germplasm
and compare their observations to those from phase 1. Finally, in phase 4, produc-
ers in the wider region who would likely benefit from the efforts of the first three
phases are identified and seed is made available to them.
A good example of PVS was in 2012, when seed from high zinc and iron
­genotypes were distributed to over 3000 farmers in Bangladesh, India, and Nepal
(ICARDA 2012). Information from these farmers will allow selection for adapted
genotypes with these high-mineral traits. Concurrently, participating farmers are
being made aware of the health benefits of eating these cultivars and that grow-
ing them also represents an opportunity for growing lentil destined for the export
pipeline.
Compared to PVS, PPB is a more costly and time consuming activity. Farmer
participation includes growing and selecting from segregating populations. It can be
viewed as preliminary work that will result in material that would be entered into a
PVS network. Currently, PPB in lentil remains a goal yet to be achieved or at least,
documented (Solanki et al. 2007). It is anticipated that when progress from PVS
nears exhaustion, PPB efforts will increase.
4 Lentil 127

8.2  Abiotic Stress Tolerance

Because plants are unable to move away from stresses, a goal of plant breeding
even more than animal breeding is adaptation, or ability to have high yield in a
particular region. However, in the case of lentil breeding adaptation also includes
making improvements of which the local producer can take advantage. Also impor-
tant is that lentil growers from different regions can have vastly disparate levels of
management resources available to them. This demonstrates why breeding objec-
tives for lentil in the developed world focus much on adapting cultivars that have
a maximum response to management inputs such as soil fertility and pesticide use.
Lentil is largely produced as a dry land crop; therefore, drought tolerance will
continue to be an important trait worldwide. In lentil, much of the effort at mitigat-
ing aridity is focused around altering crop phenology avoid drought. Drought avoid-
ance can be accomplished in both time and space. Plants have many mechanisms
for avoiding drought including deep rooting, seed dormancy, and completion of the
life cycle prior to drought, to name a few. The search for new sources of genetic
variability continues to be a specific goal for lentil researchers at ICARDA, USDA,
the University of Saskatchewan CDC, and Commonwealth Scientific and Industrial
Research Organisation (CSIRO).
Improving drought avoidance has been most successful by altering crop phenol-
ogy so that key developmental stages are completed in the absence of drought. In
many parts of the lentil-growing world, this means that the crop must also be able to
tolerate cold or freezing temperatures so that a good stand is established before the
optimal temperatures for vegetative growth is perceived. Tailoring genotypes with
combined cold tolerance and adapted phenology remains a major specific goal of
lentil researchers.
Lentil plants must perceive a particular vernalization, temperature, and photope-
riod regime to initiate reproductive growth (Summerfield et al. 1984; Erskine et al.
1989; McKenzie and Hill 1989). In the case of photoperiod, this response is known
to be under genetic control (Summerfield et al. 1984). However, temperature and
vernalization are known to influence the magnitude of the photoperiod response,
and this magnitude varies regionally and presumably genetically (Saxena and Was-
simi 1984; Erskine 1997). Clearly, the control of this aspect of lentil phenology is
complicated.
Local lentil varieties grown in the Mediterranean climates of North Africa are
sown in the spring and reproductive growth begins just before the solstice in early
summer at day lengths at or near 12 h (Shrestha et al. 2009). These lentils ideally
need to complete seed fill before the hot dry late summer months. Conversely, len-
til grown in northern Argentina, South Asia, and Australia are planted in the fall,
grow vegetatively during days of decreasing photoperiod and flower during much
shorter days of about 11 h (Erskine 1983; Shrestha et al. 2009). In this situation, the
crop will yield best if it completes seed fill just before the end of the spring rainy
season. When lentil lines from the Mediterranean/West Asian regions are grown
in South Asia, seed filling is diminished because of hot dry conditions resulting in
lower yields (Erskine and Hawtin 1983; Shrestha et al. 2009). Erskine et al. (1994)
128 T. R. Stefaniak and K. E. McPhee

reported that 49 % of the variation in yield is due to variation in flowering time,
demonstrating the efficacy of phenotypes that were able to flower and fill pods prior
to terminal drought stress. Therefore, the adaptation of local landraces has taught
us that the breeding of new high-yielding cultivars must be done within the con-
straint of appropriate phenology for the region. Considerable effort has and is being
expended to improve lentil performance using this strategy of drought avoidance.
In Morocco, the cultivar Chakkouf was released and has been observed to yield
40 % greater than the comparable check variety (Idrissi et al. 2012). At ICARDA,
‘Idlib-3’ was developed for use in Syria where it yielded 13.1 % more than check
varieties in advanced yield trials (El-Ashkar et al. 2004a).
Cold tolerance has been described as a quantitative trait in winter barley
­­( Hordeum vulgare L.; Rohde and Pulham 1960), wheat ( Triticum aestivum L.; Sut-
ka 1994), and pea ( Pisum sativum (L.) Mill.; Liesenfeld et al. 1986) among others
species. Results from studies such as these suggest that the multitude of genes that
affect this stress, do so by affecting not only physiological but also morphological
traits. Genetic studies regarding the inheritance of cold tolerance are currently a
major goal to augment breeding efforts in lentil and all the pulses.
Identification of genetic variability for cold tolerance has made possible the
development of segregating populations for quantitative trait loci (QTL) analysis.
Kahraman et al. (2004a) developed ten recombinant inbred line (RIL) populations
from cold tolerant by sensitive parents. The population was evaluated for winter
survival at one location in Washington State, and two locations in Turkey. They
estimated heritabilites ranging from 15.9 to an extremely high 90.7. In one of their
populations, they were able to map four QTL in three locations with one of them
being identified in all the environments tested. Three QTL were detected in each of
the two Turkey locations and one in Washington. Using the same population (Kah-
raman et al. 2010), a QTL for smaller leaf area that explained 20.5 % of the pheno-
typic variation was subsequently identified in the same region indicating these two
QTL could be for the same gene. The association between the two QTL was 0.75.
These are encouraging results because they corroborate the efficacy of selecting for
small leaf area to improve cold tolerance.
Some physiological mechanisms for drought tolerance have the potential for im-
provement in lentil. An increase in osmotic adjustment (OA) can enhance water use
in lentil. OA means the plants cells increase their concentration of solutes which
increases the cells’ affinity for water. The result is less water leaving the cell and
traveling through the apoplast and out of the plant through stomata. Substantial
genotypic variation for this trait has been identified (Ashraf and Waheed 1990; Cle-
ments et al. 1997; Shreshta 2009). OA is a physiological trait that can also improve
tolerance to salinity and freezing.
4 Lentil 129

8.3  Biotic Stress Tolerance

Efforts to better understand the inheritance of disease tolerance are currently high
priorities for many lentil breeders. Resistance to stemphylium blight caused by S.
botryosum Wallr. has been shown to be inherited quantitatively (Saha et al. 2010).
Disease resistance is frequently quantitative because the disease is often caused by
multiple races or pathotypes, and numerous plant structures can potentially pre-
vent infection. Saha et al. (2010) used a 206-family RIL population to study the
inheritance of resistance to stemphylium blight in two successive growing seasons
in Bangladesh. One QTL was detected in the 2006–2007 season, and three were
detected in the 2007–2008. The one QTL from 2006–2007 was common to both
years and explained 25.2 % of the phenotypic variation in that year and 46 % in the
second. They concluded that this marker could be of substantial use to breeders
upon verification in additional environments.
Many other foliar diseases infect lentil such as anthracnose caused by Colle-
totrichum trifolii, powdery mildew caused Erysiphe polygoni, and downy mildew
caused by Peronospora lentis, among others (Khare 1981; Materne and McNeil
2007). Some root diseases that infect lentil are dampening off caused by Pseudomo-
nas, Fusarium, and Pythium species and Macrophomina phaseolina among others
(Khare 1981). Aspergillus, Fusarium, and Helminthosporium species are among the
pathogens that cause seed spoilage diseases in lentil (Khare 1981). The above list
includes diseases that researchers are currently working on to mitigate though resis-
tance is by no means complete and further discussion of lentil pathology is beyond
the scope of this chapter.

8.4  Plant Architecture

Lentil producers in West Asia and North Africa are interested in mechanizing their
harvests. In fact, hand harvesting can represent up to 47 % of production costs for
these farmers (Sarker et al. 2009). To achieve this goal, cultivars must be developed
that combine the appropriate maturity of local landraces, with superior plant archi-
tecture of unadapted germplasm. In this case, superior plant architecture consists of
good standing ability and pods that are higher off the ground. ICARDA has led the
way in this effort and has reported reduction in harvest expenditures of between 17
and 20 %. Idlib-4 is a high-yielding lodging-resistant cultivar that has been released
by ICARDA for use in Syria that yielded 20 % greater than the check variety in 14
on-farm yield trials (El-Ashkar et al. 2004b). Producers in Syria in particular are
interested in using combine harvesters to reduce production costs. The quantitative
inheritance of phenology and architecture has made progress laborious and costly.
A specific goal for lentil breeders is to conduct genetic studies intended to map loci
for these traits. Tullu et al. (2008) used a RIL population with 94 lines to investigate
earliness and plant height in lentil. Phenotypic data were collected in two locations
in western Canada. QTL for both traits were mapped to six linkage groups (LG),
130 T. R. Stefaniak and K. E. McPhee

and they explained 37–46 % of the phenotypic variation for earliness and 31–40 %
for height. More encouraging results from this study were that two QTL for earli-
ness and two QTL for plant height were consistent across locations. These results
indicate that these QTL would be good candidates for marker-assisted selection
(MAS). Molecular mapping technologies are becoming less expensive daily and
this will only augment current efforts to breed for improved phenology and archi-
tecture.
Lentil seed are rich in micronutrients. Breeding for the enhancement of some
of these minerals would represent a cultivar with added value. ICARDA has been
involved in this effort and has, or is in the process of fast-tracking the release of high
iron and zinc lines to producers in Ethiopia, Bangladesh, Nepal, Syria, Turkey, Por-
tugal, and Morocco (Sarker et al. 2009). Fortunately, significant genetic variation
for these traits has been identified and are being exploited (Idrissi et al. 2012). The
introduction of these value-added cultivars not only increase the worth of the crop
but also open up new markets for producers in these countries. Additionally, these
cultivars tend to perform better in some nutrient-deficient soils.

8.5  Inheritance of Important Traits

As an ancient self-pollinated crop species, the development of adapted or novel cul-

tivars has two distinct phases: selection only followed by hybridization and selec-
tion. The first selections which defined domestication are listed above (plant height,
seed size, flower number, etc.) and were made simply to increase harvestability and
yield. Following domestication, selection took the form of selecting out of land
races genotypes that were adapted to the regions where lentil was being introduced.
This took the basic form of selecting lines that would flower and produce yield in
a given environment. It is not until the early twentieth century that formal breeding
of lentil began.

8.6  Qualitative Traits

Reports of formal breeding of lentil began with the introgression of particular coty-
ledon colors out of their “native” germplasm. In 1928, Tschermak observed a 3:1 ra-
tio of red/orange to yellow cotyledons in reciprocal crosses between these types and
reported that red/orange was dominant over yellow (Sharma 2007). In this report, it
was noted that orange seed from heterozygous F1 plants was indistinguishable from
orange seed from homozygous plants. Therefore, he deduced that red/orange was
not only completely dominant but also there was no cytoplasmic maternal effect on
the trait. This was confirmed in later work using reciprocal crosses (Slinkard 1978)
and the symbol Yc was proposed for the orange/red color class (Singh 1978). In
the same crosses, Slinkard (1978) noted that yellow was completely dominant over
green.
4 Lentil 131

The genetic control of color of the seed coat or testa is not as clear as that for
cotyledon color. The consensus presently is that there are four testa color classes:
black, brown, grey, and green (Sharma 2007). Black testa color is controlled by
one locus with either codominance (Vandenberg and Slinkard 1990) or a dosage
effect (Emami and Sharma 2000; Sharma et al. 2004) that causes heterozygotes to
be difficult to assigned to a discrete class. In either case, plants homozygous for
black testa (Blt) have completely black seed coats that are easily scored (Sharma
et al. 2004). Bltblt plants have seed that appear in a range between very dark brown
(dense black spotting) to grey or green. To the breeder, the exact dominance state of
seed color is not of practical importance because it is a monogenic trait that can be
fixed with minimal inbreeding. Some other monogenic traits in lentil are seed hard-
ness ­(Ladizinsky et al. 1984; Vaillancourt and Slinkard 1992), seedcoat spotting
(Ladizinsky 1979; Vaillancourt et al. 1986), resistance to pea seed-borne mosaic
virus (PSbMV; Haddad et al. 1978), number of flowers per inflorescence (Khosravi
et al. 2010), growth habit (Ladizinsky 1979), pod shattering (Ladizinsky 1979), and
zero seed tannin content (Vaillancourt et al. 1986).

8.7  Quantitative Traits

Many economically important traits in lentil are quantitatively inherited. These

include seed size (Abbo et al. 1991), protein content (Chauhan and Singh 1995),
winter hardiness (Kahraman et al. 2004a, b), primary branch number, seed number
per pod, and seed yield (Goyal et al. 1976). An interesting result from the crosses
studied in Goyal et al. (1976) is that as a whole the 7 × 7 diallel had highly signifi-
cant general combining ability (GCA) and specific combining ability indicating that
in some of the individual crosses, dominance and epistatic effects were important
for yield and all its components, while in others the genetic effects were mostly ad-
ditive. It is useful to have reliable estimates of genetic parameters in order to design
efficient breeding methods for these traits.

9  Breeding—Improvement Methods

9.1  Pure-Line Selection

Many of the accessions in international lentil collections that represent available

global genetic diversity are landraces, which themselves harbor substantial genetic
variation. These landraces are surprisingly heterogeneous and many include in-
dividuals that meet many breeding objectives without the need of hybridization.
Many cultivars and germplasm have been released simply by making selections
from these land race populations using pure-line selection. Pure-line selection
simply involves selecting a desirable individual plant, inbreeding until homozy-
132 T. R. Stefaniak and K. E. McPhee

gosity is reached, increasing its seed, and then replicated testing in multiple loca-
tions and years. Selection in this strategy is very subjective and typically consists
of just choosing the plant that looks best agronomically and is based on the ob-
served phenotype. Results from the replicated tests confirm plant performance as a
crop community. These results can be used by breeders to select desirable parents
(germplasm) for breeding populations. Conversely, they can be used to describe
the cultivars’ predicted performance to producers directly. Cultivars released using
this method include ‘Crimson’ (Muehlbauer 1991), Idlib-2 (El-Ashkar et al. 2003),
‘Northfield’ (Ali 1995) and ‘Bichette’ (Sakr et al. 2004). Germplasm released us-
ing pure-line selection include ILL 5582 (Erskine et al. 1996), ILL 5588 (Erskine
et al. 1996), WH8449085, WH8449090, and WH8449041 (Spaeth and Muehlbauer
1991). In all the above selections, primary attention was paid to yield and growth
habit appropriate to mechanized harvesting. Some examples of secondary selection
parameters are Bichette and ILL 5588 selected for resistance to Ascochyta blight,
Idlib-2 and ILL 5588 were selected for resistance to vascular wilt, and Northfield
and Crimson were selected for maturity and seed type.

9.2  Bulk Population Breeding

Bulk population breeding (BPB) begins with a cross between parents whose traits
the breeder would like to try to combine. After the initial cross, the progeny are har-
vested in bulk, randomly sampled, and then advanced to the next cycle. The goal is
to inbreed the progeny to a certain level of homozygosity, usually the F5 or F6, and
then make single plant selections at that time for increase. This method of breeding
is simple and requires little record keeping. By the time the population has been
advanced five or six generations, the ratio of superior to inferior yielding genotypes
has increased through natural selection. BPB is popular among lentil breeders who
are working with traits that are amenable to “natural” selection that occurs within
research plot environment. The breeder selects individual superior plants for their
desired traits such as seed size or quality once these traits are considered fixed. Rep-
licated testing then commences and breeder seed is developed. A disadvantage to
this method is that it cannot be done in greenhouses or off-season nurseries so prog-
ress may be slow. The cultivars ‘Mason’ (Muehlbauer 2002), ‘Pennell’ (Muehlbauer
and McPhee 2004), and ‘Merrit’ (Muehlbauer and McPhee 2004) were developed
using the bulk selection method. In all three of these examples, the breeders selected
for particular seed size and color.

9.3  Bulk Pedigree Selection

Bulk pedigree selection (BPS) is a breeding method that is a modification of BPB

and differs in the generation that selection is performed (Fig. 4.4). Most cultivars
released thus far were developed using BPS. Typically, F2:3 families are screened for
4 Lentil 133

Fig. 4.4   Schematic representation of modified bulk population/pedigree selection breeding

method

phenotypic response and single plants are selected. Family rows are evaluated for
four to five generations, and the best rows and plants within rows are selected and
advanced in bulk to a desired level of homozygosity, usually the F6 or F7. Prelimi-
nary replicated testing begins followed by advanced yield trials and finally multilo-
cation regional testing.
The various public institutions engaged in lentil cultivar development modify
BPS based on when familial generation pedigree selection begins and how many
generations of pedigree selection are conducted before preliminary yield trials are
conducted. Advantages of BPS are that selection of a plant in early generations can
insure that a desired allele for a required phenotype will be present in that family
and will be advanced through the program. This means that less land will be re-
quired because only those F2- or F3-derived families will need to be advanced. Dis-
advantages to this method is that the breeder must be able to observe the phenotype
for selection in the F2-derived lines using relatively few (~ 50) families. Addition-
ally, bulking F2-derived families would need to be done in the target environment
limiting the number of generation per year that can be grown.
Cultivars released using this method include ‘Emerald’ and ‘Brewer’ (Muehl-
bauer 1987), Idlib-3 and Idlib-4 (El-Ashkar et al. 2004a, b), ‘CDC Robin’ (Van-
denberg et al. 2002b), ‘CDC Vantage’ (Vandenberg et al. 2002a), and ‘CDC Plato’
134 T. R. Stefaniak and K. E. McPhee

(Vandenberg et al. 2005). The F2:3 family selected for the CDC cultivars were all cho-
sen based on Ascochyta blight and anthracnose reaction as well as seed characters.
The Idlib cultivars were selected for harvestability, disease reaction, and market traits
reinforcing the more recent desire in Syria and Lebanon for mechanized harvest.

10 Integration of New Biotechnologies in Breeding

Programs

Genetic engineering in lentil is not accepted in international markets, and this is not
likely to change; therefore, no varieties have been released with artificially intro-
duced traits. However, new molecular biology platforms and technology offer great-
er potential to develop saturated genetic maps and to study agronomically important
traits in greater detail. This potential arises from two key limitations researchers
currently encounter in mapping studies of lentil; optimal mapping ­populations have
been difficult to assemble, and the number of informative molecular markers is
small relative to other crop species.
The most recent and saturated genetic map for lentil was published in 2012
(Gupta et al. 2012). The map was a distance of 3843.4 cM which is considerably
longer than the length of the pea genome (1100–1800 cM), which is predicted to
be similar to lentil based on the two species’ close phylogeny (Laucou et al. 1998).
Additionally, this most recent map has 11 LGs and lentil is known to have a haploid
chromosome number of n = 7. Limitations of this study were that they were only
able to screen the population with 1319 markers, and 523 of these were random am-
plified polymorphic DNA (RAPD) which are anonymous dominant markers. Of the
1319 markers used, 118 were found to be informative of which 79 were RAPDs and
the rest were simple sequence repeat (SSR) or inter-simple sequence repeat (ISSR)
markers. Also, the mapped population was at the F2 generation which is particu-
larly constraining because dominant markers cannot distinguish heterozygotes from
homozygous dominant genotypes. Development of RIL populations from parents
divergent for a host of agronomic traits will in the future be used to improve map-
ping studies. Additionally, the use of biotechnological techniques to develop more
widely distributed markers, particularly SSRs and single nucleotide polymorphisms
(SNPs), will also improve future genetic maps of lentil.
Though no cultivars developed using genetic transformation have been released,
lentil has been successfully transformed with alien genes despite its recalcitrance to
transformation. Initial attempts at lentil transformation have used variations of par-
ticle bombardment or Agrobacterium tumefaciens vectors with virtually no success.
Akcay et al. (2009) described an A. tumefaciens protocol that has a transformation
efficiency of 2.3 % using improved strains of A. tumefaciens and one Turkish lentil
genotype. Chopra and Aparna (2012) described a procedure that was much less ef-
ficient (0.66 %), but that was successful using four Indian cultivars. Neither of the
above reports utilized an agronomically beneficial gene in their inserted construct.
4 Lentil 135

Khatib et al. (2011) developed a construct that included the DREB1 A gene. This
gene has been reported to improve tolerance to drought, salinity, and freezing stress
(Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000) in Arabidopsis presum-
ably by means of OA. Transformants were induced to express the DREB1 A gene
and demonstrated tolerance compared to the controls when watered with a saline
solution of 50 mM NaCl (Khatib et al. 2011). Whether this work will result in field
production of transgenic plants is uncertain. However, these techniques will be use-
ful in generating genotypes that can serve as good parents for the development of
mapping populations for QTL studies on stress tolerances.

11 Conclusions

Much opportunity exists for the breeding improvement of lentil for many reasons.
Lentil has received very little if any attention from the major agribusiness conglom-
erates that dominate cultivar development in crops such as maize, soybean, cotton,
sorghum, wheat, or barley. Substantial collections are being curated that harbor co-
pious diversity for genetic improvement. Lentil is grown in both developed and de-
veloping countries making breeding objectives numerous. Finally, as a high-quality
inexpensive source of protein, greater-yielding cultivars can diminish the need for
animal protein and improve the diets of people worldwide.

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Chapter 5
Faba Bean

Gérard Duc, Jelena M. Aleksić, Pascal Marget, Aleksandar Mikić, Jeffrey

Paull, Robert J. Redden, Olaf Sass, Frederick L. Stoddard, Albert Vanden-
berg, Margarita Vishnyakova and Ana M. Torres

1 Introduction

The faba bean (Vicia faba L.) is a rich protein grain legume belonging to the Faba-
ceae family with a long tradition of cultivation in the temperate zone of the northern
hemisphere. Sometimes also referred to as horse bean or broad bean, it is mostly
harvested as dry seeds for food or feeds, but its fresh seeds or pods can also be used
as vegetables. Faba bean provides valuable ecological and environmental services
in sustainable agriculture, diversity in cropping systems and host numerous associ-
ated organisms including pollinating insects. The capacity of this species to estab-
lish symbiosis with specific rhizobia bacteria results in biological nitrogen fixation
which reduces the input of fertilizers in arable lands. The species has an intrinsic
ability to adapt to diverse climates, but its low and unstable yields hamper its com-
petitiveness as a crop.
The area grown with faba bean was only 1.2 % of the 200 million ha of annual
grain legume cultivation in the world in 2011 (Table 5.1). Among the grain legumes,

G. Duc ()
UMR1347 Agroecologie, INRA, 17 rue de Sully, BP 86510, 21000 Dijon, France
e-mail: [email protected]
J. M. Aleksić
Laboratory for Plant Molecular Biology, Institute of Molecular Genetics and Genetic
Engineering (IMGGE), University of Belgrade, Vojvode Stepe 444a, PO Box 23,
Belgrade 11010, Serbia
e-mail: [email protected]; [email protected]
P. Marget
UMR1347 Agroecologie, INRA, 17 rue de Sully, BP 86510, 21000 Dijon, France
e-mail: [email protected]
A. Mikić
Forage Crops Department, Institute of Field and Vegetable Crops,
Maksima Gorkog 30, Novi Sad 21000, Serbia
e-mail: [email protected]
© Springer Science+Business Media New York 2015 141
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_5
142 G. Duc et al.

faba bean ranks at seventh position behind a top group including soya bean ( Glycine
max L.), groundnut ( Arachis hypogaea L.) and common bean ( Phaseolus vulgaris
L.). In the EU27, faba bean covers 21.3 % of the 1.6 million ha of grain legume
cultivation, ranking second behind pea and just ahead of soya bean. The major faba
bean-producing countries at world level are presented in Table 5.2 (FAOstat 2013;
Eurostat 2014). China, the leading producer, followed by Ethiopia, the UK, Austra-
lia and France were the five main producing countries in 2011, harvesting 72 % of
the world production. No accurate statistics are available on fresh seed production
as a vegetable, as a consequence of the predominantly small-scale gardening nature.
Research activity on faba bean can be measured by publication effort (Table 5.3).
In a CAB abstracts query over all documents 2004–2013 (23 January 2014), a 10-
year production on all grain legumes detected 63,970 papers, of which 4.8 % re-
ferred to V. faba when soya bean ranked first with 33 % of the publications. The
distribution of disciplines allocated by journals slightly differed between faba bean
and the assembly of grain legume species, by a lower proportion for genetics and
breeding and for nonfood and non-feed uses, compensated by higher investments

J. Paull
School of Agriculture, Food and Wine, The University of Adelaide, PMB 1,
Glen Osmond, SA 5064, Australia
e-mail: [email protected]
R. J. Redden
Agriculture Productivity. Department of Economic Development, Jobs, Transport & Resources,
Australian Temperate Field Crops Collection, Private Bag 260, Horsham VIC 3401, Australia
e-mail: [email protected]
O. Sass
Norddeutsche Pflanzenzucht Hans-Georg Lembke KG,
Hohenlieth-Hof 1, 24363 Holtsee, Germany
e-mail: [email protected]
F. L. Stoddard
Department of Agricultural Sciences, University of Helsinki,
PO Box 27, (Latokartanonkaari 5-7), 00014 Helsinki, Finland
e-mail: [email protected]
A. Vandenberg
Department of Plant Sciences/Crop Development Centre, University of Saskatchewan,
51 Campus Drive, Room 2C24, Saskatoon, SK S7N 5A8, Canada
e-mail: [email protected]
M. Vishnyakova
Genetic Resources of Grain Legumes, Vavilov Institute of Plant Industry,
Bolshaya Morskaya, 42, Saint-Petersburg, Russian Federation 190000
e-mail: [email protected]
A. M. Torres
Area de Mejora y Biotecnología, IFAPA Centro Alameda del Obispo,
Avenida Menéndez Pidal s/n, 14080 Córdoba, Spain
e-mail: [email protected]
5  Faba Bean 143

Table 5.1   Area and production for major annual grain legumes at the world and European Union
levels in 2011. (FAOstat 2013 data adjusted for Eurostat differences in faba bean and lupins)
World EU27
Production Area Production Area
(1000 t) (1000 ha) (1000 t) (1000 ha)
Oilseed
Soya bean 262,038 103,605 1094 386
Groundnut 40,017 24,637 9 11
Pulses (dry seeds)
Common bean 23,062 30,411 142 82
Chickpea 11,610 13,181 45 46
Cowpea 4952 10,640 0 0
Pea 9730 6141 1614 684
Faba bean 4686 2587 1168 411
Pigeon pea 4444 5863 0 0
Lentil 4404 4172 51 59
Lupin 1213 1115 237 251
Total 366,156 202,351 4360 1926
Annual grain legume area 0.145 0.016
(% of arable land)

Table 5.2   Area of cultivation (ha) and production (Mg) of faba bean in major faba bean-produc-
ing countries at world level. (Eurostat data for Europe, FAOstat 2013 data for the rest of the world)
Main countries Area (ha) Dry seeds (Mg)
(> 40,000  Mg)
Asia 919,000 1,655,000
China 872,000 1,550,000
Africa 883,000 1,312,000
Ethiopia 460,000 698,000
Egypt 55,000 175,000
Morocco 200,000 171,000
Sudan 72,000 155,000
Tunisia 57,000 73,000
Europe 410,000 1,168,000
France 91,000 345,000
The UK 125,000 419,000
Italy 50,000 96,000
Germany 17,000 61,000
Spain 35,000 55,000
Australia-New Zealand 175,000 350,000
Australia 175,000 350,000
South America 138,000 137,000
Peru 52,000 65,000
World 2,587,000 4,690,000
144 G. Duc et al.

Table 5.3   CAB abstracts (www.cabi.org) query over all documents 2004–2013 (23 January
2014). Distribution of disciplines (%) among 63,970 papers on all grain legumes and 3053 papers
on Vicia faba L.
Discipline All grain legumes Faba bean
Breeding and genetics 30.9 23.6
Physiology and biochemistry 20.6 24.5
Viral, bacterial and fungal diseases 12.9 13.4
Pests 10.6 12.6
Cropping systems 10.7 11.4
Soil biology 9.5 8.4
Forage and fodder crops 8.8 10.0
Feed composition and quality 1.8 3.8
Food composition and quality 7.7 7.2
Nonfood, non-feed uses 6.6 4.3

in plant physiology and biochemistry. Numerous papers refer to diverse ecosystem

services provided by faba bean such as the potential for diversification of crop-
ping systems, the establishment of the root symbioses with Rhizobiaceae bacteria
(Rhizobium leguminosarum bv. viciae) and with vesicular-arbuscular mycorhiza
(VAM), the impact on soil biology and fertility or with the capacity to host and feed
pollinating insects. Apart from these issues, genetics and breeding progress is the
main topic to improve yield reliability, bring competitiveness and ecosystem ser-
vices, improve nutritional value and, as a result, increase the cultivated area of this
species. This chapter reviews the advances in both faba bean genetics and breeding,
underlining the significant progress currently made in molecular genetics which
will help speed up the breeding efforts to improve targeted traits.

2  Origin and Systematics

Faba bean is among the most ancient crops of the Old World. Together with chick-
pea ( Cicer arietinum L.), grass pea ( Lathyrus sativus L.), lentil ( Lens culinaris Me-
dik.), pea ( Pisum sativum L.) and bitter vetch ( Vicia ervilia (L.) Willd.), it was one
of the factors of the beginnings of early crop domestication. Faba bean played an
important role in spreading agriculture during the Neolithic, Bronze and Iron Ages
throughout Eurasia and North Africa, with numerous archaeological deposits. Very
little is known of the exact origin and the single steps during the domestication of
faba bean (Maxted et al. 1991). Its presumed domestication is in the Fertile Crescent
with the most ancient fossils dated to 11,200 BP in Iraq (Ladizinsky 1998). Cubero
(1973) suggested the Near East as its centre of origin, with four different directions
from this centre: (1) to Europe, (2) along North African coast to the Iberian Penin-
sula, (3) along the Nile to the Ethiopian highlands and (4) from Mesopotamia to the
Indian subcontinent.
5  Faba Bean 145

Various trait analyses distinguished two main faba bean groups: the small-seeded
forms from southwestern Asia and the large-seeded forms developed in the West.
The Eastern group is very ancient and may be traced back to Neolithic culture. It
has the greatest number of endemic forms and the greatest diversity of specific
characteristics lacking in other groups, such as few to many pairs of leaflets, their
glaucous and grey-green colour, shattering or non-shattering pods, the wide range
of variation of the maturity period, seed size, colour and shape, leaflet size, stem
height and branching of stem. The Mediterranean group, with a dense geographic
concentration of the large-seeded forms, is regarded as a more recent secondary
centre of the faba bean diversity. The remains of faba bean in the archaeological ex-
cavations in the Mediterranean and Central Europe are dated between the third and
second millennia BC (Bond 1976; Cubero 1973). However, the broad bean (also
known as major types, with more than 1g per seed) is thought to have originated
only after 500 BC (Hanelt 1972).
Faba bean spread from the primary centre and from Mediterranean Europe, form-
ing an important third centre of diversity in Ethiopia. The migration of faba bean
to South America, especially to the Andes, probably occurred during the fifteenth
century along with the Spanish and Portuguese conquests. Another relatively recent
development is that of winter faba beans in the nineteenth century in Europe, bred
from Russian and French small-seeded, winter-hardy populations (Bond and Crof-
ton 1999). Finally, China seems to be another secondary centre of faba bean genetic
diversity and reproductively isolated from the European and West Asian gene pools,
especially the Chinese winter gene pool (Zong et al. 2009).
Since wild faba bean has not been found so far, and because faba bean does not
cross with other Vicia species, its wild ancestor remains unknown (Muratova 1931;
Hanelt 1972; Cubero 1982). As a consequence, it is not known how much of the
diversity in the wild progenitor of faba bean has been lost. All botanically close faba
bean relatives are diploid species with 14 chromosomes, while faba bean is a dip-
loid with 12 chromosomes. A study of the nuclear DNA amounts and the chromo-
some number of 56 Vicia species (Raina and Rees 1983) showed that faba bean has
a high content of DNA and a large metacentric pair of chromosomes, twice the size
of the remaining five pairs of acrocentrics and probably evolved from an ancestral
fusion. The size of the faba bean genome (1C = 13.3 pg) is very different from that
of Vicia sativa (1C = 2.3  pg), Vicia narbonensis (1C = 8 pg; Raina and Rees 1983)
and the model species in legume genomics, Medicago truncatula (1C = 0.48  pg).
Along with numerous archaeobotanical findings, there is rich linguistic evidence
on the antiquity of the faba bean crop (Mikić 2012). It was known by the ancestors
of nearly all present European nations, as suggested by the attested roots in the pro-
tolanguages of the existing language families. The words related to faba bean in the
modern Indo-European languages and their dialects demonstrate a high degree of
similarity, especially among and within the most abundant branches, such as Ger-
manic, Italic, and Slavic. The migrations of the Indo-European tribes produced nu-
merous derivations. The lexicological evidence is still preliminary and incomplete,
but it may provide a valuable testimony to the role faba bean and other traditional
grain legumes played in the everyday diets of the old and modern Europe peoples.
146 G. Duc et al.

3  Varietal Groups

Various levels of tolerance to winter conditions helped to distinguish between

spring-sown and autumn-sown cultivars. They differ in their level of frost hardi-
ness, vernalization requirement, day-length response and flowering time and fungal
disease resistance, but the boundary between the two cultivar types is not always
clear cut and some spring cultivars of Northern Europe may be grown in winter of
some Mediterranean zones.
It has long been recognized that faba bean falls into several distinct genetic pools.
Moreno and Martinez (1980) recognized that Mediterranean faba beans differed
from the rest. The distinction between autumn-sown and spring-sown faba beans
was also clear (Link et al. 2010). Detailed genotype-by-environment analysis has
shown that in both the autumn-sown and spring-sown classes, there are distinctions
in adaptation to oceanic and continental climates (Flores et al. 2012, 2013). Thus,
we may recognize five broad classes or gene pools of faba bean, at least in Europe:
Mediterranean, spring-sown for the oceanic zone, spring-sown for the continen-
tal zone, autumn-sown for the oceanic zone and autumn-sown for the continental
zone. The five pools differ with regard to optimum flowering time, duration of crop
growth and tolerance to cool and warm weather. Furthermore, they are exposed to
different types of changes in photoperiod, with both Mediterranean and autumn-
sown cultivars establishing during shortening days, while spring-sown cultivars es-
tablish during lengthening days, and Mediterranean types ripen during lengthening
days, while continental and oceanic cultivars ripen during shortening days. Flow-
ering initiates during lengthening days, but, in the Mediterranean region, days are
very short at the start of flowering shortly after midwinter (~ 10-h day length), and
very long in the boreal end of the continental region, where it starts about midsum-
mer (~ 18 h day length). Faba bean is generally considered day-neutral, but some
accessions require at least 12-h photoperiod in order to flower (Stoddard 1993).
Inter-pool crosses are valuable for expanding genetic diversity and bringing in some
positive alleles (Link et al. 1996), but can bring in undesirable traits through linkage
drag unless there is an appropriate amount of backcrossing and selection.
The genetic variability of the species is quite large and often described on the
basis of differences in seed weight, shape and size. Muratova (1931) cited four
botanical varieties: the large-seeded faba bean ( V. faba sp. faba var. major), with a
seed weight of more than 1g and developed in the South Mediterranean regions and
China; V. faba sp. faba var. equina, with an intermediate seed size (0.45–1.1 g per
seed), developed in the Middle East and North Africa; V. faba sp. faba var. minor
(0.2–0.5 g per seed) found in the Ethiopian highlands, Sudan and in Northern Eu-
rope; and V. faba sp. paucijuga grown in Central Asia.
Seed size is important for meeting market and farmer needs, although it is a
continuously variable property. Large-seeded cultivars are widely favoured for food
use, whether as a fresh green vegetable or, in many cultures, dry. Small-seeded cul-
tivars are preferred for the specialist market for feeding pigeons (for which ‘Maris
Bead’ is still grown in the UK) and easy drying after harvest in high latitudes such
as the Nordic region and the Canadian province of Saskatchewan. The most widely
5  Faba Bean 147

grown cultivars, mostly used for animal feed in European cultures, and for use
as dry pulses in West Asia and North Africa, have medium-sized seeds. Breeding
progress for yield tends to be fastest in this intermediate size class, perhaps because
of the balance between high seedling vigour from relatively large seeds and high
multiplication rate from relatively small seeds.
The absence of tannins in seeds, improving the protein digestibility in mono-
gastric animals resulted in a “zero-tannin” group of cultivars while low contents of
vicine and convicine in seeds, improving feed value in poultry and reducing favism
risks in humans, define a “low vicine–convicine” group. The combination of both
traits has received the generic name “fevita” (Crépon et al. 2010).

4  Genetic Resources and Utilization

Because the wild progenitor is unknown, all genetic diversity available is contained
in ex situ collections and local populations maintained by farmers in traditional ag-
ricultures. Faba bean germplasm of over 38,000 accessions is conserved worldwide
in at least 43 national gene banks as well as at the International Center for Agricul-
tural Research in Dry Areas (ICARDA). ICARDA safeguards the largest collec-
tion (10,045 accessions) in the world with materials from 71 countries with a high
percentage of unique accessions. In the global collection, 8628 accessions comprise
the international collection held in trust for the global community. Worldwide, ca.
95 % of known accessions are maintained in 17 major national or international ex
situ collections (Table 5.4).
Since the germplasm collections are large, new methods are needed for choos-
ing which accessions to screen for any given trait. Faba bean was the successful
test case for applying the focused identification of germplasm strategy (FIGS) to
abiotic stress resistance (Khazaei et al. 2013b). In this method, all available data on
the provenance of the accession, particularly climate data, are used to predict the
probability of finding appropriate germplasm.
The adequate ex situ conservation of faba bean collections is limited by the out-
crossing floral biology of the species and its low multiplication rate (Suso et al.
2011). Genetic contamination, allele losses and inbreeding should be minimized.
Large open-field populations separated or bagged to prevent intercrossing between
each other would offer a good protection of the genetic integrity of each accession,
but it is practically and economically unachievable with thousands of genotype en-
tries. Only a few collections maintain small plots under protection from pollinating
insects by net cages or by inter-plot plant barriers (Suso et al. 2006).
Large investments in the discovery of genetic variability and also in breeding
activity for traits of agronomic interest have been made for faba bean at the end of
the twentieth century in European countries and also at ICARDA, Syria, in particu-
lar for tolerance to several biotic and abiotic stresses (Bond and Poulsen 1983; Duc
1997). A large genetic variability has been identified in terms of floral biology. Cy-
toplasmic and nuclear determinisms of male sterility, various flower architectures
or colours and various levels of autofertility and attractivity for pollinating insects,
148 G. Duc et al.

Table 5.4   Major Vicia faba ex situ collections worldwide in 2014

Country Institute/city Number of accessions
Syria ICARDA/Aleppo 10,045
China CAAS/Beijing 5900
Australia Austalian Grain Gene Bank/ 2445
Victoria
Germany Gene Bank IPK/Gatersleben 1920
France INRA/Dijon 1900
Russia VIR/St Petersburg 1881
Italy Genebank/Bari 1876
Morocco INRA/Rabat 1715
Spain CNR/Madrid 1622
Poland IOPG-PA/Poznan 1258
Ethiopia PGRC/Addis Ababa 1118
Spain IFAPA/Cordoba 1091
Poland PBAI/Radzikow 856
Portugal INRB—IP/ Oeiras 788
USA USDA/Pullman 750
The Netherlands DLO/Wageningen 726
Bulgaria IIPGR/Sadovo 692
Total world More than 43 known 38,360 accessions
collections
ICARDA International Institute for Agricultural Research in Dry Areas, CAAS Chinese Academy
of Agricultural Sciences, IPK Institute of Plant Genetics and Crop Plant Research, INRA Institut
national de la recherche agronomique, CNR Consiglio Nazionale delle Ricerche, IOPG Institute of
Packaging, Ghana, PGRC Plant Genetic Resources Centre, IFAPA Instituto de Investigación y For-
mación Agraria y Pesquera. Anterior, PBAI Plant Breeding and Acclimatization Institute, INRB
National Institute of Biological Resources, USDA US Department of Agriculture, DLO Dienst
Landbouwkundig Onderzoek, IIPGR Institute for Introduction and Plant Genetic Resources

were described; this could help to manipulate the levels of outcrossing (Link et al.
1994; Duc 1997).
As a result, current national breeding programmes are delivering higher-yielding
new cultivars with improved combinations of disease resistances and a stronger em-
phasis on market quality. The success of plant breeding highlights the importance
of ex -situ gene banks in collecting and preserving local landraces with their associ-
ated ranges of adaptations to respective crop environments.

4.1  Plant Height and Branching

A set of ca. 5000 faba bean accessions from the National Genebank of China was
randomly sampled and analysed for plant height, which averaged 78 cm (Zong
et al. 2006). The shortest accession was only 10.3 cm in height, and the tallest was
201.5 cm. The number of effective branches (with seeded pods) ranged from 1.1 to
11.4. In the Institut national de la recherche agronomique (INRA)-F reference col-
lection of 250 genotypes, plant height ranged 40–210 cm, and 20 % of accessions
5  Faba Bean 149

had a single main stem, whereas 70 % carried 1–3 basal branches (Duc and Magnin-
Robert pers. com.). Winter genotypes are more likely to develop basal branching.

4.2  Yield-Related Traits

The accessions from the National Genebank of China were sampled as well for
characters of yield component analysis (Zong et al. 2006), the number of effective
pods ranged from 1.1 to 93.7. The number of mature seeds per pod is from 0.8 to
6.1. The 100-seed weight of dry grain ranged from 6 to 240 g and the dry grain
yield per plant from 1.2 to 127.0 g. The mean dry pod length was 6.5 cm (range
1.2–18.8 cm) and the width 1.6 cm (range 0.7–3.5 cm; Zong et al. 2006).

4.3  Flower and Seed Colour

Flower colour (Fig. 5.1) is subject to oligogenic determination of major traits. A black

dot is often present on the wing petals, and the flowers can be pure white or with dif-
fuse pigments on all petals (purple or dark brown; Picard 1976). Recessive alleles at
either of two genes ( zt-1 and zt-2) can determine the pure white flower trait and have
a pleiotropic effect on the seed coat composition determining the absence of tannins.
As shown in Fig. 5.2, faba bean seeds display large genetic variation in seed coat
colour and pattern (spotted, marbled), hilum colour and cotyledon colour (yellow or

Fig. 5.1   Coloured flowers

150 G. Duc et al.

Fig. 5.2   Variation in seed

coat colour and pattern

green). Major oligogenic determinations have been described with a strong relation-
ship with tannin content (Picard 1976; Duc et al. 1999).

4.4  Abiotic and Biotic Stress Resistance

Genetic resources are continuously under evaluation for escape or protection mech-
anisms against these stresses (see reviews by Khan et al. 2010; Duc et al. 2011).
Major biotic stresses differ in diversity and damage according to geographic zones
and date of sowing. Ascochyta blight, chocolate spot, rust, downy mildew, Fusari-
um spp., broomrapes, nematodes, aphids, sitona weevil and bruchids are the major
parasites or pests so far reported for which sources of genetic resistance are needed.
The history of wide dispersion of faba bean has resulted in regional adaptations
to abiotic stresses and variation in disease resistances, and genetic resources col-
lections are being evaluated to detect resistance sources. Resistance to chocolate
spot has been found in Ecuador, tolerance to heat stress in Bangladesh, frost toler-
ance in winter types from Europe, drought tolerance in the Mediterranean region,
plus genetic resistances to Ascochyta blight, nematodes, bean yellow mosaic virus
(BYMV) and bean leaf roll virus (BLRV) and broomrapes. Genetic resistances of
faba bean to its major fungal diseases in temperate regions: chocolate spot, As-
cochyta blight, rust, downy mildew (reviewed by Sillero et al. 2010) and to its
important parasitic plant Orobanche (Fernandez-Aparicio et al. 2012; Rubiales and
Fernández-Aparicio 2012) have been identified. The use of these resistance sources
in breeding programmes is described in Part 6 of this chapter.

4.5  Nutritional Composition

The nutritional composition of 1828 faba bean accessions from the National Gene-
bank of China was determined (Zong et al. 2006). On a total seed dry matter ba-
5  Faba Bean 151

sis, the crude protein content ranged 17.6–34.5 %. The total starch content ranged
33.2–53.4 %, while amylose content of starch ranged 6.0–27.9 %. The lipid content
ranged 0.52–2.80 %. A similar range of variations was described for these main
seed constituents in a European seed collection (Duc et al. 1999), where mean seed
coat proportion ranged 11.0–14.8 %, (the zero tannin cultivars being 2 % lower than
tannin-containing ones), mean neutral detergent fibre ranged 13.4–21.7 %. In a set
of 72 accessions, amylose content ranged 17–29 % of starch, with a significant ten-
dency for lower content in larger-seeded accessions.
By contrast with soya, trypsin inhibitor activity is generally low in faba bean
seeds with a range of 0.3–5.3 units/mg measured on a European collection (Duc
et al. 1999). In this collection, white-flowered genotypes were tannin-free, whereas
coloured-flower genotypes contained 6–10 g/kg of condensed tannins, concentrated
in the seed coat. An allele ( vc-) of the VC gene has been discovered (Duc et al.
1989) which reduces by 10–20 times the content of vicine and convicine in the seed.
Mean vicine–convicine content in conventional genotypes is 6–12 g/kg of seed dry
matter (DM) when it is close to 0.5 g/kg in vc-homozygotes. These two products
are concentrated in cotyledons and are responsible for favism risk in humans and
lower production performances in chickens or laying hens (Arese and De Flora
1990; Crépon et al. 2010).

5  Major Breeding Achievements

The effort allocated to faba bean breeding is relatively small, and it is strongly as-
sociated with its relatively small area of cultivation. Major breeding programmes
are located in several European countries, the ICARDA network, Egypt, Morocco,
China, Canada and Australia. In the 2014 edition of the list A of the European cata-
logue, only 130 faba bean cultivars are listed in contrast to 397 field pea, 363 soya
bean and 2109 bread wheat cultivars. This reflects the small size of the breeding
programmes on faba bean in comparison with other major agronomic species. These
programmes are mostly maintained by public institutions. In 2014, in Europe, fewer
than ten private breeding programmes for faba bean were identified. Beyond the
genetic improvement achieved by farmers through empiric mass selection over pre-
vious centuries, a burst of research and breeding activity occurred in Europe in the
1980s after a severe plant protein supply deficit occurred in 1973, which represents
a relatively short history of breeding.
During the past 50 years, remarkable steps of improvement can be identified
with representative genotypes which at a time have developed reduced stem growth
preventing lodging and associated with an improved harvest index, earliness, yield
stability and harvestability (cv. Aguadulce in the broad bean types, cvs Alfred and
Espresso in the minor types); winter types with increased frost tolerance and vari-
ous levels of earliness adapted to Northern Europe (cvs. Maris Beagle, Hiverna,
Diva, Castel, Irena, and Wizard); winter types adapted to the Mediterranean zone
(cvs. Alameda and Baraca); low vicine–convicine content in the seed (cv. Melodie
152 G. Duc et al.

and its successors); high tolerance to Ascochyta blight (cv. Irena); tolerance to Oro-
banche (Egyptian cv F402); outstanding spring types with short stems, low tannin
and high earliness (cvs. Optica and Kompacta) or with small seeds (cv. Maris Bead;
Duc 1997; Flores et al. 2012).

6  Specific Goals in Current Breeding

The traditional goal for a plant breeder is yield and yield stability. There are many
ways of achieving that, considering phenology, adaptation and stress resistance.
Moreover, the product has to be marketable, so key quality attributes have to be
met. Lodging resistance, or standing power, is one of the top objectives and is af-
fected by stem strength and stem length. The available dwarfing gene, used in the
garden broad bean cultivar Sutton’s Dwarf, reduces internode length too much for
use in field-grown, machine-harvested cultivars. The Japanese autumn-sown cul-
tivar ‘Rinrei’ has relatively short internodes and strong stems, but appears fairly
unique in this regard. Reducing stem length by use of the terminal-inflorescence
gene ti has been associated with too great a loss in yield in most environments,
although it is used in Spanish cultivar ‘Retaca’ grown for vegetable use.
The indeterminate habit allows the crop to produce more reproductive nodes
but is relatively sensitive to poor growing conditions, in comparison to other crops.
This sensitivity equates broadly to stress susceptibility, so the various stresses are
discussed next.
A major goal in sustainable crop production is intensification of the use of grain
legumes in crop rotation for environmental contributions of nitrogen fixation and
reduction of inputs. At the same time, the global discussion is expanding on the is-
sues of food and nutritional security. Faba bean can play a significant role in both
these areas, and it can also make a contribution to the expansion of the vegetable-
based protein supply in temperate production regions. Breeding programmes will
need to keep in mind the requirements for increased yield relative to other protein
crops, while maintaining a focus on reduced input cost, on suitability for mecha-
nised harvest and on improvement of protein quality and reducing vicine–convicine
in seeds.

6.1  Biotic Stresses

Faba bean is subject to a range of diseases, parasites and pests, and resistance breed-
ing is an important issue with priorities differing from region to region. As part of
the levers for integrated disease management (reviewed in Stoddard et al. 2010),
the resistances to pathogenic fungi reduce the need for application of fungicides
but provide incomplete resistance. Therefore, they should be combined with prac-
tices such as crop rotation (duration and choice of non-host species), with sanitary
5  Faba Bean 153

control of certified seeds in the case of aschochyta blight, with reduction of relative
humidity in the canopy (by lower sowing rate, good soil drainage, choice of plant
architectures and prevention of lodging), with targeted period of sowing and plant
cycle and with prevention of nutrient deficiencies, frost damage and weed infesta-
tions (Stoddard et al. 2010).
Of the pathogenic fungi, chocolate spot disease, caused by Botrytis fabae Sard.,
is the one on which there is the most literature (Stoddard et al. 2010). It is present in
all regions where faba bean is grown and, because its expression depends particu-
larly strongly on environmental conditions, it is one of the most difficult diseases to
handle in a breeding programme. When conditions are just right for the fungus for
about 48 h, with damp leaves, high atmospheric humidity and temperatures close to
20 °C, it grows rapidly from its small, brown spots scattered across leaves until the
whole plant is dead. In any location, such conditions may occur only once every 10
years but when they do, the farmer has only a brief opportunity to use a fungicide
before the crop is past saving. Resistance is quantitative, and the sources used by
ICARDA breeding programmes are from South America (Tivoli et al. 2006), where
faba bean has been cultivated for only five centuries.
Ascochyta blight, caused by Ascochyta fabae Speg. (teleomorph Didymella fa-
bae Jellis and Punithalingam), is prevalent on autumn-sown faba bean, including
Mediterranean climates. Breeding for resistance to this disease has been more suc-
cessful than against chocolate spot, with major and minor genes identified (Kohpina
et al. 2000) along with quantitative trait loci (QTLs; Román et al. 2003; Avila et al.
2004). Some accessions have shown stable resistance across environments, whereas
others were more resistant in either the Mediterranean or continental environment
(Rubiales et al. 2012).
Rust disease, caused by Uromyces viciae-fabae (Pers.) J. Schröt., is prevalent
in warmer conditions than either of the preceding two. Rust resistance is generally
quantitative or incomplete, but a hypersensitive response that slows rather than stops
the spread of the disease has also been reported (Sillero et al. 2000). Combined dis-
ease resistance is obviously valuable, and it has been possible to select resistances
to chocolate spot disease and rust disease together (Villegas-Fernández et al. 2011).
Resistance to downy mildew, caused by Peronospora viciae f. sp. fabae (Berk.)
Caspary, has been identified in the UK germplasm (Thomas and Kenyon 2004) and
resistance to Cercospora leaf spot, caused by Cercospora zonata G. Winter, is an
objective in Australia (Kimber and Paull 2011).
Faba bean is susceptible to several generalist pathogens including Fusarium
oxysporum Schl.; Rhizoctonia solani, J.G. Kühn; Alternaria alternata (Fr.) Keissl.;
and Sclerotinia sclerotiorum (Lib.) de Bary; but there have been few advances in
breeding for resistance to these. It is also attacked by the stem nematode ( Dity-
lenchus dipsaci (Kühn) Filipjev species complex), root-knot nematodes ( Meloido-
gyne incognita (Kofoid and White) Chitwood and Meloidogyne javanica (Treub)
Chitwood) and root-lesion nematodes (several species of Pratylenchus). These are
generally managed by rotation rather than resistance breeding, although it has been
noted that some cultivars of faba bean are non-hosts of Pratylenchus neglectus
154 G. Duc et al.

(Rensch) Filipjev and Schuurmans Stekhoven (Yunusa and Rashid 2007), and this
may be a valuable avenue for further investigation.
Breeding for parasitic insects resistance has not been so far developed due to
the lack of solid resistance sources. In the Mediterranean basin, broomrapes are
the major limitation to growing faba bean. The main species of these root parasites
are crenate broomrape ( Orobanche crenata Forsk.), fetid broomrape ( Orobanche
foetida Poir.) and Egyptian broomrape ( Phelipanche aegyptiaca Pers.) Pomel (syn.
Orobanche aegyptiaca Pers.). All can attack other host species as well, but crenate
broomrape is currently the most important and widespread. Field testing for resis-
tance is especially difficult for a root parasite, as its seeds are unevenly distributed
in the soil and individual host plants commonly escape infection. The resistance to
O. crenata is generally quantitative (Díaz-Ruiz et al. 2010) and has been used in
breeding programmes since the release of the Egyptian accession ‘F402’ in 1982.

6.2  Abiotic Stresses

Faba bean, like all crops, has optimum temperatures, water and mineral require-
ments for growth, and conditions outside these ranges cause stress. Climate change
is likely to increase the frequency of heat and water deficit stresses, but may also
affect the exposure to other stresses. Recovery from a transient stress is as important
as survival, but it is less often examined in experiments.
Drought, or water deficit stress, is considered by many workers to be the major
abiotic stress for most crops. In almost all conditions, transient water deficit occurs
at some stage during the growing season, and, particularly in Mediterranean cli-
mates, terminal water deficit is normal. Drought responses are usually characterised
as escape, avoidance and tolerance. Early maturity in a Mediterranean climate is an
example of escape, improved water metabolism through better root systems and
stomatal closure represents avoidance and protection of cell metabolism through an
osmoprotective substance such as glycine betaine or proline provides tolerance. Es-
cape is not a useful mechanism against transient, mid-season water deficit, and the
other approaches are necessary. There is direct evidence for wide variation in sto-
matal traits, and there is indirect evidence from the same experiments for variation
in the ability of roots to obtain water (Khazaei et al. 2013a). Canopy temperature
depression is an effective, rapid and economical way to determine the water status
of a faba bean plant (Stoddard et al. 2006; Khan et al. 2007, 2010; Khazaei et al.
2013a). Osmotic adjustment through the synthesis of protective substances has not
been identified in faba bean.
Faba bean is well adapted to cool temperatures and seldom shows damage from
temperatures down to − 6 °C, although flowers are not tolerant of frost (Link et al.
2010). Autumn-sown beans in oceanic and especially continental regions have to
withstand much harder frosts than this, often in repeated freeze–thaw cycles, and in
some regions prolonged snow cover as well. The assemblage of traits required for
winter tolerance is complex (Link et al. 2010) and has seen some attention to indi-
vidual components such as frost tolerance (Arbaoui and Link 2008; Arbaoui et al.
2008) and tolerance to snow cover (Fukuta and Yukawa 1998).
5  Faba Bean 155

There is little literature on heat stress as separate from water deficit stress in faba
bean, but it has been suggested that selection for one would also select for the other.
Calibration of the agricultural production systems simulator (APSIM) crop growth
model suggested that the optimum temperature for growth in this species is 23 °C,
whereas 27 °C was used for the CROPGRO model (Boote et al. 2002). The tem-
perature at which processes such as pod setting fails ranges from 34 to 45 °C (Boote
et al. 2002), and this implies that selection for heat tolerance may be required at
many latitudes as climate changes.
Transient waterlogging may occur after a heavy downpour, during prolonged
rainy periods, after snowmelt or as part of the difficulties associated with com-
pacted soil. Faba bean was the most tolerant of seven grain legume species in a
pot-based test at 35–42 days after sowing, and there was intraspecific variation in
tolerance as well when tested 21–30 days after sowing (Solaiman et al. 2008).
Oxidative stress is a part of all abiotic stresses. General stress resistance (or
avoidance) may therefore be conditioned by better methods of responding to oxi-
dative stress. Superoxide dismutase (SOD) is one of the enzymes involved, and it
is inducible by stress in this species, but no report showing significant genotypic
variation in SOD activity has come to light (Khan et al. 2010). There are many
articles on the induction of SOD activity by various metal ions (Cd, Co, Al, La and
Pb, among others), and this method may be a simpler and more consistent way of
inducing stress than extremes of water supply or temperature.
Early maturity is a desired attribute in other zones besides Mediterranean cli-
mates. Faba bean often matures after the small-grained cereals, whether spring
sown in boreal climates or autumn sown in oceanic climates. There is a fundamental
conflict between the aims of earliness and high yield, because the more radiation
that a crop can intercept through a longer growing period, the greater its potential
biomass production. Thus, the correlation between growing degree days and yield is
often positive and significant (Fig. 5.3). Breeding progress for yield is easily made
along the trend line, but is more worthwhile when it moves the line upward. Some
of this increase presumably comes from improvement in radiation use efficiency,
but there is remarkably little literature on this subject.

6.3 Targets Against Abiotic Stresses Associated

with Climate Change

A reliable symbiotic activity in situations of abiotic stresses (drought, waterlogging

and salt in particular) is crucial to assure stable yields. Rhizobial and VAM symbio-
ses, each use 4–16 % of the assimilate produced by a faba bean plant, but the crop
generally compensates for this by increasing its photosynthetic rate, so there is no
net yield penalty to either of these symbioses (Kaschuk et al. 2009).
This indicates that faba bean yield is more sink limited than source limited, which
has repercussions for other breeding objectives such as competitive ability and seed
yield in a situation of stress, but it does not exclude that particular plant–symbiont
combinations may be selected and monitored to improve the stress tolerance.
156 G. Duc et al.






6HHG\LHOGWKD










        
Thermal time from sowing to maturity (°C-d)

Fig. 5.3   Correlation between growing degree days and yield

Nutrient use efficiency is another important trait for the future, as we try to make
crops thrive on reduced inputs. Faba bean releases citrate, malate and protons that
solubilize many nutrients but gradually acidify the soil. Phosphorus uptake ability
ranged threefold in a set of 50 faba bean accessions, attributed to variation in root
exudates (Rose et al. 2010). A dedicated search should uncover variation in phos-
phorus utilization efficiency as well as uptake efficiency to be used in breeding
programmes.
Winter faba bean offers the pertinent agronomic advantages of winter crops,
namely higher probability of good sowing conditions, longer growth cycle, good
root establishment, earlier flowering and seed-filling period, escape from the main
risk of summer drought stress and early maturity. With the advent of climate change
and some improvement of freezing resistance and general winter hardiness (Ar-
baoui and Link 2008), the zone of adaptation of winter faba bean is expected to
expand to more continental regions in Europe.
European winter-hardy faba bean cultivars are minor types, some examples in-
clude Diva, Diver, Gladice, Hiverna, Husky and Wizard. Their level of resistance
to freezing depends very much on (1) the basic resistance without hardening and on
(2) the response to hardening. Hardening is also well known as cold acclimation and
results from exposure to low, nonfreezing temperatures. For faba bean, about 5 °C
and short day length (~ 8–10 h) were effective for hardening (Herzog 1987; Arbaoui
and Link 2008). A slow dehardening will also be a key breeding trait required to
tolerate more irregular winter temperatures.
Tolerance to drought, heat stress and salinity has been extensively investigated at
ICARDA and elsewhere and were recently reviewed by Duc et al. (2011). Drought
5  Faba Bean 157

may be escaped by a deep root system together with earliness that concludes the
reproductive phase before a terminal drought. Water loss is also minimized through
stomatal control. Inbred line ILB938/2 and cultivar Melodie were the two best ac-
cessions at avoiding dehydration in this way (Khan et al. 2007). These two acces-
sions differ at two loci affecting stomatal activity (Khazaei et al. 2014). The broad
adaptation of the crop including its cultivation in tropical and subtropical regions,
such as Bangladesh, suggests that many accessions of the species are tolerant of
relatively high temperatures as long as water is in sufficient supply. Notable oc-
currences of salinity tolerance was found in accessions from China, Greece, Syria,
Morocco and Australia (Leonforte at al. 2013). The mechanisms and genetic basis
of these tolerances still remain to be analysed for their practical use in breeding.
Interestingly, grain legumes have the potential to decrease the greenhouse gas re-
lease in agriculture by reducing the production and use of chemical nitrogen fertilis-
ers. Some rhizobia associated with legumes produce enough nitrous oxide reductase
that greenhouse gases release cannot be detected (Sameshima-Saito et al. 2006),
showing another way in which this environmental hazard can be reduced.

7  Breeding Methods and Specific Techniques

7.1 Evaluation

A high level of genotype × environment interaction has been demonstrated for faba

bean, particularly for comparisons between germplasm pools and across mega-
environments. There is also variation in the degree of adaptation across environ-
ments among cultivars of a germplasm pool, for example, autumn-sown cultivars
for medium-duration winters and spring-sown cultivars where winters are long and
severe, as in China. Much of this variation can be attributed to variation in the
optimal phenology for different geoclimatic regions, but variation in stresses, such
as the dominant diseases, between regions are also important. Trials conducted in
Germany and ICARDA demonstrated very poor yields of Central European cul-
tivars in Syria, whereas the Mediterranean cultivars yielded well, and were early
flowering, in both environments (von Kittlitz et al. 1993). Similarly, the three Euro-
pean germplasm pools, minor, major and Mediterranean were adapted to different
regions in trials in German and Mediterranean environments (Schill et al. 1998),
while Mediterranean, spring and winter germplasm showed adaptation to specific
regions in trials conducted in Mediterranean and continental European environ-
ments (Annicchiarico and Iannucci 2008). European spring faba bean production
regions could be assigned to three mega-environments: continental, oceanic and
Mediterranean, based on the genotype plus genotype × environment yield response
of European spring faba bean cultivars across a range of test environments (Flores
et al. 2013). These results demonstrate the importance of selection of trial locations
158 G. Duc et al.

that represent the target environments of the breeding programme when evaluating
and selecting breeding lines. Genome-wide association studies combined with high
throughput phenotyping studies for traits of interest represent new tools to evaluate
genetic resources and to detect genes of interest for breeding.

7.2  New Variation

As previously described, faba bean is reproductively isolated from all other Vicia
species. This limits the genetic variation available for crop improvement to exist-
ing germplasm collections, to new collection missions or to de novo approaches
to generate genetic diversity. The two major methods to introduce novel variation
in faba bean are mutation and transformation. Experiments undertaken by Sjodin
(1971) with a range of mutagens, including X-rays, generated numerous mutant phe-
notypes, most of which were determined by recessive alleles against wild allele.
Oldach (2011) listed 19 mutant faba bean cultivars released in the period 1959–2009.
The mutant traits included early maturity, plant architecture, yield, dwarfism, protein
content, disease resistance and lodging resistance. The major trait developed through
mutagenesis and used in breeding programmes was the determinate or terminal-in-
florescence growth habit conditioned by the recessive allele of the ti gene.

8 Integration of New Biotechnologies in Breeding

Programmes

8.1 The Difficulty of Adaptation of In Vitro Methods

to Vicia faba

The application of biotechnology in plant breeding requires an efficient in vitro re-

generation procedure. Despite the intensive investigations since the 1960s, a reliable
regeneration system for faba bean has been difficult to establish. Gnanasambandam
et al. (2012) reviewed the attempts to cultivate faba bean in vitro, mainly focused on
the optimal growth of callus tissue or suspension cultures rather than the induction of
shoot morphogenesis and plant regeneration. Regeneration via organogenesis using
in vitro seedling as explants by micro-cutting were followed by axillary bud prolif-
eration or meristem tips as explants. Despite the use of different media compositions
and explant sources, and optimizing the in vitro culture conditions, the morphogenic
potential of faba bean cells was relatively low, and all attempts to initiate shoot re-
generation remained unsuccessful (Gnanasambandam et al. 2012).
The major constraint in the process was the deterioration of the explants and
cultivated tissues due to the action of phenolic compounds. Therefore, the effect of
various chemical and physical factors in axillary shoot cultures was examined, re-
vealing that low temperatures limit the formation of phenolics. Furthermore, plant-
5  Faba Bean 159

let regeneration from explants lacking pre-existing shoot meristems was reported.
These achievements were followed by the regeneration of faba bean plants from
protoplasts isolated from leaves and shoots, and from cell suspensions. Subsequent-
ly, somatic embryogenesis in callus and suspension cultures derived from immature
cotyledons of faba bean and an improved protocol combined with the Agrobacte-
rium tumefaciens-mediated gene transfer were also achieved (Gnanasambandam
et al. 2012 and references herein). An efficient in vitro regeneration protocol has
been reported using single cotyledon explants with half of the embryo axis (Anwar
et al. 2011).
One of the most limiting factors to obtain successful faba bean regeneration is the
difficulty of root induction from regenerated shoots. Root growth does not always
occur in the earlier stages in plant cell culture, and it is an essential requirement
for successful plant growth after the micropropagation procedure. This challenge
seems to be overcome, as the in vitro root induction of faba bean was achieved from
regenerated shoots (Ismail et al. 2011). Grafting techniques can also overcome the
rooting difficulties. The efficient regeneration protocol reported so far allows for
successful micropropagation of faba bean, a key issue for future genetic improve-
ment of plants via transformation protocols.

8.2  Mutants and Transformants

The lack of an efficient protocol for the regeneration of transgenic plants was the
main obstacle to faba bean transformation. The most widely used method to transfer
foreign genes into dicotyledonous plant is Agrobacterium. However, only a few
reports on faba bean using Agrobacterium-mediated transformation for producing
transgenic plants based on stem segments, mature embryo discs or cotyledonary
nodes are available (Bottinger et al. 2001; Hanafy et al. 2005). A regeneration and
microprojectile-mediated transformation system was established using the biolistic
bombardment gene delivery system (Metry et al. 2006).
Although the major obstacles in generating transgenic faba bean plants seem
to be circumvented, the number of transgenes introduced so far is still limited.
Two successful studies used this approach to improve protein quality (reviewed in
Gnanasambandam et al. (2012)) and Hanafy et al. (2013) reported the first evidence
that PR10a, a gene-enhancing salt and/or drought tolerance in potato, transferred
into faba bean by A. tumefaciens-mediated transformation system, caused the same
effect in this crop.

9  Molecular Characterisation of Genetic Resources

A burst in development of diverse molecular tools for molecular characterisation

of V. faba germplasm, especially over the past decade, was followed by an increas-
ing number of empirical studies analysing larger numbers of genotypes and/or ac-
160 G. Duc et al.

cessions representing geographically broader diversity of V. faba (e.g. Kwon et al.

2010; Wang et al. 2012), its botanical varieties (e.g. Link et al. 1995; Sanz et al.
2007; Ouji et al. 2012) and/or winter versus spring forms of this crop (e.g. Zong
et al. 2009, 2010; Wang et al. 2012). However, overall insights into the geographic
structure of nuclear genetic diversity in V. faba, heterozygosity levels, delineation of
core collections and broad utilization of these tools in breeding efforts and marker-
assisted selection are still to come, and future large-scale surveys will provide in-
formation on the cytoplasmic diversity of the V. faba gene pool as well. Chloroplast
DNA (cpDNA) and mitochondrial DNA (mtDNA) genomes, which are predomi-
nantly maternally inherited, are not only useful for genetic diversity surveys but are
also markers of choice for both phylogenetic and phylogeographic studies that can
shed more light on the origin and domestication of V. faba.

9.1  Molecular Characterisation of Cytoplasmic Diversity

Work on the V. faba chloroplast genome during the 1980s was mainly oriented
towards understanding the evolution and organization of the genome (e.g. Ko et al.
1987), gene organization (e.g. Mubumbila et al. 1984) and gene mapping (e.g. Ko
et al. 1983). The most striking discovery was that the V. faba chloroplast genome,
whose size has been estimated to c. 123 kb (Raina and Ogihara 1994), does not have
the quadripartite structure present in most angiosperms, that is, a large and a small
single-copy region separated by a pair of c. 25-kb long inverted repeats comprising
ribosomal RNA genes. One set of ribosomal RNA genes in an inverted repeat seg-
ment closest to the psbA gene and a quarter of the ancestral small single-copy region
have been lost, and genetic organization of V. faba cpDNA genome was established
via three inversions after the deletion event (Ko et al. 1987). Findings of a similar
organization of the cpDNA genome in temperate legumes (e.g. P. sativum, M. trun-
catula and L. sativus), but not in tropical Glycine max and Lotus japonicus provide
evidence that two large clades in Fabaceae are characterised by contrasting patterns
of cpDNA evolution. Interestingly, the structural differences between the cpDNA
of these two clades correspond to two contrasting nodulation types (Wojciechowski
et al. 2004) resulting from plant-rhizobia coevolution.
The focus of studies on the Vicia cpDNA genome has shifted over the past 20
years towards utilization of nucleotide cpDNA diversity in phylogenetic surveys.
Raina and Ogihara (1994) were the first to employ restriction-site variation (restric-
tion fragmentation length polymorphisms, RFLPs) for assessing interspecific rela-
tions in the genus Vicia and delineation of closest wild relatives of V. faba. Fennell
et al. (1998) expanded research to sequences of the trnL intron, while Potokina et al.
(1999) studied the Restriction Fragment Length Polymorphism Analysis of PCR-
Amplified Fragments (PCR-RFLP) of five chloroplast genes. Haider et al. (2012)
demonstrated that PCR-RFLPs on nine chloroplast regions and a set of 12 consen-
sus chloroplast simple sequence repeats (ccSSRs) can be used for identification of
diverse Vicia species. Schaefer et al. (2012) provided the most comprehensive phy-
5  Faba Bean 161

logenetic inference of the tribe Fabeae based on both chloroplast and nuclear data.
Unfortunately, instead of providing unambiguous answers regarding the progenitor
and allies of V. faba, these studies as well as molecular surveys based on nuclear
and/or mitochondrial genomes (e.g. Potokina et al. 1999; Sanz et al. 2007) were
largely incongruent with traditional morphology-based taxonomies and systematics
(more than 20 since Linnaeus, reviewed in Maxted 1993) because morphologically
similar species, such as V. faba and species from the Narbonensis complex, appar-
ently are not close at the DNA level.
Furthermore, available molecular phylogenies are not consistent in delineating
the closest relatives of V. faba. Potential reasons include convergent or homoplastic
morphology and/or molecular characters, hybridisation followed by introgression,
backcrossing following secondary contact, incomplete lineage sorting in recently
diverged taxa or obstacles related to the methods used and/or low resolution of data
sets of various sizes. The unquestioned outcome of diverse studies is the distinctive-
ness of V. faba, supported by cytological, karyological, hybridisation, biochemical
and other studies and the justification for treating Vicia paucijuga as a subspecies of
V. faba (Potokina et al. 1999; Schaefer et al. 2012).
The features of the Vicia cpDNA genome advantageous for phylogenetic surveys
have posed a limitation on studies on the intraspecific level (Shiran and Mashayekh
2004; Haider et al. 2012). Diverse studies led to the conclusion that V. faba origi-
nated from a single ancestral maternal lineage that either possessed little cpDNA
diversity at the origin or lost much of its variation during domestication through a
severe bottleneck (Shiran and Mashayekh 2004). However, further studies based on
broader samples are required for more general conclusions on evolutionary unfold-
ing of Vicia and phylogeography of V. faba.
MtDNA diversity in V. faba, on the other hand, was observed rather early. Unlike
cpDNA, angiosperm mtDNA genomes are rather dynamic and very variable in size
(200–2500 kb), structure and sequence content. MtDNA mutations/rearrangements
that can cause cytoplasmic male sterility (CMS) were observed in V. faba during
the 1980s (e.g. Scalla et al. 1981; Nikiforova and Negruk 1983 among many other
examples), and it has been shown that the nuclear context participates in the control
of mitochondrial plasmid sequence, appearance, and copy number (Flamand et al.
1992).
Reports on organization and variability of V. faba mtDNA genes (e.g. MacFar-
lane et al. 1990) were confirmed when the sequence of the complete mtDNA ge-
nome of this crop became available (Negruk 2013). The size of the mitochondrial
genome of cultivar Broad Windsor was estimated at 588 kb with 45.04 % guanine,
cytosine (GC) content, with c. 40 % similarity with mtDNA of the legumes Lo-
tus japonica and Millettia pinnata (Kazakoff et al. 2012). Furthermore, 45 % of V.
faba mtDNA sequences were found to be homologous to the Medicago truncatula
nuclear genome.
The availability of the complete mtDNA sequence of V. faba and other legumes
opens a new chapter in phylogenetic and phylogeographic studies that may provide
answers on the origin and domestication of V. faba. Using total mtDNA as a probe
against mtDNA from 52 accessions of 12 Vicia species, Van de Ven et al. (1993)
162 G. Duc et al.

were able to distinguish species but not cultivars representative of the different bo-
tanical varieties of V. faba. Furthermore, Scallan and Harmey (1996) found consid-
erable mtDNA diversity at the species level, as they were able to divide nine V. faba
commercial cultivars of the major type, which were all Western cultivars from a nar-
row geographical range, into two cytoplasmic types and at least six groups. These
findings are supported by work in progress (Aleksić et al. personal communication).
Two major independent and spontaneous sources of cytoplasmic male sterility
were identified in faba bean (350 and 447 cytoplasms), both of them displaying
instability features incompatible with a hybrid seed production scheme (Duc et al.
1992). Markers of the male sterility trait at the level of mtDNA and RNA could not
be detected (Flamand et al. 1992). Independently of mitochondria, RNA-containing
particles present in the cytoplasm appeared to be accurate markers of the 447 de-
terminism of male sterility (Scalla et al. 1981). A high content of plant tissues in
these cytoplasmic particles was also a good marker of the stability of the 447 male
sterile phenotype.

9.2  Genomics of Nuclear Genome

Recently, various molecular markers have been successfully used to characterise

genetic diversity in faba bean accessions. Link et al. (1995) examined three groups
of faba bean inbred lines from European and Mediterranean gene pools by ran-
domly amplified polymorphic DNA (RAPD) assays. Amplified fragment length
polymorphism (AFLP) diversity analysis of 22 V. faba elite cultivars from Europe
found most to share genetic similarity among the spring types, with one winter cul-
tivar classified as separate, and the closest relationship being between two advanced
lines from the same parents (Zeid et al. 2001). A diverse collection of 20 faba bean
accessions from a gene bank in Spain that were assayed with transposon-based
molecular markers sorted into groups mostly unrelated to geographic origin or mor-
phology (Sanz et al. 2007). Twenty Greek landrace populations were analysed with
inter-simple sequence repeat (ISSR) by Terzopoulos and Bebeli (2008), with sepa-
ration of small-seeded minor types from medium- to large-seeded Mediterranean
types which were divided into two subgroups. Winter ecotype (Zong et al. 2009)
and spring ecotype (Zong et al. 2010) faba bean germplasm sets from China were
separately compared with accessions from the rest of the world using AFLP, reveal-
ing significant separations between Chinese accessions and the accessions from
outside China. Kwon et al. (2010) evaluated a world collection of 151 faba bean
accessions with target region amplification polymorphism (TRAP) markers, and
showed a strong association with geographic origin, with five major groups identi-
fied with a dendrogram. These studies have thus shown that when studies include
cross-continental sources of accessions, associations of genetic diversity with geo-
graphic diversity tend to be found, especially with spring and winter habits of land
races reflecting the agriculture practices in respective diverse environments and
geographic origins.
5  Faba Bean 163

The genetic diversity and relationships of 802 faba bean accessions from dif-
ferent geographical locations of China and rest of the world (Asia except China,
Europe and Africa) were examined using 11 ISSR primers (Wang et al. 2012). Ac-
cessions from northern China showed highest genetic diversity, while those from
central China showed low diversity. Chinese spring faba bean germplasm was clear-
ly separated from Chinese winter faba bean, based on principal component analy-
sis and unweighted pair group method with arithmetic mean (UPGMA) clustering
analysis. Winter accessions from Zhejiang (eastern China), Jiangxi (eastern China),
Sichuan (southwestern China) and Guizhou (southwestern China) were quite dis-
tinct from those from other provinces in China. Great differentiation between Chi-
nese accessions and those from rest, of the world was shown with a UPGMA den-
drogram. Analysis of molecular variance (AMOVA) analyses demonstrated large
variation and differentiation within and among groups of accessions from China.
As a continental geographic group, accessions from Europe were genetically closer
to those from North Africa. Based on ISSR data, grouping results of accessions
from Asia, Europe and Africa were obviously associated with their geographical
origin. The overall results clearly indicated that the genetic relationship of faba bean
germplasm was closely associated with their geographical origin and their ecologi-
cal habit. China is likely to be another secondary centre of diversity for faba bean,
especially the Chinese winter gene pool, which has been reproductively isolated
from the European and West Asian gene pools (Zong et al. 2009; Wang et al. 2012).

10 Integration of New Biotechnologies in Breeding

Programmes

Complete exploitation of genomic and transcriptomic approaches and marker dis-

covery to faba bean breeding is still to be achieved, but results obtained so far are
promising. One of the main reasons for slow progress is the extraordinarily large
size of the faba bean genome. Nevertheless, comprehensive genomic and post-ge-
nomic tools are being developed. These include several types of molecular marker
sets, saturated genetic maps bearing relevant genes or QTLs and both cDNA librar-
ies and transcriptome datasets for reverse genetics. A combination of approaches,
candidate gene and colinearity with model legumes, is being pursued for the iden-
tification of genes underlying agronomically important traits. Finally, a functional
consensus map of faba bean has been constructed integrating all the markers, genes
and QTLs previously published (Román et al. 2004; Satovic et al. 2013). This sec-
tion provides a review of the genetics and genomics tools available so far in this
crop. New genomic advances, together with conventional breeding tools, will push
forward future genomic-based faba bean breeding programmes.
164 G. Duc et al.

10.1  Molecular Marker Development

The development of linkage maps in faba bean has been parallel to the progress
and availability of different types of molecular markers. The first faba bean linkage
groups only included morphological and isozyme loci but, with the advent of PCR-
derived markers, RAPDs, microsatellites and, more recently, sequences or gene-
derived markers based on single-nucleotide polymorphisms (SNPs), are part of the
genetic linkage maps reported in the crop (reviewed in Torres et al. (2012)).
The first set of microsatellites (or simple sequence repeats; SSRs) from chro-
mosome I DNA libraries were developed by Pozarkova et al. (2002). This number
was enlarged by Zeid et al. (2009) and Gong et al. (2010). The recent screening of
expressed sequence tags (EST) sequences in public databases led to the develop-
ment of new SSR loci that have been validated among several faba bean accessions
(Ma et al. 2011).
Faba bean has now achieved notable advances in marker repertoire, thanks to
the synteny among closely related species. In the last decade, significant progress
has been made in developing genomic resources in model species ( M. truncatu-
la), major legumes (soya bean and common bean, peanut and pea), and a number
of so-called orphan crops (such as chickpea, cowpea, lentil and pigeon pea). As a
result, reference genome sequences are now available for some of these species
together with saturated genetic and physical maps. The availability of ESTs from
Medicago or related legume crops constitutes a novel source of markers physically
associated with coding regions that are being extensively exploited in faba bean for
gene discovery and the macro- and microscale genomic comparison. A number of
intron-targeted amplified polymorphic (ITAP) markers mapped in M. truncatula,
Glycine max and Lupinus were used to develop the first gene-based genetic map
of faba bean using a recombinant inbred line (RIL) population derived from cross
Vf6 × Vf27 (Ellwood et al. 2008). This genetic map was further used to identify
and validate QTLs controlling flowering time and other yield-related traits (Cruz-
Izquierdo et al. 2012) and to study macrosyntenic relationships between V. faba and
other related legume species. Within the framework of the Grain Legumes Integrat-
ed Project (GLIP), a large set of additional expressed sequence tag (EST)-derived
markers were developed and mapped in the progeny of two faba bean inbred popu-
lations (Vf6 × Vf136 and 29H × Vf136) segregating for both broomrape and A. fabae
resistance (Díaz-Ruiz et al. 2009, 2010; Gutierrez et al. 2013).
At present, one of the main pillars of genomic breeding is the development of
high-throughput DNA sequencing methods (known as next-generation sequencing
(NGS)). These technologies provide the mass sequencing of genomes and transcrip-
tomes and deliver a vast array of genomic information for discovering genes and
their positions. The lower cost, compared to traditional sequencing methods, is ac-
celerating the analysis of less studied species, such as faba bean. Thus, using the
Roche 454 GS FLX Titanium Platform sequencing, a genome library with 125,559
putative simple sequence repeat (SSR) sequences was constructed and character-
ised for repeat type and length from a mixture of spring- and winter-sown faba bean
genotypes (Yang et al. 2012). A set of 94 primer pairs produced amplicons polymor-
5  Faba Bean 165

phic among 32 faba bean genotypes selected from diverse geographical locations.
Using the same technology, cDNA samples obtained from various faba bean tissue
types allowed annotation of more than 18,000 faba bean unigenes. Primer pairs for
SSR-containing expressed sequence tags (ESTs) were designed and a subset of 96
EST–SSR markers was screened for validation across different cultivars (Kaur et al.
2012). The high quality of novel SSRs developed via next-generation sequencing
technology is a valuable source for future map construction and QTL mapping.
A new allele-specific PCR SNP genotyping system developed at KBiosciences
(KASPar) has been also assayed to identify putative SNPs between sequenced seed-
ling transcriptomes from two contrasting inbred lines of faba bean. Assays were
designed for more than 800 loci, 85 % of which (757) showed robust amplification
and were validated on a test panel of 33 inbred lines (Cottage et al. 2012). The ap-
proach was used to build a medium-density genetic map that will facilitate trait
dissection in future faba bean breeding programmes and has been used to identify
QTLs associated with stomatal response in a population of recombinant inbred lines
(Khazaei et al. 2014).

10.2  Gene Tagging of Qualitative Traits

Some faba bean seed quality traits along with the hypersensitive resistance to rust
are regulated by single genes. In these cases, bulk segregant analysis (BSA) has
proved to be an effective method for the identification of markers tightly linked to
the corresponding responsible genes (Avila et al. 2003; Gutierrez et al. 2006, 2007,
2008).
The bioactive compounds mostly targeted in faba bean breeding programmes are
condensed tannins and vicine–convicine, two classes of antinutritional factor pres-
ent in seeds. Two recessive genes (zt-1 and zt-2) control the absence of tannins, and
vicine–convicine content is decreased due to the presence of the vc- allele linked to
the colourless hilum gene (Duc et al. 1989). BSA has been used to develop molecu-
lar markers tightly linked to the desired alleles (Gutierrez et al. 2006, 2007, 2008).
These markers might facilitate gene pyramiding when producing new faba bean
cultivars with improved nutritional value.
Avila et al. (2003) used BSA as well to detect RAPD markers tightly linked to
one gene causing the hypersensitive resistance to rust (race 1) in the inbred line
2N52.
The candidate gene approach has also been used in faba bean. This methodology
allows the development of disagnostic markers based on differences between alleles
of the gene responsible for the trait. These markers have the significant advantage of
being completely linked with the selected trait, thus providing 100 % efficiency in
the selection. The strategy was used for the selection of the determinate growth habit,
a trait suitable for the mechanical harvesting of the crop. A single gene, recognized as
CEN/TFL1-homologous or CEN/TFL1-like gene, is responsible for the character and
several orthologues have been described. Using this information, Avila et al. (2006,
2007) developed several PCR-based markers suitable for the unambiguous selection
166 G. Duc et al.

of determinate growth habit genotypes. Marker research for qualitative traits de-
veloped so far in faba bean has still not been adopted in any breeding programme.
Nevertheless, the identification of candidate genes underlying these target traits is
underway (Khazaei et al. 2015; Webb et al. 2015; Gutiérrez et al. 2015).

10.3  Genetic Maps

Genetic linkage maps are important tools for a wide range of genetics and breed-
ing applications. The availability of high-density maps enhances breeding progress
through the application of association analysis, map-based cloning or marker-as-
sisted selection (MAS) methods. The approach is based on the selection of markers
linked to the desired genes in segregating populations to trace or pyramid-favour-
able alleles in a given genome. As the introgression of target genes using conven-
tional breeding strategies is long and complicated, MAS provides simplicity and
reliability in identifying target individuals at early seedling stages, saving both time
and resources.
So far, 14 relevant genetic maps have been developed in this crop (reviewed
in Torres et al. 2012). Less accurate maps with dominant markers in F2 were fol-
lowed by more precise maps in the corresponding RIL populations, using codomi-
nant gene-based markers. Recently, an F2 population from a large-seeded Chinese
landrace and small-seeded inbred line was used to develop a map using exclusively
SSR markers (Ma et al. 2013).
The major drawback of these maps, and the limitation to their practical applica-
tion, is the small number of common markers and relatively low map density. The
relationships between the maps of various populations had not been well studied,
so a consensus genetic map has been lately constructed integrating most of the
markers, genes and QTL information (Satovic et al. 2013). The map contains 729
loci and covers 4602 cM, representing around a threefold increase in the number
of mapped markers and coverage, as compared with previous maps. The approach
facilitated including a more consistent locus order, assisted in the assignment of
anonymous linkage groups to chromosomes and updated the position of stable faba
bean QTLs controlling resistance and yield-related traits (Satovic et al. 2013). This
new map with the largest collection of molecular markers currently used in faba
bean genome analyses constitutes a valuable resource for comparative genomics,
genome organization and evolution studies, and it will provide a significant step
forward for faba bean breeding and genomics.

10.4 QTL Analyses: Biotic, Abiotic Stresses and Yield-Related

Traits

Disease resistances are important targets for MAS in view of the difficulties of
manipulating these traits through conventional approaches. Major biotic constraints
5  Faba Bean 167

under multigenic control such as the resistance to crenate broomrape ( O. crenata)
and ascochyta blight ( A. fabae) have been the subject of particularly intensive QTL
studies (reviewed in Torres et al. 2010, 2012). Breeding for resistance to broomrape
is challenging owing to the complexity of the disease evaluation and the polygenic
nature of the trait. This holoparasitic angiosperm is considered the most damaging
and widespread disease in the Mediterranean basin, and the identification of genes
controlling resistance against it is a key subject in faba bean breeding.
Two progenies segregating for both pathogens (crosses Vf6  × Vf136 and
29H × Vf136), have been used for the identification and validation of relevant QTLs
in different environments and genetic backgrounds. First, a set of 196 F2 individuals
derived from the cross Vf6 × Vf136 allowed detection of three putative QTLs ( Oc1,
Oc2 and Oc3) on chromosomes I, VI and II, respectively (Román et al. 2002). In
order to validate both the presence and location of the F2 QTLs, 165 RILs from
this cross were used to construct a new linkage map (Díaz-Ruiz et al. 2010). RILs
used in multi-environment trials increase the power for testing differences between
genotypic classes and the precision of trait measurement compared with other types
of progenies. This fact is of special significance for broomrape resistance, a trait
highly influenced by the environment, resulting from the infectious ability of the
parasite in a specific location together with a range of escape factors and resistance
mechanisms acting at different levels of the infection process.
Validation experiments were carried out assessing the resistance of the RILs in
three different environments. The major QTL, Oc1, was not detected in the RILs
probably due to its overdominance in the F2. In contrast, both Oc2 and Oc3 were
consistent in F2 and F6 generations (Díaz-Ruiz et al. 2010), as were detected in at
least two of the three environments, thus pointing to their suitability as targets for
MAS. Two additional QTLs ( Oc4 and Oc5) with small effects were detected in chro-
mosome I in one of the environments. A new RIL population derived from the cross
29H × Vf136 allowed identification of seven QTLs for O. crenata ( Oc7 to Oc13;
Gutierrez et al. 2013). QTL Oc7, located on chromosome VI, may correspond to the
previously reported Oc2. The addition of common markers in the 29H × Vf136 map
is being performed in order to establish a clear homology between both regions. The
outcomes achieved so far point to Oc7 as a promising candidate for future MAS
for broomrape resistance in faba bean. Saturation of this region will refine the QTL
position and identify candidate genes underlying this resistance.
Similarly to crenate broomrape, the F2 populations from both crosses were used
to detect QTLs affecting ascochyta blight response (being Vf136 susceptible, Vf6
partially resistant and Vf29H resistant). Two QTLs ( Af1 and Af2) were detected
on chromosomes III and II in the Vf6 × Vf136 F2 population (Román et al. 2003).
Avila et al. (2004) studied the resistance on leaves and stems in the F2 population
of cross Vf29H × Vf136, using two pathogenically distinct Ascochyta isolates. The
study revealed six QTLs ( Af3 to Af8), and Af1 (Román et al. 2003) and Af3 (Avila
et al. 2004) were homologous. QTL validation was undertaken next (Díaz-Ruiz
et al. 2009) by studying 165 RILs from cross Vf6 × Vf136. The new linkage map
confirmed two zones of putative QTL action, Af1 and Af2, on chromosomes III and
II, respectively. Fifteen common markers between maps facilitated the compari-
168 G. Duc et al.

son of the homologous regions. The RIL population of cross Vf29H × Vf136 has
recently been developed and the corresponding genetic map is being constructed
to confirm the relevant QTLs across generations, environments and genetic back-
grounds (Avila personal communication).
Recently, Kaur et al. (2012) developed a preliminary faba bean transcriptome
and identified a large collection of EST-derived SNPs that were further used to
develop a genetic map in a population varying for ascochyta blight resistance (Kaur
et al. 2014). Four QTLs controlling resistance were identified in this map, one of
which (QTL-3), appears to be identical with Af 2 identified in prior studies (Díaz-
Ruiz et al. 2009).
Frost and drought tolerance have been as well the subject of QTL studies. An
RIL population derived from the cross between two frost-tolerant lines, Côte d’Or
1 and BPL4628 was used to identify putative QTLs associated with frost tolerance
and auxiliary traits (Arbaoui et al. 2008). Similarly, screening of an RIL popula-
tion derived from two drought-tolerant lines, Melodie 2 × ILB938/2, identified two
QTLs 100 cM apart on chromosome II that affected stomatal activity. In both cases
candidate genes were identifiable from the synteny with the M. truncatula genome
(Khazaei et al. 2014). Finally, an RIL population (Vf6 × Vf27) has been used to map
orthologous legume cross-species markers. The study revealed the macrosyntenic
relationships between V. faba, M. truncatula, L. culinaris and other related legume
species and was used to identify and validate for the first time, QTLs controlling
flowering time and other yield-related traits (Cruz-Izquierdo et al. 2012).
The prospects of MAS for quantitative traits in faba bean breeding programmes
will depend on the extent to which QTL results can be extrapolated and validated
among environments and genetic backgrounds. Mapping additional common anchor
markers is a prerequisite to prove this conservation and determine a QTL homology
or the existence of different resistance loci acting in each of the crosses. Moreover,
marker density remains rather low for the identification of responsible genes and
use of MAS approaches. Comparative genomics and transcriptomic analyses (see
below) are providing new resources to saturate syntenic regions bearing relevant
QTLs. Mapping new genes from Medicago, pea, lupin, lentil and soya bean will
provide new anchor points for comparing the maps, refining the QTL positions and
unravelling positional candidate genes responsible for these traits.

10.5  Transcriptomic Analysis

Recent advances in transcriptomics are emerging as promising genomic resources

for breeding applications in faba bean. Using the 454 Roche GS FLX Titanium
technology, Kaur et al. (2012) sequenced cDNA samples from various tissues and
genotypes for a de novo assembly and gene annotation of a faba bean transcriptome.
A total of 304,680 reads were assembled to generate a set of 60,440 unigenes. In
comparison to M. truncatula and soya bean coding sequences, 10,179 and 19,497
unique hits, respectively, were obtained. A total of 18,052 faba bean unigenes was
subsequently annotated from GenBank. Further comparison to the genome of soya
5  Faba Bean 169

bean resulted in 16,497 unique hits for faba bean, corresponding to ca. 30 % of the
known gene space (Kaur et al. 2014).
Genome-wide transcription profiling by deepSuperSAGE has been used for
quantifying the early transcriptional changes elicited by A. fabae in the resistant
inbred line 29H and to identify key resistance factors steering plant responses to this
stress (Madrid et al. 2013). A total of 1,313,009 tags were obtained, representing
51,485 unique sequences (UniTags) of which 2222 were expressed differentially
between inoculated and control leaves. After gene ontology (GO) annotation, only
2143 of these matched database sequences. The most enriched GO terms corre-
sponded to tags related with photosynthesis metabolism or structural components.
Ten were associated with plant defence, due to their association with responses to
the jasmonic acid pathway, pectin esterase activity, or gene silencing. Validation of
the SuperSAGE data by qPCR of ten differentially expressed UniTags confirmed
a rapid increase or decrease in messenger RNA (mRNA) 8–12 h after inoculation
in most of the upregulated tags and, less consistently, in the downregulated ones
(Madrid et al. 2013).
These studies provide a foundation for the identification of regulators associated
with the host–pathogen interaction and also potential targets for molecular breeding
in this crop.

11  Seed Production

Compared to other major crops, farmers need a relatively large amount of seed per
ha for the establishment of the faba bean crop, so the seed costs are relatively high
and farmers pay particular attention to quality when buying seed. The seeds of faba
beans have a few specific features that make it a special challenge to produce vig-
orous seeds with a high level of germination. The high cost of seeds also explains
why, in some countries, about 50 % of the seeds required for sowing are self-pro-
duced by farmers who accept the risks of lower sanitary control, lower germination
quality and loss of genetic integrity.
Among the cultivated crops in most temperate farming systems, faba bean has
the highest individual seed size. The thousand-seed weight (TSW) within breeding
material can easily vary between 300 and 2000 g (China and Japan), and in released
cultivars for Western Europe it ranges from 450 to 650 g. TSW is highly heritable,
but the conditions during ripening determine whether the expression of the genetic
seed size is at the higher end (optimal conditions) or the lower end (stress, drought).
The variation within one cultivar can differ up to 150 g depending on the conditions
of the production site. The large seed of faba bean offers a large surface area that
is exposed to mechanical impact, and a large diameter that requires adequate time
for moisture to move from the interior of the seed to the testa where it is released.
For companies involved in the seed production of faba bean seed, many specific
requirements in the different steps of production need to be addressed. The partial
allogamy of faba bean results in both self- and cross-fertilised seed on a single
170 G. Duc et al.

Table 5.5   Regulations for seed production of faba beans in Germany. (Source: Sorten-und Saat-
gutrecht, 12th edition)
Basic seed Certified seed
Parameters for the seed
Minimum germination 85 % 80 %
Maximum moisture content 15 % 15 %
Technical purity 98 % 98 %
Other species in 1000 g 0.30 % 0.50 %
sample
Wild oats in a 1000 g sample 0 seeds 0 seeds
Rumex spec in a 1000 g 2 seeds 5 seed
sample
Living adults of Bruchus 0 0
rufimanus
Maximum number of Dity- 5 5
lenchus dipsaci in 300 seeds
Maximum percentage of A. 30 % 30 %
fabae on germinating seeds
Parameters for the field
Minimum distance to others Field smaller 2 ha: 200 m Field larger 2 ha: 100 m
V. faba fields
Maximum number of offtypes 5 15
on 150 m2
Number of plants infected 10 30
with A. fabae in 150 m2
Number of plants infected 10 30
with viruses in 150 m2

plant and of plants of an individual breeding line when grown in open-pollinated

conditions such as in a yield trial or in fields of the seed producer or the farmer. The
importance of cross-fertilisation on the progeny is greater for traits that are under
recessive genetic control (such as zero-tannin), and uncontrolled pollination will
rapidly result in a loss of genetic integrity. Residual seed of the initial multiplication
should be retained to enable further multiplications, and distances (from 300 m to
1 km) between multiplication fields must be assured.

11.1  Regulations and Practical Implications

The major regulations and thresholds which have to be observed in Germany are
summarized in Table 5.5. In other countries, these figures may differ, but the table
might serve as a template for the relevant parameters. The key factor for the han-
dling of faba bean seeds is the moisture content. The threshold for seeds sold to
the farmer is 15 %, but the optimal moisture content with respect to elasticity and
tolerance to mechanical input is between 17 and 19 %. This means that harvest
and transporting to the store should take place before the seed in the field is fully
5  Faba Bean 171

mature (15 % or higher). Especially under hot and dry conditions, the farmer must
take care to start the harvest early enough and not wait until it is fully dry. If they
miss this stage, it is often recommended to wait until the seed moisture content
has been increased by either a little rain or early-morning dew. In western Canada,
growers often apply harvest aids to faba bean crops to harvest, typically with diquat
or other approved desiccants. This allows more rapid and uniform drying of stalks
and seeds.
Once in the store, the commercial seed must be dried slowly down to the desired
storing and packing moisture 14–15 % (which is higher than the targeted 7 % values
frequently used by gene banks to allow long-term conservation). It is crucial that
the drying process is slow and does not get beyond this crucial point of moisture
content. It is strongly recommended to use air temperatures not higher than 35 °C.
This usually allows a slow and continuous drying process. Especially if the seed
arrives with moisture content above 20 %, it is recommended to allow for a break
of at least 12 h during the drying process rather than aiming for the final moisture
content in one step.

11.2 Machinery

Very often, equipment that is used for production of small grains such as cereals is
not suitable for faba bean and needs adjustment or replacement. The setup specific
for faba beans can be used for cereals but usually not vice versa. This results in spe-
cific investments for faba bean seed production. The sowing machinery must have
sufficiently large coulters to allow uniform flow of large seeds, and the combine
harvester should have its threshing concave opened wide to avoid broken, cracked
and crushed seeds. As the pod shells open very easily, the drum speed should be
set to the lowest possible speed. Driving during harvest should be relatively fast
in order to get more biomass into the machine, which also has a buffer function
for the seed inside the machine. The most gentle setup would be a combine with a
tangential drum. Adjustments for the emptying auger should be set to run at lower
revolutions per minute (RPM) in order to avoid seed damage. In addition, post-
harvest handling systems must be adjusted to avoid drops from great height onto
hard ground that would bruise the embryo or break the seed. Therefore, the belts
for moving seeds should ideally be made of rubber, and special handling systems
should be used in bins to avoid seed breakage during filling.

11.3  Sanitary Concerns

For seed production, the following diseases and pests are relevant:
• Black bean aphid ( Aphis fabae, other species) transmit viruses in some environ-
ments, so control by suitable chemicals is recommended.
172 G. Duc et al.

• Bean seed weevil (Bruchus rufimanus) larvae develop within the seeds and leave
a hole in the seed. While this has relatively little impact on germination, adult
weevils remain in the holes in the seeds, and removal will require chemical fu-
migation. Therefore, a strategy to control this pest is strongly recommended.
• The presence of stem nematode (D. dipsaci) precludes use of the seed. Close
monitoring in the field together with appropriate crop rotation helps to control
the nematodes.
• Ascochyta blight (A. fabae) disease starts on the leaves but can move to the pods
and seeds. Infested seeds transmit the disease to the next field. Strict control by
chemicals is highly recommended.
• Chocolate spot ( B. fabae) fungus is not transmitted by seed. Severe infection can
lead to early loss of the canopy resulting in small and shrivelled seeds. Timely
fungicide application is recommended for control.

12 Conclusion

Faba bean breeding has a large number of objectives, and the priorities depend on
regional stress factors and market forces. As a result of climate change, stronger
demands exist for genetic resistance to abiotic stresses (early or late drought, heat,
frost, waterlogging) as well as to pests and diseases ( O. crenata, sitona weevils, leaf
miners, bruchids and pathogenic fungi) in new geographic zones. Potential novel
uses in foods also strengthen demands for low vicine–convicine, high-protein con-
tent and sometimes low tannin genotypes. Inevitably, when the breeder needs to add
more objectives to an existing programme, either more resources have to be added
to cover it or overall progress slows down. In addition to large, characterised and
structured genetic resources collections, rapid breeding methods and translational
omics will help to streamline the breeding progress, so more of the objectives can
be met.
Progress in faba bean genomics is still behind that in other legume crops. Nev-
ertheless, a wide range of genomic and post-genomic resources is being developed
to boost genetic research and breeding applications. More than 14 mapping popula-
tions have been established to study the inheritance of important agronomic traits.
Different molecular marker sets have been developed and used to construct more
saturated linkage maps in order to identify genes or QTLs controlling major stress
responses. Current efforts are focussed on the development of highly accurate selec-
tive breeding tools, using NGS methods. Transcriptomic analysis (SuperSAGE and
cDNA libraries) is enriching the scarce faba bean EST libraries and provides ad-
ditional resources for refining the maps with functional markers. Moreover, transla-
tional genomics, based on the collinearity with model and related species, opens the
possibility to identify candidate genes underlying agronomically important traits.
Finally, a faba bean functional consensus map has been constructed integrating all
the genes and QTLs previously published. The combination of these and new tools
together with a close link between academic research and commercially focused
5  Faba Bean 173

breeding programmes may help researchers to find genes of interest to speed the
release of more competitive faba bean cultivars in the near future.

Acknowledgments  This research was partly supported by the following projects: (1) FP7
LEGATO, European Union; Research Grant 173005 of the Ministry of Education, Science, and
Technological Development of the Republic of Serbia, and the project of bilateral collabora-
tion between the Republic of Serbia and the Republic of France “Pavle Savić”, FABAGRALE
2012–2013, number 680-00-132/2012-09/15, (2) ECONET- PAVLE SAVIC projects, Ministry
of Foreign Affairs—France and PARI-1 Agrale 6-2010-2013, Burgundy Council-France, (3)
RTA2010-00059 and IPT-2011-1259-010000 Spanish projects, cofinanced by Fonds Européen de
Développement Économique et Régional (FEDER) funds, (4) Grains Research and Development
Corporation, Australia.

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Chapter 6
Lupins

Wojciech Święcicki, Magdalena Kroc and Katarzyna Anna Kamel

1 Introduction

The genus Lupinus is characterized by particularly broad diversity in terms of

growth and development, colors, environmental requirements, distribution regions,
number of chromosomes, type of usage, etc. Lupins are not only annual herbs but
also trees grown from the tropics to Alaska. Fifteen-thousand-year-old seeds of
­Lupinus arcticus still germinating have been found, as well as seeds germinating
from under ash after the eruption of the Mt. St. Helens volcano in 1980 (Findley
1981; Porsild et al. 1967). Some species are beautiful ornamentals; seeds can be
used for feeding man and animals (32–45 % of protein), and the green mass can be
used as animal fodder or organic manure. Therefore, lupins can be of great practical
importance and also an intriguing research material.
Lupins were used by man in ancient times—Andean lupin ( Lupinus mutabilis
Sweet) in South America and white lupin (Lupinus albus L.) in the Old World. Even
ancient man was aware of lupin’s advantages—for soil improvement and soaked,
debittered seeds (Cowling et al. 1998b).
The use of lupins in agriculture was caused by some factors. First of all, 50 % of
the area of Poland and Belarus, the former Prussia, is covered by sandy soils. More-
over, the consequences of the Thirty Years’ War, the collapse in yields in 1772, and
the increasing population resulted in the introduction of new, leafy crops (beets, clo-
ver, potatoes, rape) to improve soils. The Prussian King, Friedrich II (1740–1784),

W. Święcicki () · M. Kroc · K. A. Kamel

Department of Genomics, Institute of Plant Genetics, Polish Academy of Sciences,
Strzeszyńska 34, 60-479 Poznan, Poland
e-mail: [email protected]
M. Kroc
e-mail: [email protected]
K. A. Kamel
e-mail: [email protected]
© Springer Science+Business Media New York 2015 179
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_6
180 W. Święcicki et al.

and the land owner, Carl von Wulffen, started (1816) to grow white lupin to improve
soils (seeds were imported from Italy). Unfortunately, due to its soil requirements
and late ripening, both projects collapsed. The successful introduction of yellow
lupin as a green manure was effected for the first time in 1840 by J. N. Borchardt.
He multiplied his own seeds and produced a higher yield of rye as a successive crop
after lupin. The popularity of yellow lupin was transferred from Prussia to Poland
and Russia (Brummund and Święcicki 2011).
Lupin domestication was connected with the removal of the hard seed coat,
dehiscing pods, and decreasing alkaloid content (Święcicki and Święcicki 1995;
Święcicki et al. 2000a). Elaboration of a screening method by von Sengbusch en-
abled the selection of the first “sweet” plants of yellow lupin (1927), narrow-leafed
lupin (1927–1928), white lupin (1930–1931), and Lupinus polyphyllus (1931). The
first, low alkaloid yellow lupin cv. “von Sengbusch Munchberger Gelbe, Grünfutter
Süsslupine” is considered the beginning of fodder lupins. The importance of this
type of use led to a growth in its popularity as a result of increasing demands for
protein in feeding pigs and poultry. It was impossible to ignore plants with 32–45 %
protein in seeds, useful for cropping on light soils in moderate climatic conditions.
There were some important steps in the improvement of lupin cultivars, for ex-
ample, breeding of cultivars resistant to Fusarium, fast growing, thermoneutral,
self-completing (restricted branching), and recently with improved resistance to an-
thracnose. The employment of molecular biology and biotechnology also enabled
achievements in contemporary breeding programs. The history of lupin in agricul-
ture was summarized by Brummund and Święcicki (2011). In addition to Germany,
large lupin breeding projects were implemented after the Second World War in Be-
larus, Chile, Poland, Russia, Ukraine, and Australia (Kubok 1988; Kurlovich 2002;
Lukashevitch et al. 2011; Kuptsov 2000). The last example should be underlined
particularly (Cowling et al. 1998a). The narrow-leafed lupin in Western Australia
stopped a decrease in the yields of wheat grown in monoculture and the acreage
increased up to 1.0–1.5 bln of hectares in the nineteens of the twentieth century.
The world lupin area harvested in 2012 is presented in Table 6.1. A substantially
important role in the propagation of lupin use and breeding progress is performed
by the International Lupin Association. An excellent source of information on these
crops are proceedings of 13th International Lupin Conferences organized in differ-
ent continents and in different countries between 1980 and 2011.
What is the main factor distinguishing lupins? The process of transformation of
most species from a wild plant to a crop lasts hundreds or thousands of years. In the
case of lupin, it took only 1–2 generations of breeders. Outstanding people and their
contributions were discussed by Brummund and Święcicki (2011).

2  Origin and Systematics

The genus Lupinus belongs to the Papilionoideae subfamily of legumes where it is

classified among the tribe Genisteae within an early-branching phylogenetic clade
Genistoid (Lavin et al. 2005; Cardoso et al. 2013). It is generally accepted that
6 Lupins 181

Table 6.1   The lupin area harvested (FAOSTAT 2012, http://faostat.fao.org/)

Country Area (ha)
Argentina 110
Australia 689,064
Austria 98
Belarus 20,735
Chile 21,467
Ecuador 4500
Egypt 764
France 2553
Germany 17,900
Hungary 54
Italy 5000
Lativa 100
Lebanon 50
Lithuania 5100
Peru 9656
Poland 49,221
Russian Federation 17,800
Slovakia 80
South Africa 12,000
Spain 6700
Switzerland 49
Syrian Arab Republic 13
Ukraine 24,000
Total 887,014 (100 %)
South America 35,733 (4 %)
Australia 689,064 (78 %)
South Africa 12,000 (1.4 %)
Other Africa 764 (0.09 %)
Europe 149,390 (17 %)

Lupinus evolved out of the tropical and subtropical representatives of the primitive
papilionoid tribe Sophoreae living in tertiary (Gladstones 1998a; Lavin et al. 2005).
Lupinus is a diverse and widespread genus comprising 275 annual and perennial
species found in both lowland and mountain regions (Cardoso et al. 2013; Drum-
mond et al. 2012). There are two geographically isolated groups within the genus:
the Old World and New World lupins (Cowling 1999). The genus Lupinus origi-
nated in the Old World and subsequently dispersed to the New World (Aïnouche
et al. 2004; Drummond et al. 2012).
The group of the Old World lupins is composed of 13–15 species (Table 6.2)
found in the Mediterranean region and northern Africa (Cowling et al. 1998a;
Święcicki et  al. 1996; 1999; Pascual 2004; Gladstones 1974). These are all an-
nual herbaceous, mostly autogamous species characterized by digitate leaves (Aï-
nouche et al. 2004). Twelve Old World lupin species were described by Gladstones
(1974), and one of them, Lupinus somaliensis, is probably extinct. Two species—
182 W. Święcicki et al.

Table 6.2   Old-world lupin species list with the division according to the seed coat texture
No Species Chromosome no
Smooth-seeded group
1 L. albus L. 48
2 L. angustifolius L. 40
3 L. hispanicoluteus Swiec. et Swiec. 52
4 L. hispanicus Boiss. & Reut 52
5 L. luteus L. 52
6 L. micranthus Guss 52
Rough-seeded group
7 L. anatolicus Swiec. et Swiec.
8 L. atlanticus Glads 38
9 L. consentini Guss 32
10 L. digitatus Forsk 36
11 L. palaestinus Boiss 42
12 L. pilosus Murr 42
13 L. princei Harms 38
14 L. somaliensis Baker (extinct)
Intermediate species
15 L. mariae-josephi H. Pascual 52

Lupinus anatolicus (Święcicki et  al. 1996) and Lupinus mariae-josephi (Pascual
2004; Pascual et al. 2006)—have been fairly recently discovered, and one species—
Lupinus × hispanicoluteus (Święcicki et al. 1999)—was created as an interspecific
hybrid of two Old World lupins. The group of the Old World lupins has been fur-
ther divided into two sections according to seed coat texture: the smooth-seeded
(section Malacospermae) and the rough-seeded (section Scabrispermae) species
(Table  6.2; Gladstones 1974). Representatives of the Scabrispermae could suc-
cessfully be crossed, confirming their genetic relationship beyond similarities in
terms of morphological, chemical, and cytological characteristics (Aïnouche et al.
2004; Naganowska et al. 2003; Przybylska and Zimniak-Przybylska 1995; Gupta
et al. 1996; Wolko and Weeden 1990; Gladstones 1974). They also form a strongly
supported monophyletic group in phylogenetic analyses despite their variable ge-
nome size and chromosome number (Aïnouche et al. 2004). The smooth-seeded
lupins form a heterogeneous group of species separated by major genetic barri-
ers (Cowling et al. 1998a; Plitmann and Pazy 1984). The three cultivated lupin
species—L. angustifolius, L. albus, and L. luteus—are the smooth-seeded lupins.
Two Old World lupins do not fall into the classic division scheme. One of them is
L. anatolicus, morphologically similar to Lupinus micranthus and Lupinus pilosus
and described as a smooth-seeded species on the basis of its macroscopic seed coat
texture (Święcicki et al. 1996, 2001). The distinctness of the Anatolian lupin has
been supported by chemotaxonomical analyses, including protein, oil, and alkaloid
profiles as well as isozyme and seed globulin patterns (Przybylska and Zimniak-
Przybylska 1995; Świecicki et al. 2001). Additional studies have shown that based
on micromorphological seed coat structure, L. anatolicus is clearly related to the
6 Lupins 183

Scabrispermae section, while internal transcribed spacers (ITS) sequence data have
moreover revealed its explicit relationship to L. pilosus and Lupinus palaestinus.
At the same time, the differences of L. anatolicus ITS sequence from the latter two
species exhibiting the same ITS sequence are evident, indicating the divergence of
the Anatolian lupin (Aïnouche and Bayer 2000). On the other hand, there have also
been reports questioning the distinctness of this new species. Clements et al. (1996)
considered it a smooth-seeded accession of L. pilosus, as such a mutation was found
in the natural L. pilosus population. These authors report successful crosses be-
tween the Anatolian lupin and the related L. pilosus. The second new species, L.
mariae-josephi, is intermediate between the smooth-seeded and rough-seeded lu-
pins based on the micromorphological seed coat texture which also differs from any
known New World species. Phylogenetic data show that it is a distinct line within
the Old World lupins without clear affinity to either smooth-seeded or rough-seeded
groups. Another unique feature of this species is its occurrence which is restricted to
calcareous basic soils, exceptional within the genus (Mahé et al. 2011).
The majority of species within Lupinus occur in the New World. They are dis-
tributed in both North and South America, with the highest concentration of species
being endemic to mountain habitats (Aïnouche et al. 2004; Drummond 2008). The
New World species are both annuals and herbaceous and woody perennials with
unifoliolate or compound leaves (Aïnouche et al. 2004; Drummond et al. 2012).
Phylogenetic analyses based on nuclear and chloroplast DNA have shown that the
New World lupins exhibit the highest known speciation rate among land plants
(Hughes and Eastwood 2006; Drummond 2008). Further studies have also proved
that the rapid diversification within the genus was promoted by evolutionary transi-
tion from annuality to perenniality coinciding with colonization of montane territo-
ries and dispersion from North America to South America and Mexico (Drummond
2008; Drummond et al. 2012).

3  Varietal Groups

For ordering of species variation, a division to groups of different types is quite

often used, for example, systematic (for species with a rich variation, botanical va-
rieties are separated on the basis of monogenic differences) or genetic divisions (for
monogenic characteristics not considered in taxonomy for different reasons). In the
genus Lupinus, an additional reason are the regions of species origin and some char-
acteristics which appeared during domestication and cultivar improvement. These
divisions have a great practical importance in lupin gene resource characterization
and usage.
The first division was effected on the basis of origin and distribution regions
(New World lupins/American continents and Old World lupins/the Mediterranean
basin). The next division considers seed characteristics—size and seed coat struc-
ture. New World lupins have small seeds, with the Andean lupin ( L. mutabilis) be-
ing an exception. All Old World lupins have large seeds. These species are divided
184 W. Święcicki et al.

into two groups, on the basis of the type of seed coat—smooth seeded ( L. albus, L.
angustifolius, L. luteus, L. hispanicus, L. micranthus) and rough seeded ( L. cosen-
tinii, L. palaestinus, L. pilosus, L. atlanticus, L. princei, L. digitatus; Cowling et al.
1998a).
For increasing food and fodder value during lupin domestication, decreasing al-
kaloid content in seeds was very important. Seeds of wild lupins can contain up
to several percent in dry mass. There is no clear cut level of alkaloids from which
seeds can be used in feeding. At the beginning, a level of 0.1 % in dry mass was
accepted for fodder lupins. According to Römer and Jahn-Deesbach (1988), sweet
lupins should contain less than 0.05 % of alkaloids, but Gladstones (1988) sug-
gested 0.02 % in cultivar seeds as being reasonably safe for feeding. Today, dif-
ferent minimum levels are accepted in different countries, but the newest cultivars
contain even less than 0.01 %. This criterion still exists in practice, and lupins are
divided into two groups using popular names—high alkaloid (bitter) and low alka-
loid (sweet) lupins.
There are some other important characteristics of variety ideotype which divide
lupins into groups, for example, vegetation self-completing (restricted branching),
thermoneutrality, and the type of usage (for seeds and green forage; Święcicki and
Święcicki 1995; Święcicki et al. 2000a).

4  Genetic Resources and Utilization

The main aim of collections and gene banks is to maintain the genetic variation (to
protect against genetic erosion) of crop plants and their wild relatives. In the case of
the genus Lupinus, influencing factors are present needs.
The large number of taxa inside the genus Lupinus, for example, differentiated
regions of origin and secondary distribution, and also the environmental require-
ments of four lupin crops have led to a broad interest in these genetic resources as
a material for basic research and applied breeding. This has generated a particular
interest in the enlargement and valorization of resources as a result of the increasing
importance of lupins—a relatively new, modern crop (Brummund and Święcicki
2011). As a consequence, some large and active collection institutions work in close
contact with breeders.
For the genus Lupinus (annual and perennial species, from acaulescent to tree-
like shrubs), two centers of origin and secondary distribution are considered—
American continents (the New World—200–300 small-seeded species and one lu-
pin crop, large-seeded the Andean lupin, L. mutabilis) and the Mediterranean basin
(13–15 large-seeded species including three crops—yellow lupin/L. luteus, narrow-
leafed lupin/L. angustifolus, and white lupin/L. albus). The first collecting mission
for the genus Lupinus was the Vavilov’s expedition to the Mediterranean basin in
1926. His, and also further, collections are maintained at the Vavilov’s institute at
St. Petersburg, Russia (including original paper bags with Vavilov’s handwriting),
but the availability of information is restricted by the Russian alphabet.
6  Lupins 185

Of substantial importance for Lupinus genetic resource, users are the Interna-
tional Lupin Association, the Biodiversity/International Plant Genetic Resources
Institute, and the lupin project of the Department of Western Australia/University
of Western Australia at Perth. These organizations have strongly supported not only
the inventorization and elaboration of descriptors (IBPGR 1981) and databases but
also the collection, characterization, and valorization of samples. At the Fifth In-
ternational Lupin Conference in Poznań (Poland), in 1988, managers for collection
databases of respective lupin crops were elected (Święcicki and Leraczyk 1994).
Then, during the first meeting of the Legume Working Group of the European Co-
operative Programme (ECP)/Genetic Resources (GR) at Copenhagen (Denmark) in
1995, the manager for the European Lupinus Collections Database was elected. The
first version of this database was presented during the Ninth International Lupin
Conference in Klink (Germany) in 1999 (Święcicki et al. 2000b) and the updated
version at the meeting of the Legume Working Group of the ECP/GR in Novi Sad
(Serbia) in 2013. Permanently useful for lupin collection curators and users are
the references of Gladstones (1974) and Cowling et al. (1998a) on taxonomy, re-
gions of origin, and distribution of the Old World lupins as well as gene resource
characterization, collection gaps, and agronomy problems. The Australian lupin
project is a model example of how direct practical benefits can be obtained thanks
to investment in gene resources. One result was a dramatic increase in the narrow-
leafed cropping area from 97,000 ha in 1981 to 1,151,000 ha in 1994. Wolko et al.
(2011) summarized the world holdings of Lupinus germplasm accessions on the
basis of the International Plant Genetic Resources Institute (IPGRI) Directory of
Germplasm Collections. Data cover 36,854 lupin accessions gathered in 42 centers
in 20 countries including the Americas and Australia. For the common, passport
database of lupin collections, data from 13 European centers (57 % of accessions),
Australia, and the USA (43 % of accessions) were obtained for 13,964 accessions
(Tables 6.3 and 6.4). Table 6.4 shows the numbers of accessions gathered in respec-
tive countries and for respective Lupinus species. As many as 96 % of accessions
are collected in six countries, 76 % are lupins of the Old World and 71 % belong
to lupin crops (including wild populations as well as genotypes created by man).
Wild populations of lupin crops protected in Spain and Portugal are very important
as, in particular, is the Australian collection resulting from collecting expeditions
together with a characterization of collection sites and their environmental condi-
tions (Cowling et  al. 1998a). Table  6.4 also suggests directions and regions for
collecting expeditions and shows the restricted availability of the New World lupin
gene resources and some wild species of the Old World. On the one hand, we have
a resistance to Colletotrichum lupini and alkaloid content analyzed in hundreds of
accessions of lupin crops within Wiatrowo’s collection, and on the other hand, there
is no accession for Lupinus somaliensis or just one accession of L. anatolicus and
six accessions of L. princei.
186 W. Święcicki et al.

Table 6.3   Donor institutions of the Lupinus collections passport data

Donor institutions
AGRITEC, Ltd. Sumperk, Czech Republic CZE
INRA—Dijon, France FRA
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, DEU
Germany
Centre for Genetic Resources (CGN), University and Research Centre, Wagenin- NLD
gen, The Netherlands
Poznan Plant Breeders Ltd., Wiatrowo, Poland POL
Instituto Superior de Agronomia, Departamento de Botânica e Engenharia PRT 1
Biológica (DBEB), Tapada da Ajuda, Lisboa, Portugal
Instituto Nacional de Recursos Biologicos (INRB) INIA—Oeiras, Quinta do PRT 2
Marquês, Portugal
Estacao Nacional de Melhoramento de Plantes, Elvas, Portugal PRT 3
ISOPlexis—Banco de Germoplasma, Centro de Estudos da Macaronésia, Uni- PRT 4
versidade da Madeira, Campus da Penteada, Funchal, Portugal
Plant Production Research Center, Piešťany, Slovak Republic SVK
Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, Centro ESP
Nacional de Recursos Fitogeneticos, Alcala de Henares, Madrid, Spain
Western Regional Plant Introduction Station, Pullman, Washington, USA USA
University of Western Australia, Centre for Legumes in Mediterranean Agricul- AUS
ture, Crawley, Western Australia

5  Major Breeding Achievements

In the familiar speech of a farmer, the term “lupin” in the singular or plural is often
conversely used to name the crop. Meanwhile, there are at least four lupin crops
each with clearly differentiated characteristics, including environmental and agro-
nomic requirements. Each of them has a different number of chromosomes; they do
not cross with each other; and two are self- and two cross-pollinating. For example,
white and yellow lupin are as different as wheat and rye. As a consequence, the
progress of breeding for respective species has been different in different coun-
tries. Obviously, these crops are closely related, resulting in the chance to select a
Vavilov’s homologous order of desirable, hitherto not existing characteristics (e.g.,
the mutation of the rb gene). Seeds of a white and Andean lupin were used in the
distant past, but the real transition from a wild plant to a modern crop was achieved
by a maximum two generations of breeders (Brummund and Święcicki 2011). De-
spite the genetic improvement of the above four lupins, the domestication of some
others has also been conducted.

5.1  Andean Lupin, Tarwi ( L. mutabilis Sweet)

Breeding achievements for this crop are clearly smaller than in Old World lupin
crops. Species domestication is a more appropriate term than breeding improvement
Table 6.4   Lupinus species collected in gene banks
Species NLD SVK FRA ESP USA DEU AUS CZE POL PRT1 PRT2 PRT3 PRT4 Total
affinis 1 4 5
6  Lupins

affinus 1 1
albicaulis 5 1 6
albifrons 5 1 6
albo-coccineus 1 1
albus 13 28 253 732 347 233 979 39 362 375 305 11 3677
anatolicus   1 1
andersonii 3 3
angustifolius  8 542 190 279 2165 17 361   41 291 3894
arboreus 4 3 1 8
arbustus 13 13
arcticus 1 1
argenteus 48 48
arizonicus 2 2
arizonicus 1 1 2
atlanticus 4 4 153 166
benthamii 1 1
bicolor 11 1 4 16
bracteolaris 1 1
campestris 1 1
citrinus 1 1
coccinnus 3 3
cosentinii  17 5 11 251 5 22 311
cryptantus 1 1
densiflorus 3 1 4
digitatus 3 4 1 8
187
Table 6.4  (continued)
188

Species NLD SVK FRA ESP USA DEU AUS CZE POL PRT1 PRT2 PRT3 PRT4 Total
douglasi 1 1
elegans 2 1 3
exaltus 2 2
excubitus 1 1
formosus 1 1
garfieldensis 2 2
gibertianus 1 1
graecus 10 10
gredensis 107 107
havardii 2 2
hirsutissimus 2 1 2 5
hirsutus 2 2
hispanicus 102 45 48 98 16  3 103 415
hispanicoluteus 7 7
hybrid 5 5
hybridus lem. 1 1
interspecific cross 8 8
latifolius 7 1 8
lepidus 20 20
leucophyllus 55 1 56
liniifolius 4 4
littoralis 6 6
longifolius 1 1
luteolus 4 4
luteus 56 18 303 86 132 463 26 354 78 283 1799
W. Święcicki et al.
Table 6.4  (continued)
Species NLD SVK FRA ESP USA DEU AUS CZE POL PRT1 PRT2 PRT3 PRT4 Total
mariae-josephi H. 2 2
6 Lupins

Pascual
mexicanus 8 5 6 19
micranthus 12 20 51 1 10 94
microcarpus 10 3 13
multiflorus 1 1
mutabilis 20 79 30 221  2 17 150 519
nanus 5 3 1  1 3 13
nootkatensis 15 2 17
pachylobus 1 1
palaestinus 3 10 6 19
paniculatus 1 1
parviflorus 1 1
perennis  1 1 1 3
pilosus 61 14 189 6   1 271
polycarpus 1 1 2
polyphyllus  1 49 28 12 17 2 109
princei 6 6
pubescens 15 1 1 17
pusillus 3 3
rivularis 6 1 7
rothmaleri 58 58
rotundiflorus 1 1
sericeus 60 2 62
sparsiflorus 1 1 2
189
Table 6.4  (continued)
190

Species NLD SVK FRA ESP USA DEU AUS CZE POL PRT1 PRT2 PRT3 PRT4 Total
stiversii 3 3
subcarnosus 8 8 2 18
subvexus 1 1
succulentus 4 10 3 1 1 19
sulphureus 11 11
texensis 2 1 3
truncatus 1 6 7
variicolor 1 1 2
lupinus sp. 1 4 75 1905 13 1 7 2006
Total 69 54 254 1843 1293 2767 4665 107 1169 648 1077 11 7 13,964
W. Święcicki et al.
6 Lupins 191

(von Baer 2002). Moreover, achievements in a center of diversity (Andean coun-

tries) are different in comparison to other, particularly European countries (Römer
and Jahn-Deesbach 1988). The main, exciting advantages of the species are the high
protein (over 50 %) and oil (16–20 %) content in seeds. Disadvantages include the
high alkaloid content in seeds and late maturity under long photoperiod and humid
weather conditions. A definite difficulty in breeding is cross-pollination—above
10 % (Römer and Jahn-Deesbach 1988) or even 18 % (Gnatowska et al. 2000).
In Andean countries with extensive agriculture, the technology of seed debitter-
ing (decreasing the alkaloid content from 3 to 0.003 %) was more popular than the
breeding of low alkaloid cultivars. In these conditions, the selection of local variet-
ies was more effective, although one undoubted achievement was the breeding of
the low alkaloid (0.0075 %) cultivar Inti in Chile, than was the case with participa-
tion in breeding projects in other countries (Römer and Jahn-Deesbach 1988).
In European countries interested in the advantages of the Andean lupin, a serious
disadvantage is the indeterminate growth habit resulting in endless flowering under
humid weather conditions during the ripening period. Early maturity was looked for
in different ways—collections of genetic resources were analyzed, distant crosses
were performed (using L. albus. L. elegans, and L. polyphyllus), and mutations were
induced. The restricted branching mutant described by Römer (1994) seems to be
promising.
A comparison of Römer and Jahn-Deesbach (1988) and von Baer (2011) presen-
tations allows the following achievements in the improvement of Andean lupin to
be mentioned:
• A high protein and oil content in seeds was combined with a low alkaloid con-
tent, but this is difficult to maintain because of the polygenic inheritance and
high degree of cross-pollination,
• Hybrids L. polyphyllus × L. mutabilis (von Baer 2002),
• A valuable source of earlier maturity is the restricted branching mutant selected
and described by Römer (1994).
Despite the above achievements, valuable cultivars useful for modern agriculture
have not been bred so far. Natural species adaptation to environmental Andean con-
ditions resulted in barriers which are difficult to overcome. Andean lupin cultivars
will not quickly be able to compete with European and Australian narrow-leafed
and yellow lupin cultivars.

5.2  White Lupin (L. albus L.)

White lupin is grown on a limited area, although it could be an important fodder

crop. Seeds contain 32–36 % protein and 8–14 % oil. It requires better soil than yel-
low and narrow-leafed lupin, but seed yields are higher (3–4 t/ha). In east-central
Europe, it is used as a spring crop, but in southern countries, it is a winter crop. The
main disadvantages are the long vegetation period and susceptibility to anthrac-
nose. Long vegetation results in late harvesting, and seeds are infected by fungi and
192 W. Święcicki et al.

mycotoxins. The small acreage is a result of limited cultivar value, and vice versa
breeding is less advanced (a limited number of breeding centers) because of the lim-
ited usage of the crop. In spite of the above, hitherto breeding work has resulted in
remarkable effects. For example, a breeding project in Poland started in 1946, and
the first cultivars were released in 1965 (cv. Kali) and 1976 (cv. Kalina). In succes-
sive cultivars, that is, cv. Wat (1978) and cv. Hetman (1980), the harvest index was
substantially improved (Kubok 1988).
In white lupin breeding, some achievements should be underlined: decrease in
alkaloid content in seeds (although not as much as in narrow-leafed and yellow
lupin), improvement of harvest index (vegetative growth decreased), shortening of
vegetation period, selection of restricted branching, and partially thermoneutral mu-
tants and finally introducing these characters to cultivars (Święcicki 1986; Boersma
et al. 2007b). The newest Polish cultivars, Butan (normal growth, released in 2000)
and Boros (restricted branching, released in 2003), are suitable for modern farming,
but a radical increase of white lupin acreage depends on a substantial shortening of
vegetation and improving resistance to anthracnose (C. lupini). The difficulty lies
in the selection of both characteristics from genetic resources and their transfer to
cultivars (Cowling et al. 1998a; Adhikari et al. 2009). Work on white lupin suitabil-
ity to winter cropping in south European countries was conducted by Huyghe and
Papineau (1990). Cold resistance is the most important characteristic. Experiments
on the role of different parts of plants in cold resistance show that a large root and
root parenchyma are required. High vernalization needs are necessary but not suf-
ficient condition. The ability to acclimatize to the cold, which partially depends on
the above characteristics, is very important. The above research was extended by
Annicchiarico et al. (2011).

5.3  Yellow Lupin ( L. luteus L.)

The species is one of the most important legumes for sandy soils. N fixation, an
improvement of soil fertility and high protein content in seeds (40–46 %), caused a
large interest in east-central and northern European countries and in Western Aus-
tralia. Yellow lupin was sown for the first time in Germany in 1840 as green ma-
nure, and then it spread rapidly in Prussia and north-central Europe as a forecrop
to fertilize sandy soils and increase the yields of successive crops (Brummund and
Święcicki 2011). It appeared in modern farming after 1945 together with chemical
weed control, defoliation, and the use of combine harvesters. Improved cultivars
were adopted to two types of usage—for seeds and green mass (as green manure
and forage).
The first achievements which were important for modern breeding were the re-
moval of wild characteristics (hard seed coat and pod shattering), selection of low
alkaloid plants/seeds by Sengbusch in 1927, and then sowing 2 ha of sweet lupin
culture in 1931 (Brummund and Święcicki 2011). Great breeding progress achieved
in a relatively short period was presented by Święcicki (1986), Brummund (1988),
6 Lupins 193

and Kubok (1988). Święcicki et al. (2000a) described the range of expression and
the practical value of 43 alleles at 20 loci controlling alkaloid content, pod shatter-
ing, growth rhythm, color of plants, flowers and seed coat, plant branching patterns
and resistance to diseases and abiotic stresses. From the above review, it is possible
to select some characteristics; the introduction of which to the cultivar ideotype was
an outstanding achievement in breeding.
In the 1950s/1960s, a serious menace for yellow lupin cropping was the Fusar-
ium. Sometimes, an outbreak completely destroyed the crop in a field. Fortunately,
genetic sources of resistance controlled by the gene Fus1 were available. The first
Fusarium-resistant cultivar, Refusanova bred in Germany in 1965, became a source
of resistance in further breeding (Cowling et al. 1998b). In a relatively short time,
new resistant cultivars were introduced to cropping. This achievement should be
underlined. In the breeding of other crops, there are not so many examples of stable
resistance to a disease (that still exists), caused by a pathogen with a few races and
controlled by a single gene.
The next milestone was the introduction of thermoneutrality to cultivars. Yellow
lupin plants need vernalization in an early growth stage (2–4 °C for about 2 weeks)
to reach the generative stage and produce a high seed yield. Selected plants with
lower thermal requirements have a shorter initial growth stage, start to flower ear-
lier, and mature earlier and more uniformly (Święcicki et al. 2000a; Adhikari et al.
2012; Fig. 6.1). Thanks to a faster plant development, an escape resistance to bean
yellow mosaic virus is observed (incompatibility between aphids and host plant
development).

Fig. 6.1   Selection of thermoneutral yellow lupin genotypes (flowering plots) in late sowing con-
ditions (without vernalization)
194 W. Święcicki et al.

The cultivar ideotype used for seeds and green forage was the same for a long
period of time. In the case of seed harvesting, a disadvantage is indeterminate plant
growth. Successive branches (secondary or even tertiary laterals) cause nonuniform
seed maturation and make harvesting more difficult. In the 1970s, a spontaneous
mutation with only one main shoot and single flowers instead of branches was se-
lected (Troll 1967). This appeared to be controlled by the recessive gene rb (restrict-
ed branching). The rb gene creates a new cultivar ideotype—early and uniformly
maturing (the so-called self-completing cultivar) cropped for seeds (Święcicki and
Święcicki 1994). The strongly reduced vegetative mass (improved harvest index)
should give an increased seed yield, particularly in regions with a short vegetation
season and higher rainfall. The first rb cultivar, that is, Manru (released in Poland
1990), yielded higher than controls in state trials.

5.4  Narrow-Leafed Lupin ( L. angustifolius L.)

The reviews by Gladstones (1988), Święcicki and Święcicki (1995), and Brum-
mund and Święcicki (2011) discuss the most important achievements in the breed-
ing of this crop, particularly when we take into account the uncommon career in
Western Australia where its acreage increased from 97,000 to 1,151,000 ha between
1981 and 1994.
Similar to the yellow lupin, a crucial role was played by the selection by von
Sengbusch in winter 1927/1928 of low alkaloid seeds/plants as sources of the trait
for modern cultivars. Investigations have revealed that a low alkaloid content in
seeds is controlled by three genes: iuc, depr, and es. Suggestions of breeders and
the Australian National Health and Medical Research point to a “normal” cultivar
level of about 0.020 % for reasonable safety (Gladstones 1988). However, use of
gas chromatography in alkaloid content screening allowed cultivars to be bred with
less than 0.01 % seed dry mass.
Local varieties and the first cultivars were characterized by pod shattering, par-
ticularly in dry and hot weather during maturation and harvest. This led to as much
as an 80 % yield loss. In removing or at least clearly improving this character, two
genes were used. The gene le ( lentus) causes more elastic walls preventing their
shrinking at drying, and the gene ta ( tardus) is responsible for strong sutures.
The importance of restricted branching is differently estimated depending on
growing conditions. In the Middle East and North Europe, it is similar to yellow
lupin. In narrow-leafed lupin, there are broader possibilities because more cases
of mutation, spontaneous and included, were selected with different levels of
lateral branch reduction, controlled by alleles of the rb gene or by another locus
(Gawłowska et al. 1999). Restricted branching cultivars were bred yielding tradi-
tional but earlier and more uniform ripening (Fig. 6.2). However, the winter grow-
ing season in south-west Australia shows that a fully determinant type is too re-
stricted in its branching as ripening is premature, and the pod fills poorly.
In the review by Święcicki and Święcicki (1995), the importance of 35 genes
controlling important plant characters was presented. For example, in cv. Svalöf
6 Lupins 195

Fig. 6.2   Narrow-leafed lupin—improved harvest index and earliness of restricted branching
(right) versus traditional cultivar (left)

Böre, a spontaneous mutant was found with very early flowering and lower vernal-
ization requirements. Gladstones (1977) defined this trait as thermoneutrality con-
trolled by the gene Ku which then was introduced to Australian and Polish cultivars.
Ku cultivars have faster initial growth and show the so-called escape resistance to
cucumber mosaic virus (CMV).
At the end of the last century, the CMV and leaf spot (Stemphylium vesicari-
um) were still considered the most important diseases. Anthracnose (C. lupini) was
deemed less significant in Central Europe. Unfortunately, a highly virulent strain
of C. lupini has spread around the world in lupin (Cowling et al. 1998a). This dis-
ease appeared to be the most important in breeding because it caused a substantial
acreage decrease. Additionally, sources of resistance and pathogen biology were
unknown. Intensive work by breeders and pathologists and then crop rotation and
seed production monitoring resulted in resistant cultivars and hopefully elimination
of the epidemic.

6  Specific Goals in Current Breeding

Despite a number of common characteristics, different goals can be determined

for each lupin species. Thanks to biotechnological and molecular techniques, these
goals should be achieved faster.
196 W. Święcicki et al.

Andean lupin in regions of natural distribution (Peru, Bolivia, Equador) should

be adopted to extensive agriculture. Therefore, the morphotype of the local variety
should not be changed drastically. To decrease alkaloid content, the technological
procedure of debittering was suggested by Römer and Jahn-Deesbach (1988). It is
also possible to improve cultivars genetically using available sources of low alka-
loid content, but a high degree of cross-pollination and unmonitored wild Andean
lupin plants could cause difficulties in maintaining the stability of characteristics.
The next, important aim even for local varieties is resistance to anthracnose.
There are two ways: breeding improvement similar to narrow-leafed lupin or using
2-year-old seeds for sowing, because storing seeds for 18 month results in practical
elimination of infection (Cowling et al. 1998a).
An exciting target seems to be a European crop made from the Andean lupin.
Over 50 % protein and about 20 % oil in seeds would result in an excellent com-
ponent for feeding pigs and poultry. Unfortunately, the adaptation of this plant to
European environmental conditions, drastic improvement of the harvest index, and
resistance to anthracnose are extremely difficult aims and are unlikely to be realized
in the near future.
White lupin (32–36 % protein, 8–14 % oil, and potentially the highest yield of
European lupins) provides a more realistic possibility. The cultivar ideotype should
contain high seed yield with low alkaloid content (below 0.02 %), possibly the high-
est content of protein and oil and, most importantly, resistance to anthracnose plus
a vegetation period shortened by at least 10–14 days. Present cultivars give satis-
factory seed yield with 0.02–0.03 % alkaloids and 8–11 % oil content. The biggest
disadvantages are late maturing and susceptibility to anthracnose. An improvement
of both characteristics is the main condition for an increase in commercial crop
importance and acreage. Collections of genetic resources contain valuable initial
material for most of the required characteristics (Cowling et al. 1998a; Adhikari
et al. 2009; Kurlovich 2002). For similar results to those obtained in L. angustifo-
lius, a large project of resistance breeding against C. lupini is indispensible. More
difficult for another reason (no source of earliness) seems to be the shortening of
vegetation for earlier and more uniform ripening. A precise review of available gene
resources (Cowling et al. 1998b; Święcicki et  al. 2000b) and mutation breeding
is necessary. The gene rb (restricted branching) could be useful, as this strongly
changes the harvest index value. First rb cultivars yield about 10 % lower than tra-
ditional ones. However, this gene influences earlier and more uniform maturity. It
must be remembered that the introduction of strong, even monogenic changes into
a cultivar ideotype quite often disturbs genotype balance, and its reconstruction is
time-consuming. For example, the afila characteristic in pea was described in 1954.
Then, a period of pea cultivar improvement in Poland using the afila gene from
releasing the first, high-yielding cultivars Sum and Wasata to all afila cvs. in the
national register lasted from 1979 to 2002.
Achievement of the above mentioned goals in white lupin breeding justifies ev-
ery expense and is much more practicable than an introduction of Andean lupin to
European agriculture.
6 Lupins 197

Fig. 6.3   Left and Right, symptoms of anthracnose (Colletotrichum lupini) on lupin plants

For yellow and narrow-leafed lupin, specific goals are partially similar, but the
scale of importance is somewhat different. In yellow lupin improvement, the most
important is a resistance to anthracnose, which is clearly worse than in narrow-
leafed lupin (Fig. 6.3). Moreover, it is necessary to decrease the alkaloid content
in seeds and exclude the gramine which in some cultivars is present in substantial
quantities.
In narrow-leafed lupin breeding, an important goal is resistance to Fusarium
up to a level similar to that in yellow lupin. In both lupin crops, yield stability is
very important. Here, of undoubted influence are resistance to draught and hitherto
unknown factors causing flower abortion—the most important disadvantage for all
legumes.
Lupin fields are quite often destroyed by wild animals (stags, deer and hares).
Therefore, if possible, it would be desirable to breed cultivars with high alkaloid
content in their green mass and low alkaloid seeds.
Among the specific goals for future breeding, attention must be drawn to two
subjects. Increased yields have been hitherto achieved via improving harvest in-
dex. Without any doubt, unused reserves are available in activity/efficiency of plant
physiological processes. Additionally, mobile equipment for mass screening in the
field is available. However, in terms of seed quality, improvement of the content
of protein and antinutritional compounds (alkaloids) is considered exclusively. Ex-
perts should establish if more substances are present in lupin seeds where the im-
198 W. Święcicki et al.

provement could increase seed value or at least feeding efficiency. For example, the
most recent literature data show the particular properties of a white lupin protein in
curing diabetes and atherosclerosis.
Trials are undertaken also to domesticate new wild lupins, for example, L. atlan-
ticus, L. cosentinii, L. hispanicus, L. pilosus, or L. polyphyllus (Cordero et al. 1988;
Buirchell 2000a, b; Campos-Andrada et al. 2000; Kurlovich et al. 2008). The aim is
to use their adaptability to untypical or even extreme environmental conditions. For
example, L. pilosus is the most lime tolerant of the lupin species.

7  Breeding Methods and Specific Techniques

The domestication period of lupin crops is over. In lupin breeding, most important
is the cross-pedigree method. In a breeding procedure, a larger size of family (an
offspring of a single plant) and later beginning of selection in segregating progenies
(F3 the earliest) must be considered because of lupin polyploidy. Cross-pollination
of L. luteus and L. mutabilis causes some technical difficulties—the isolation of
single plants selected from a segregating progeny or the isolation of a whole breed-
ing material under a net tent. In the final stage of maintaining breeding, the homoge-
neity/purity of a given strain/cultivar can be maintained in a spatial isolation.
The selection of a plant morphotype, growth and development, as well as yield
creating factors is conducted in field-natural conditions. For certain characteris-
tics, it is indispensable to test samples in parallel (representing progenies sown in a
field), and these are then destroyed (e.g., estimation of alkaloid content or resistance
to pathogens with artificial infection). A pedigree scheme allows us to estimate the
value of a plant material sown in a field on the basis of the laboratory or greenhouse
testing of a sister part of the progeny.
The initial material is of substantial importance for breeding effectiveness. Col-
lections of gene resources as well as genotypes obtained in pre-breeding are con-
sidered to be a so-called breeding gene pool. Exchange between lupin companies
is not easy and is restricted by the standard material transfer agreement (SMTA).
Moreover, there are not so many lupin centers in the world. Therefore, characteriza-
tion and valorization of gene resources are important, as are convergent crossing
programs (unfortunately, these are multiyear) for combining new characteristics
on a valuable genotypic background. For example, sources of desirable and avail-
able characteristics/genes in lupin collections are given by Święcicki and Święcicki
(1995), Święcicki et al. (2000a), and Kurlovich (2002).
Sources of genetic variation are mutations and recombinations. The natural vari-
ation of lupins is not too broad from a breeding point of view. Therefore, in the
event of a lack of desirable characteristics, a useful tool is mutation induction. For
an efficient project of mutation breeding, the introductory work is important—mu-
tagene and dose optimization and suitably large pedigree population for mutant
selection (= a number of families/offsprings of treated seeds × number of plants per
family). Because of lupin polyploidy, the number of plants per family must be big-
6 Lupins 199

ger than for diploids, and the selection should start in M3, at the earliest. In the
case of looking for one particular characteristic, it is possible to sow a mixture in
a suitable generation (instead of a pedigree population), but when a mutant is low
yielding or lost, there will be no possibility of its reselection.
Different genotypes have been treated by different mutagenes in lupins, and dif-
ferent results have been obtained (Klamroth et al. 2011; Rudloff 2011; Stawinski
and Rybinski 2000; Micke and Święcicki 1988; Święcicki and Olejniczak 1999).
The most spectacular seems to be a selection of restricted branching mutants—
induced in Andean, white, and narrow-leafed lupins (Micke and Święcicki 1988;
Römer 1994; Gawłowska et al. 1999) and spontaneous in yellow and narrow-leafed
lupin (Gawłowska et al. 1999; Troll 1967).
In lupin breeding, similar to other crops, mass-screening and selecting tech-
niques are very important. These techniques should accept micro samples and not
destroy them. Desirable is also mobile field equipment allowing selection in early
stages of plant growth. For certain characteristics (e.g., resistance to diseases and
unfavorable environmental conditions), selection is often conducted under condi-
tions with a great intensity of a stress factor (10 years of continually cropping lupins
or very late sowings).
Lupins have their own, particular environmental requirements and show weak
growth in greenhouse conditions including in vitro regeneration. Therefore, effec-
tive methods to shorten multiyear breeding cycles are not available. Surma et al.
(2013) tried to elaborate the single-seed descent (SSD) technique using in vitro cul-
tures of lupin embryos ( L. luteus and L. angustifolius). It appeared that lupin roots
and shoots grow well in vitro, but further plantlet acclimatization ex vitro is rather
difficult. Nevertheless, it is possible to obtain 2.5–3 generations per year.
In recent years, a lot of results have been published on the possibilities of using
molecular markers in the selection of agricultural characteristics (see the next sec-
tion).

8 Integration of New Biotechnologies in Breeding

Programs

8.1 Biotechnology

The progress in lupin breeding is undeniable, although many problems affecting

yield still apply and could be solved by combining genotypes with desired traits
in specific sexual hybridization between species (Rybczynski and Podyma 1993a).
The high level of species incompatibility within the genus Lupinus significantly
limits the production of viable seeds through interspecific crosses using conven-
tional hybridization methods (Święcicki et al. 2000a). Employment of biotechnol-
ogy in contemporary breeding programs may allow the creation of new germplasm,
significantly broadening the genetic variability. Plant tissue culture is also useful in
200 W. Święcicki et al.

vegetative propagation to increase the number of individuals with the same geno-
type. Although research towards tissue culture manipulation and transformation has
long been conducted, in the case of lupins, the scope of achievements is still lim-
ited (for a review, see also Sator (1990), Atkins and Smith (1997), Święcicki et al.
(2000a), and Wolko et al. (2011)).
Like many large-seeded legumes, lupins are considered difficult to manipulate
in culture (Nadolska-Orczyk 1992). The significant variation in the morphogenic
potential of lupin species has been emphasized by Rybczynski and Podyma (1993b)
and Zgagacz and Rybczynski (1996). The key requirement for successful plant tis-
sue culture is an optimal media composition and a protocol developed for the spe-
cies/genotype of interest. Therefore, the attention of many scientists has been drawn
to the adjustment of proper culture conditions to the culture aim. The research un-
dertaken concerned mainly micropropagation, somatic embryogenesis, embryo
rescue, protoplast culture, androgenesis and transformation. Various lupin explants
derived from different stages of plant ontogenesis have been analyzed.
Experiments on lupin plant restoration ( L. albus) were started by Ball (1946,
1960). Sroga (1983) successfully produced callus for L. angustifolius hypocotyl
explants which was further used for the induction of suspension cultures. The re-
search on regeneration from leaf and hypocotyl explants of L. polyphyllus, L. hart-
wegii, L. angustifolius, and L. luteus resulted in successful callus induction but very
limited plant regeneration (Sator 1985b). In contrast, organogenesis into complete
plants via callus culture in L. angustifolius and L. polyphyllus was reported by Sroga
(1987), although these results could not be repeated by Nadolska-Orczyk (1992).
Callus culture formation from seedling explants of L. mutabilis was also investigat-
ed by Phoplonker and Caligari (1993) who, by testing different culture conditions
and protocols, concluded that stem and hypocotyl explants produced the greatest
quantity of callus.
Regeneration and propagation of plants bypassing callus formation have also
been explored in lupins. Micropropagation of L. texensis hypocotyl explants has led
to shoot formation while the rooting is rather weak (Upadhyaya et al. 1992). On the
other hand, complete, blooming and seed-setting plants of L. luteus have been ob-
tained from hypocotyl segments (Daza and Chamber 1993). Rybczynski and Pody-
ma (1993a) regenerated root systems of the shoot tip explants of L. albus, L. luteus,
L. angustifolius, L. hispanicus and L. polyphyllus. Vegetative propagation based on
apical meristem cultures was successful for L. hispanicus and using cotyledonary
node explants for L. hispanicus and L. albus. The plantlets were further successfully
adapted to in vivo conditions (Rybczynski and Podyma 1993a). The complete long-
term micropropagation protocol for the four lupin crops ( L. angustifolius, L. albus,
L. luteus, and L. mutabilis) through axillary meristem multiplication was fairly re-
cently reported (Pniewski et al. 2002). Regenerated shoots could be cultured for at
least ten passages in good plant conditions. Moreover, further rooting experiments
carried out on material from the second regeneration round demonstrated the good
rooting ability of all the species. Since the potential of root formation decreased in
succeeding regeneration rounds, grafting was performed as an alternative method of
transferring plants to greenhouse conditions (Pniewski et al. 2002).
6 Lupins 201

In vitro plant multiplication might also be carried out through somatic embryo-
genesis. Successful direct somatic embryogenesis from immature cotyledons of L.
angustifolius, L. albus and L. mutabilis has been achieved, although the authors
give no details on plant regeneration (Nadolska-Orczyk 1992). Similar studies have
been carried out for L. albus, L. luteus, L. angustifolius and L. hispanicus presenting
effective somatic embryogenesis for L. albus only; however, the obtained plant-
lets were further easily adapted to greenhouse conditions (Rybczynski and Podyma
1993b). The response of immature embryo-derived explants to different culture me-
dia was also tested by Zgagacz and Rybczynski (1996). The experiment performed
on L. albus, L.mutabilis and L. hispanicus showed that the most responsive part of
the immature embryo was cotyledons and depending on the species only somatic
embryos or shoots could be observed in culture (Zgagacz and Rybczynski 1996).
Embryo rescue culture might be an alternative approach to overcome crossabili-
ty barriers. Several attempts at embryo rescue and its modification have been under-
taken in the case of lupins. An embryo rescue protocol and culture condition have
been evaluated for L. luteus, L. angustifolius, L. albus, L. mutabilis and L. polyphyl-
lus as a basis for future development of interspecific hybrids (Wilson et al. 2008;
Kasten and Kunert 1991). In the trial of interspecific crosses between L. albus, L.
mutabilis and L. angustifolius, hybrid embryo rescue was carried out and its fur-
ther in vitro culture led to the development of plants (Przyborowski 1997). Embryo
rescue has also been applied to successfully produce F1 plants of L. mutabilis × L.
hartwegii (Schaefer-Menuhr et al. 1988; Clements et al. 2008; Kasten et al. 1991),
L. angustifolius × L. luteus (Kasten et al. 1991) as well as L. mutabilis × L. mexicans
and L. arizonicus (Clements et al. 2008).
In vitro research in lupins also targets protoplast culture, focusing not only on
plant regeneration attempts but also on protoplast fusion. Babaoglu (2000) inves-
tigated various cultivation systems and factors on the yield, viability and division
of protoplasts from L. mutabilis, but the progress in determining optimum explant
and culture conditions was limited. Sinha et al. (2003/2004) optimized the proto-
col for routine production of highly viable cotyledonary protoplasts of white lupin
and further elaborated the plating environment stimulating protoplast elongation
and division (Sinha and Caligari 2005). Their results established benchmarks for
future regeneration and hybridization studies. The procedure for protoplast isola-
tion derived from hypocotyls, cotyledons and young leaves, as well as the effects of
various culture conditions on protoplasts development have also been elaborated for
yellow lupin by Wiszniewska and Pindel (2009, 2013). These authors also reported
protocolony formation in yellow lupin protoplast culture (Wiszniewska and Pindel
2009). Successful protoplast fusion of L. angustifolius and L. subcarnosus as well
as subsequent shoot regeneration of hybrid callus was recently reported by Sonntag
et al. (2009).
Androgenesis plays an important role in double haploid (DH) line development.
Fast recovery of fully homozygous inbred lines could significantly accelerate mod-
ern breeding in new cultivar production (Ormerod and Caligari 1994). Up until now,
several studies on androgenesis induction in lupins have been reported, but no DH
lines have yet been produced (Skrzypek et al. 2008). Callus from another culture
202 W. Święcicki et al.

of L. polyphyllus was obtained by Sator et al. (1983). Unfortunately, the chromo-

some count of plantlets regenerated afterwards showed their diploid characteristic
(Sator 1985a). Ormerod and Caligari (1994) presented the results of spontaneously
released microspores in liquid medium of L. albus anthers and further regeneration
of embryo-like structures from microspores. A protocol for potentially large-scale
production of L. albus and L. angustifolius pro-embryos (multicellular structures)
has also been reported (Bayliss et al. 2004). Androgenetic induction to anther-de-
rived callus of L. luteus, L. angustifolius, and L. albus has been obtained, however,
no plant regeneration has been achieved (Skrzypek et al. 2008). Further studies to
determine the optimum conditions for another culture initiation in L. angustifolius
have concerned not only the localization and size of buds, color, and size of anthers
but also the influence of different factors on microspore embryogenesis induction
(Kozak et al. 2012). Moreover, for all tested L. angustifolius genotypes, callus could
be obtained that further continued its growth and produced roots (Kozak et al. 2012).
An alternative method to DH aiming at receiving homozygous lines is the “SSD
technique,” which might significantly shorten the breeding cycles. A combination
of the SSD method and in vitro culture of embryos was recently proposed for lupins
(Surma et al. 2013). As a first step towards that goal, culture conditions for L. an-
gustifolius and L. luteus embryos dissected from fully developed, green seeds have
been established. Regeneration of shoots and roots has been observed. Moreover,
some of the regenerated plants have survived the transfer to greenhouse conditions
where they flowered and set pods/seeds. These results constitute a very optimistic
step towards the rapid attainment of succeeding generations via the SSD technique
(Surma et al. 2013).
Transformation systems might serve as a method of direct introduction of agro-
nomically useful genes into the genomes of interest. Several successful transfor-
mation protocols have been published for lupins. Mugnier (1988) obtained hairy
root cultures of L. albus and L. pollyphyllus by inoculation with Agrobacterium
rhizogenes. Routine transformation of L. angustifolius to herbicide resistance was
performed by Pigeaire et al. (1997). The shoot apices transformation was medi-
ated by Agrobacterium tumefaciens carrying the bar gene, and transformants were
regenerated from axillary buds (Pigeaire et al. 1997). T1 and T2 plants were stable
in a glasshouse, and molecular analyses confirmed foreign gene integration and
its stable expression (Pigeaire et al. 1997). A trial at improving narrow-leafed lu-
pin nutritive value by means of expressing a sulfur-rich, sunflower seed albumin
(SSA) gene was undertaken by Molvig et al. (1997). The authors demonstrated
a stably transformed line with an SSA gene expressed at high level in seeds. The
SSA accounted for 5 % of the extractable seed protein, and the conducted feeding
trial showed an increase in the nutritive values of transgenic versus wild-type seeds
(Molvig et al. 1997). A. tumefaciens-based transformation has also been attempted
to improve narrow-leafed lupin resistance to fungal pathogens (Wijayanto et al.
2009). Several transgenic lines expressing the anti-apoptotic baculovirus gene p35
have been characterized with reduced disease symptoms (Wijayanto et al. 2009).
An attempt to increase narrow-leafed lupin yield by preventing flower abortion was
also undertaken with the aid of transgenic approach (Atkins et al. 2011). Lines sta-
6 Lupins 203

bly transformed with a gene involved in cytokine synthesis from A. tumefaciens

were obtained, and some of the transgenic lines were characterized by a higher pod
number (Atkins et al. 2011). The first transgenic plants of L. mutabilis were reported
by Babaoglu et al. (2000). A. tumefaciens-mediated transformation of shoot apices
led to the development of kanamycin-resistant plants expressing β-glucuronidase
(Babaoglu et al. 2000). Transgenic L. luteus plants with herbicide resistance were
obtained by infection of apical meristem explants with A. tumefaciens (Li et al.
2000). Agrobacterium-based transformation of yellow lupin was also optimized by
Pniewski et al. (2006) with the aim of a callus/tumor tissue generation capable of
hepatitis B virus (HBV) surface antigen production useful in oral immunization.
Achievements in genetic transformation have also been attained in the case of white
lupin. In the course of study on the formation and function of cluster roots formed
as a consequence of adaptation to phosphorous deficiency, A. rhizogenes-mediated
root transformation systems were established (Uhde-Stone et al. 2005; Cheng et al.
2011).

8.2  Molecular Breeding and Marker-Assisted Selection

Field-based selection for agricultural traits is often time-consuming, inconvenient,

or even simply not possible. Marker-assisted selection (MAS) is a very promising
tool to improve efficiency and accelerate selection in breeding programs, although
its potential is still under-evaluated. The prospect of being able to select desirable
lines based on genotype using simple molecular markers at the seedling stage in
early generations and parallel screening of multiple traits is very attractive to plant
breeders (Young 1999; Holland 2004).
Development of molecular markers linked to genes controlling desirable traits is
the main challenge in MAS. In order to routinely use a molecular marker in breed-
ing selection, the following requirements should be met: (1) the marker is closely
linked or co-segregates with a gene of interest, (2) the screening techniques should
be efficient in a large population and highly reproducible and (3) the application
of the marker should be more economical and user friendly than conventional se-
lection methods (Gupta et al. 1999). Nowadays, simple polymerase chain reaction
(PCR)-based markers, sequence-tagged microsatellite site (STMS), sequence-char-
acterized amplified region (SCAR), sequence-tagged site (STS), and allele-specific
PCR (AS-PCR) markers best conform to the marker requirements for selection in
plant breeding (Wolko et al. 2011).
In recent years, a large expansion of next-generation sequencing (NGS) tech-
nologies enabling polymorphism identification and marker development can be
observed. NGS methods have also been successfully applied for lupins including:
restriction-site associated DNA sequencing—RAD-seq (Yang et al. 2012, 2013a)
and transcriptome sequencing—RNA-seq (Parra-Gonzalez et al. 2012). A great ad-
vantage of using NGS technology is the possibility of the easy conversion of candi-
204 W. Święcicki et al.

date markers into simple and inexpensive PCR-based markers useful in molecular
breeding (Yang et al. 2012).

8.2.1  Narrow-Leafed Lupin

Narrow-leafed lupin, as the most popular lupin crop, is characterized with rich
molecular achievements including draft genome sequence (Yang et al. 2013b) and
well-saturated genetic maps (Nelson et al. 2006, 2010; Kroc et al. 2014; Yang et al.
2013b). In that species, the microsatellite-anchored fragment length polymorphism
(MFLP) technique plays a pivotal role in selection marker development (Yang et al.
2001). MFLP markers have been used in conversion into many simple PCR-based
markers for MAS (Li et al. 2011, 2012b; Yang et al. 2008, 2010; Boersma et al.
2007b). Moreover, NGS technologies, especially the RAD-sequencing approach,
have also recently been applied in the process of marker generation (Yang et al.
2012, 2013a).

8.2.1.1  Pod Characteristics

The trait of the pod-shattering in narrow-leafed lupin is regulated by two major

genes: lentus and tardus (Święcicki and Święcicki 1995). Only plants possessing
recessive alleles of both genes are fully non-shattering, since each of them controls
only partially reduced pod shattering. The recessive allele of the lentus gene modi-
fies the orientation of the sclerified endocarp of the pod, which manifests itself in
a change in internal pod pigmentation. This modification results in a reduction of
torsional forces upon drying and causes reduced pod shatter (Gladstones 1967).
Three simple PCR-based molecular markers closely linked to lentus gene have been
developed for narrow-leafed lupin (Boersma et al. 2007c; Li et al. 2012b). Two of
them are dominant markers (LeM1 and LeM2), unable to discriminate between ho-
mozygous and heterozygous genotypes (Boersma et al. 2007c). Recently, Li et al.
(2012b) reported the development of a new codominant marker (LeLi) flanking the
lentus gene. This marker is more useful than the previous ones in the discrimination
of heterozygous genotypes in the lentus locus. The overall matching rate between
the LeLi marker genotype and plant phenotype was 60.67 % on tested material,
including Australian cultivars and accessions of the Australian Lupin Collection (Li
et al. 2012b).
The second gene necessary to determine the fully non-shattering effect in nar-
row-leafed lupin is the tardus gene. The recessive allele of this gene affects the
sclerenchyma strips of the dorsal and ventral pod seams, fusing the two pod halves
in a way that their separation is impeded. Its expression is dependent on environ-
mental factors, especially temperature and humidity (Gladstones 1967). In 2009,
Boersma et al. (2009) developed three locus-specific markers (TaM1, TaM2 and
TaM3) closely linked to the tardus gene. These markers showed only a 24–39 %
6 Lupins 205

correlation between plant phenotypes and marker genotypes on wild accessions of

the Australian Lupin Collection, so further development of markers linked to the
tardus gene was still needed. A new codominant PCR-based marker flanking tardus
gene was designed by Li et al. (2010). The marker TaLi had a very high association
(94 %) between marker genotype and phenotype on tested domesticated cultivars
and the lupin core collection and may be widely used for selection in breeding pro-
grams (Li et al. 2010).

8.2.1.2  Structure of Seed Coat

A hard seed coat, impermeable to water, prevents germination and causes dormancy
of seeds. This adaptation is a very important strategy, ensuring species survival, es-
pecially to long drought periods lasting for several months (Święcicki and Święcicki
1995). A single recessive gene, mollis, resulting in soft seed coat development has
been identified so far (Mikołajczyk 1966). Up until now, two PCR-based markers
flanking the mollis gene have been designed (Boersma et al. 2007a; Li et al. 2012a).
The first one (MoA) was a codominant SNP-based marker, although the detection
method based on single-strand conformation polymorphism (SSCP) was very time-
consuming and the marker was not routinely used in MAS (Boersma et al. 2007a). A
recently published molecular marker (MoLi) closely linked to the mollis gene had a
very high correlation (91.3 %) between phenotypes and marker genotypes on tested
Australian cultivars and accessions of the core collection. Moreover, the marker
MoLi is a codominant, length-polymorphism marker, which makes its detection
more effective and the marker in general more useful in narrow-leafed lupin large-
scale selection (Li et al. 2012a).

8.2.1.3  Earliness of Flowering

Many of the wild type narrow-leafed lupins require vernalization in early growth
to promote flowering (Gladstones 1970). This requirement is not desired in do-
mesticated cultivars, especially in areas with short growing seasons. It is therefore
necessary to introduce a single dominant Ku gene responsible for the early-flow-
ering effect in worldwide modern lupin breeding (Brien et al. 2000; Gladstones
and Hill 1969). A sequence-specific, size-based marker (KuHM1) closely linked
to the Ku gene was reported by Boersma et al. (2007b). A correlation between the
markers for genotype and phenotype was tested only on parental lines and 106 F8
recombinant inbred lines (RILs) of the narrow-leafed lupin mapping population
(83A:476 × P27255) and it showed perfect compatibility (Boersma et al. 2007b).
Unfortunately, a matching test has not been carried out on cultivars and accessions
of the lupin core collection, so the application of the KuHm1 marker in breeding
selection is limited.
206 W. Święcicki et al.

8.2.1.4  Alkaloid Content

A characteristic trait of lupins is a production of quinolizidine alkaloids. Due to

their toxicity and bitter taste, a decrease in alkaloid content is especially impor-
tant for propagation of lupins in animal feed and human consumption (Gladstones
1998b). Three recessive genes—iucundus, esculentus, and depressus—controlling
low alkaloid content are known in narrow-leafed lupin (Święcicki and Święcicki
1995; Kurlovich 2002). It is also recognized that alkaloid content is dependent on
environmental factors such as weather or growing conditions (Barbacki 1952) and
soil nutritional imbalances including phosphorus and potassium deficiency (Gremi-
gni et al. 2003). Conventional measurement methods assessing general alkaloid
content only (high/low) require the usage of the Dragendorff reagent (Harrison and
Williams 1982). On the other hand, more sensitive gas chromatography enables
both quantitative and qualitative alkaloid estimation, but due to the equipment re-
quirements and quite long procedure it is not widely used by breeders (Ruiz 1978;
Wink et al. 1995).
The major gene regulating seed alkaloid content is the iucundus gene (Gremigni
et al. 2003). The first sequence-specific size-based marker (IucLi) closely linked to
the iucundus gene was reported by Li et al. (2011). Application of this codominant
marker enables the recognition of homozygous and heterozygous genotypes, which
is not possible using conventional breeding methods. Reported compatibility be-
tween genotypes and phenotypes of tested cultivars and accessions of the Australian
Lupin Collection was 88.67 % (Li et al. 2011). The number of “false-positive” re-
sults encountered in the analyzed material indicates that the marker is not anchored
but only linked to the iucundus gene, which may limit its routine application in
breeding selection (Li et al. 2011).

8.2.1.5  Fungal Disease Resistance

Anthracnose caused by the fungal pathogen C. lupini is a worldwide problem in

lupin cultivations, with yield losses up to 30 % (Nirenberg et al. 2002). Therefore, a
creation of anthracnose resistant cultivars is one of the most important aims in lupin
breeding. One single dominant gene Lanr1 determining resistance to anthracnose
has so far been identified and extensively applied in Australian breeding programs
(Yang et al. 2004).
To date, three MFLP-derived, sequence-specific markers (AntjM1, AntjM2, and
AnManM1) linked to Lanr1 gene have been developed (Yang et al. 2004, 2008;
You et al. 2005). Unfortunately, a certain genetic distance between the markers and
the gene of interest has often caused “false-positive” results, thus limiting their us-
ability (Yang et al. 2008). Recently, Yang et al. (2012) reported five new sequence-
specific PCR-based markers (AnSeq1, AnSeq2, AnSeq3, AnSeq4, AnSeq5) tightly
linked to the Lanr1 gene developed using the RAD-sequencing approach. Three of
those new markers (AnSeq1, AnSeq3, and AnSeq4) were more closely linked to the
6 Lupins 207

target gene than all those previously published and replaced former markers in the
Australian national lupin breeding programs (Yang et al. 2012).
Another important disease of lupins is phomopsis stem blight (PSB) caused by
the fungal pathogen Diaporthe toxica (Shankar et al. 1996). Two PSB-resistance
genes—Phr1 (breeding line 75A:258) and Phr2 (Merrit cultivar)—are known
(Shankar et al. 2002). The Phr1 gene has been found to be flanked by two MFLP-
derived PCR-based markers (Ph258M1 and Ph258M2; Yang et al. 2002). Owing to
the fact that this gene has not yet been integrated into commercial cultivars in Aus-
tralia, further PBS-resistance sources and linked markers are still desirable (Yang
et al. 2013a).
In 2013, Yang et al. developed seven new simple PCR-based markers (PhtjM1,
PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7) using RAD-sequencing
technology. One of these markers (PhtjM3) was co-segregating with the putative
PSB-resistance gene R (cultivar Tanjil) and the other six were closely linked to this
gene. All the new markers were tested on historical and current Australia cultivars.
The marker PhtjM3 showed high compatibility with resistant cultivars, but had sev-
eral “false-positive” results. Three other markers (PhtjM4, PhtjM5, and PhtjM7)
showed perfect correlations (100 %) between markers for genotype and plants phe-
notype, however, these require further verification on the wider gene pool, includ-
ing accessions of the Australian Lupin Collection (Yang et al. 2013a).

8.2.2  White Lupin

The research towards marker development for practical MAS is much less advanced
in white lupin. Up until now, only four MFLP-derived simple PCR-based markers
closely linked to agricultural traits have been reported (Lin et al. 2009; Yang et al.
2010).

8.2.2.1  Alkaloid Content

In the white lupin, nine recessive genes—exiguus, mitis, nutricius, pauper, primus,
quintus, reductus, suavis, and tertius—determining low alkaloid content are known
(Kurlovich 2002); however, all the Australian cultivars possess the pauper gene for
maintaining low alkaloid levels (Lin et al. 2009). One sequence specific codomi-
nant PCR-based marker (PauperM1) linked to the pauper gene conferring low alka-
loid content was reported by Lin et al. (2009). This marker was able to distinguish
the pauper gene from the other low alkaloid genes exiguus and nutricius, however,
during the marker validation process, a few “false positive” were also observed (Lin
et al. 2009). Furthermore, the pauper marker detection format demands the use of a
radioisotope or fluorescence labeling primers, which precludes its routine applica-
tion in MAS.
208 W. Święcicki et al.

8.2.2.2  Anthracnose Resistance

Field disease resistance tests have suggested that anthracnose resistance in the white
lupin is polygenically controlled. Moreover, the influence of environmental fac-
tors has also been observed (Yang et al. 2010). Two quantitative trait loci (QTLs)
controlling anthracnose resistance have been identified on two linkage groups, LG4
and LG17, explaining 31 and 26 % of the phenotypic variance (Phan et al. 2007). In
2010, Yang et al. (2010) developed three codominant sequence-specific PCR-based
markers (WANR1, WANR2, and WANR3). The marker WANR1 was linked to a
major anthracnose resistance QTL, while the second QTL was flanked by markers
WANR2 and WANR3. Utilization of these three markers jointly explains 53 % of
phenotypic variation and has been applied in MAS since 2008 in Western Australia
(Yang et al. 2010).

9  Seed Production

Seed production is a part of the seed industry in which several institutions and
organizations are involved. There are breeding and seed companies, organizations
(e.g., STV in Germany, SICASOV in France and the Seed Agency in Poland) which
monitor royalties accrued by seed companies on behalf of breeders, and seed pro-
ducers and institutions dealing with cultivar registration and protection as well as
accepting seeds as sowing material. The breeder has an exclusive right to the culti-
var, its multiplication, seed processing, selling, export/import and storage and can
authorize a seed company to multiply, process, and sell cultivars. However, the aim
of seed production is to transfer a biological progress from a cultivar (breeder’s
seeds) to seeds obtained by a farmer. Breeder’s seeds are multiplied into pre-basic
(PB), basic (B) and then qualified seeds (C1), and sometimes to C2. Cultivar and
sowing material usage are monitored by the respective institutions and regulations
at a national and international level. One of them, the Organisation for Economic
Co-operation and Development (OECD), defines the rules for seed trade. The In-
ternational Union for the Protection of New Varieties of Plants (UPOV) establishes
rules for cultivar usage (the cultivar is the exclusive property of a breeder with
two exceptions: (1) it can be used for crossings, (2) the farmer is allowed to use
seeds produced by himself on his own field by paying half of the requisite royal-
ties) and, among other activities, elaborates guidelines for the conduct of tests for
the distinctness, uniformity and stability (DUS) of the cultivars of a given species.
The Community Plant Variety Office (CPVO) in Europe adjudicates on the law to
protect cultivars and manages its own catalogue, and the International Seed Testing
Association (ISTA) elaborates methods for laboratory seed testing. However, regu-
lations related to the production and quality of seeds (for field and laboratory use)
obliging in seed turnover are determined in the EU by other appropriate guidelines.
The European Seed Certification Agencies Association (ESCAA) also partici-
pates in the regulation of the seed industry. The ESCAA states that the lupin seed
6 Lupins 209

production area in Europe in 2013 was about 6330 ha with Poland and Germany
being the biggest producers. Unfortunately, no data are available on east European
countries (Belarus, Russia, Ukraine) or Australia where large breeding projects ex-
ist and where the total harvested area of lupin in 2012 was 20,735, 17,800, 24,000
and 689,064 ha, respectively (the Food and Agriculture Organization Corporate
Statistical Database, FAOSTAT, Table 6.1). There are further countries with larger
areas of lupin harvest, for example, Chile (21,467 ha), South Africa (12,000 ha),
Peru (9656 ha), Spain (6700 ha), and Lithuania (5100 ha) giving a global harvested
area of lupin of about 900,000 ha. To sow this area, 10 % (about 90,000 ha) needed
to be set aside for seed production.
UPOV guidelines to estimate the DUS of three European lupins (Andean lupin
is not considered) cover 20 quantitative and qualitative characteristics describing
plant alkaloid content, the color of leaves, stems, flowers and seeds, the size of
leaves, pods and seeds, growth type and plant height in different growth stages, the
height of the insertion of the first inflorescence and the earliness of some phases
of plants growth and development. The technical questionnaire for cultivar regis-
tration as a rule covers fewer characteristics (alkaloid content, color of stem and
flower wings, term of flowering, and growth type) and additional factors in which a
new cultivar differs from similar ones. These characteristics are inherited in differ-
ent ways, can be mono- or polygenic and different variants of a characteristic can
be controlled by multiple alleles of one locus. The influence of the environment on
character expression can be different, and the mode of inheritance of all lupin char-
acteristics is not yet known (Święcicki and Święcicki 1995; Święcicki et al. 2000a).
Investigations into the expression of a single gene in segregating progeny show how
strong the influence of genotypic background and/or environment can be on a given
character expression controlled by a single allele, even in the case of a marker gene/
character (Święcicki 1989, 2001). However, for obvious reasons, cultivar DUS es-
timation carries substantial importance in field as well as laboratory classification.
The above should be considered by a breeder before cultivar registration to avoid
complications during the qualification of seed materials. For example, in lupins,
difficulties can be caused by the distribution and density of ornamentation on seed
coats. These are characteristics which are very difficult (sometimes impossible) to
keep uniform, even when inherited from one gene. Sunshine can influence their
expression (color intensity and density of ornamentation). Pod position in relation
to sunshine causes a different ornamentation on both seed sides. Ornamentation
uniformity can be more complicated when a cultivar genotype includes additional,
pleiotropic genes. A similar phenomenon exists in pea (e.g., expression of genes
Obs, U, F, Fs; Blixt 1972) and probably in other legumes with colored seed coats.
Here, a question arises as to whether marker characteristics for a cultivar should be
considered in the breeding process. For example, an untypical color for a seed coat
can be a useful cultivar marker, but it would be very convenient to have all cultivars
with white, non-ornamented seed coats.
In the estimation of growth type (determinate–indeterminate), the mode of in-
heritance of the restricted branching characteristic and its differing expression in
210 W. Święcicki et al.

a respective lupin crop (particularly in narrow-leafed lupin) must be considered

(Gawłowska et al. 1999).
UPOV guidelines divide lupin cultivars into two groups based on the presence of
bitter substances (alkaloids) in seeds—with or without—listing the example culti-
vars for a given lupin species. For the narrow-leafed lupin, an example cultivar with
bitter substances is Azuro, and without such substances—Bordako. The division is
based on the grain-cut method (Eggebrecht 1949). Current lupin breeding achieves
the lowest possible alkaloid content based on gas chromatography. This method
shows that the bitter cv. Azuro contains 1.381 % alkaloids in seeds and sweet cv.
Bordako 0.016 %.
Precise requirements are elaborated for producing/multiplying lupin sowing ma-
terial. They define the lowest seed grade (C/2), the number of field inspections
(first—in full flowering, second—in pod setting), type of forecrop (minimum 3
years break in lupin cropping), spatial isolation (200 m for basic seeds of a cross-
pollinating yellow lupin and 2 m minimum as a technological belt for self-polli-
nating narrow-leafed lupin), cultivar (1 plant/30 m2 of basic material) and species
purity, the presence of weeds (qualifying fields should be free from weeds, the seeds
of which are difficult to remove during purification process), and the presence of
diseases and pests. The monitoring of the spatial isolation in a cross-pollinating
yellow lupin is very important for maintaining the DUS. However, there are no pos-
sibilities for crossings between fields with different lupin species (spatial isolation
is not needed). The interspecific hybrid between yellow and narrow-leafed lupin
would be a sensation and success, most probably worth the disqualification of the
seed material. In the case of diseases and pests, there are general requirements (a
disqualification can be justified when the presence of diseases and pests reduces
lupin seed development or makes field inspection difficult) or more precise ones for
dangerous fungi diseases (anthracnose, fusariose) defining the maximum number of
infected plants or seeds. It is worth considering that to protect against C. lupini in-
fection, it is sufficient to sow 2-year-old seeds. According to Cowling et al. (1998a),
storing seeds for 18 months results in the practical elimination of infection.
A difficulty in lupin seed production is the maintenance of the high energy of
seed germination. Before harvesting, it is necessary to avoid overdrying seeds, for
example, when in branching cultivars one waits too long for seeds to ripen on sec-
ondary branches, seeds on the main stem will be much too dry (overdried) and
sensitive to mechanical damage during threshing and cleaning.
The above section has provided some conclusions on the current state of lupin
seed production. Global lupin seed production is rather limited; at least, no precise
data are available. It is important to consider that there are three different lupin
crops (narrow-leafed, white and yellow lupin, no data on Andean lupin seed produc-
tion in South America) and they do not cross with each other and to some extent
have different biology and genetics. This must be considered in cultivar description
and DUS estimation. An excellent example of using genetics in enhancing the pre-
cise identification and classification of cultivars was presented for pea by Winfield
and Green (1986).
6 Lupins 211

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Chapter 7
Cowpea

Ousmane Boukar, Christian A. Fatokun, Philip A. Roberts, Michael

Abberton, Bao Lam Huynh, Timothy J. Close, Stephen Kyei-Boahen,
Thomas J.V. Higgins and Jeffrey D. Ehlers

1 Introduction

Cowpea ( Vigna unguiculata (L.) Walpers) is a commonly grown and consumed

grain legume in sub-Saharan Africa (SSA). It is particularly well adapted to the dry
savanna region of SSA where many other crops could fail or perform very poorly
due to water stress caused by irregular and short duration rainfall as well as poor
soil fertility. The grains, which are the main product of the crop, contain between
22 and 30 % protein thus making it a good source of quality food especially among
the rural dwellers and urban poor. Cowpea grains are consumed in different forms.
They are eaten boiled, fried (as akara), or steamed (as moi moi). In addition to the
high protein content, cowpea grains are high in complex carbohydrates. Cowpea
haulms (dried leaves, stems, and pod walls) are also a good source of quality fodder
for livestock especially ruminants. In some parts of East Africa, notably Kenya and
Tanzania, young succulent leaves of cowpea, also characterized by high protein and
mineral nutrient contents, are picked and eaten as pot herbs.

O. Boukar ()
International Institute of Tropical Agriculture, Cowpea Breeding Unit,
Sabo Bakin Zuwo Road, Kano, Kano 3112, Nigeria
e-mail: [email protected]
C. A. Fatokun
International Institute ot Tropical Agriculture (IITA), PMB 5320, Ibadan, Oyo State, Nigeria
e-mail: [email protected]
P. A. Roberts
Department of Nematology, University of California—Riverside, 900 University Avenue,
Riverside, CA 92521, USA
e-mail: [email protected]
M. Abberton
International Institute of Tropical Agriculture, PMB 5320, Ibadan, Nigeria
e-mail: [email protected]
© Springer Science+Business Media New York 2015 219
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_7
220 O. Boukar et al.

Cowpea is grown mainly as an intercrop along with sorghum and millet in the
dry savannas but is also intercropped with maize in the moist savannas. Only very
few farmers in SSA grow cowpea as a sole crop. It is grown in wide spacing when
intercropped such that plant population density is usually low, perhaps around 1000
plants/ha or even less. However, when grown sole the population density is much
higher and this is reflected in higher grain yield. Cowpea, like many other legumes,
is able to contribute to the sustainability of the soil in SSA farmers’ fields. Being a
legume, cowpea is capable of fixing atmospheric nitrogen in its root nodules hence
it has little or no need for nitrogen fertilizer application. It can fix up to 240 kg/ha
and leave between 60 and 70 kg/ha in the soil after harvest (Rachie 1985). The fol-
lowing crop can therefore benefit from this left over nitrogen.
Cowpea is grown in no less than 45 countries across the globe on about
14.5 × 106 ha. A total of 6.2 million metric tons (MMT) of grains are produced annu-
ally implying an average yield of 454 kg/ha. Nigeria and Republic of Niger produce
about 45 and 15 % of total world cowpea followed by Burkina Faso with about 6 %.
The bulk of cowpea production as well as consumption are in West Africa. Another
major producing country is Brazil, but the quantity they produce is not correctly
reported in the Food and Agriculture Organization (FAO) statistics. The projected
annual production rate of growth for cowpea in SSA is expected to be 3 %, which
means 8 × 106 t by 2020 (Abate et al. 2012). Demand, however, will increase at the
rate of 5 % per year in West Africa and this has implications for the people in West
Africa especially Nigerians. Demand for cowpea grain is expected to decline in Ke-
nya and South Africa during this same period (Abate et al. 2012). Cowpea does not
feature in international trade but trade between neighboring countries such as Niger

B. L. Huynh
Department of Nematology, University of California—Riverside,
3401 Watkins Drive, Boyce Hall 1463, Riverside, CA 92521, USA
e-mail: [email protected]
T. J. Close
Department of Botany and Plant Sciences, University of California,
900 University Avenue, Riverside, CA 92521-0124, USA
e-mail: [email protected]
S. Kyei-Boahen
International Institute of Tropical Agriculture (IITA), Av. Eduardo Mondlane 326,
2nd Floor Room 210, Nampula 709, Mozambique
e-mail: [email protected]
T. J.V. Higgins
CSIRO Agriculture Flagship, Clunies Ross Street, Black Mountain, Canberra,
ACT 2601, Australia
e-mail: [email protected]
J. D. Ehlers
Department of Agricultural Development, Bill and Melinda Gates Foundation,
500 N. 5th Ave N, Seattle, WA 98102, USA
e-mail: [email protected]
7 Cowpea 221

and Nigeria takes place. There is an annual deficit of over 0.5 × 106 t in Nigeria and
supplies from Niger and Cameroon have made up for this shortfall. There is a need
for expansion in the production of cowpea if the projected deficit is to be adequately
forestalled. The bulk of the growth in cowpea recorded over years is attributable to
increase in land area cultivated to the crop. Technologies that will lead to increased
productivity per unit area of land now need to be developed and promoted if food
security is to be ensured.
There is a need for the application of agrochemicals especially insecticides to
the cowpea crop. Farmers who grow cowpea in intercrop usually do not give any
protection to the crop against insect pests and apply no fertilizer. However, the
few farmers who grow cowpea as a sole crop try to apply insecticides to provide
protection against insect pests that otherwise cause significant grain yield losses.
In many instances, the insecticides applied may not be effective against all of the
insects that limit the crop’s productivity. Different insects attack cowpea plants at
various stages of the crop’s life cycle. Aphids ( Aphis craccivora) attack cowpea and
cause the most damage when the plants are in the seedling stage while flower bud
thrips ( Megalurothrips sjostedti) cause flower buds to abort prematurely thereby
preventing them from reaching anthesis. The legume pod borer ( Maruca vitrata),
the most cosmopolitan of cowpea insect pests, damages flowers and developing
pods and seeds. A complex of pod-sucking bugs (e.g., Clavigralla tomentosicollis,
Anoplocnemis curvipes, and Riptortus dentipes) feeds on both mature and immature
pods and seeds leading to shrinking, deformity, and nonviability of the seeds. Such
deformed seeds are not fit for consumption and therefore not marketable. Cowpea
weevil ( Callosobruchus maculatus) feeds on stored seeds, which is why most farm-
ers sell off the seeds shortly after harvest at fairly low and noncompetitive prices
to avoid storage losses caused by the weevil. From the foregoing, it is obvious that
insects are capable of wreaking immense damage to productivity of cowpea if not
adequately controlled. For now, the application of insecticides seems to be the only
method for control of some of the cowpea pests.
Generally, the traditional farmers’ cowpea varieties are late maturing (> 90 days
to flowering) and characterized by spreading growth habit. On the other hand, most
of the improved varieties are erect to semierect in growth habit and could be early
(60–65 days) or medium maturing (75–80 days). The early maturing erect cowpea
lines are well suited to sole cropping and could be planted at high population den-
sity, while the spreading type seems to be more adapted to intercropping systems.
Studies have shown that some spreading-type cowpea lines such as ‘Dan Ila’ are
able to withstand shading better than non-spreading types (Terao et al. 1997). Farm-
ers in the dry savanna areas still grow their traditional varieties because even when
insects have caused grain yield losses these varieties still are able to get fodder
which they harvest and sell for income or use as quality feed for their livestock.
The response of cowpea plants to photoperiod has been described as being typi-
cal of quantitatively short-day implying that photoperiod beyond a critical value can
only delay but not prevent flowering (Njoku 1958; Lush et al. 1980). While most
of the farmers’ traditional varieties belong to this category, that is, day-length sensi-
tive, there are some lines which are day neutral (i.e., length of days does not influ-
222 O. Boukar et al.

ence time to flower). Most of the improved cowpea varieties being grown presently
are day neutral in addition to being erect or semierect in growth habit.

2  Origin and Systematics

Cowpea is an indigenous crop in SSA. It has been reported that the immediate
progenitors of cultivated cowpea such as V. unguiculata ssp. dekindtiana/spontanea
are widely distributed across Africa including Madagascar (Ng and Singh 1997).
Ng and Maréchal (1985) suggested that cultivated cowpea moved from West to
East Africa from where it was taken to Europe. It was recognized by the Romans
as far back as 2300 before present (BP). It probably moved from Europe to India in
2200 BP and to the Americas by Spanish and Portuguese traders in the seventeenth
century. The greatest amount of genetic diversity in cultivated cowpea has been
found to exist in West Africa especially the dry savanna regions of Cameroon, Ni-
ger, Nigeria, Burkina Faso, Benin, and Togo. However, the origin of wild cowpea
has been traced to southern Africa particularly the area covering from Namibia,
Transvaal to Swaziland. It is in this subregion of Africa that the highest amount
of genetic diversity for wild V. unguiculata and V. rhomboidea has been detected
(Ng and Singh 1997). The wild cowpea lines found in southern Africa usually have
small seeds when compared with those found in West Africa such as subspecies de-
kindtiana/spontanea, which have slightly larger seeds. This probably confirms the
claim that V. unguiculata ssp. dekindtiana/spontanea is an immediate progenitor of
cultivated cowpea that is characterized by a large-seed size. The different wild cow-
pea relatives were previously regarded as independent species but Maréchal et al.
(1978) merged all of them into a single species ( V. unguiculata). Further, taxonomic
efforts have subdivided these into various subspecies and varieties. Examples are
V. unguiculata ssp. unguiculata var. spontanea, ssp. dekindtiana var. dekindtiana,
ssp. pubescens, and ssp. protracta var. protracta among others. The taxonomy of
the genus Vigna and particularly cowpea and its relatives was recently reviewed
by Pasquet and Padulosi (2012). There is yet to be a well-defined and universally
agreed classification of the wild cowpea relatives. So far, there are no distinct clas-
sifications into primary, secondary, and tertiary gene pools for cowpea. Varying
levels of difficulties are encountered when crossing some of the wild V. unguiculata
subspecies with cowpea and even among themselves. For example, embryo rescue
was necessary for a successful cross between a cowpea line and a line of V. unguicu-
lata ssp. pubescens (Fatokun and Singh 1987). That the wild cowpea relatives have
hardly been used in the genetic improvement of the crop may have contributed to
the low interest in defining the crop’s gene pools. Desirable genes conferring resis-
tance to many insect pests that cause damage to cowpea yield are present in some
wild Vigna species such as V. vexillata, which have resistance to aphids, Maruca pod
borer, and some others. However, strong incompatibility barriers prevent successful
crossing of vexillata and cowpea thus making it impossible to transfer such useful
genes to cowpea through conventional breeding methods.
7 Cowpea 223

3  Varietal Groups

Cultivated cowpea and its cross-compatible wild relatives belong to the section
Catiang of the genus Vigna. All of the cultivated cowpea lines are classified as
V. unguiculata subspecies unguiculata. Cultivated cowpea is subdivided into four
cultivar groups (cv.-gr.), namely, Biflora, Textilis, Sesquipedalis (yard-long- bean),
and Unguiculata/Melanophthalmus (Westphal 1974; Marechal et al. 1978). Each of
these cultivar groups is distinct from the others. For example, Textilis is character-
ized by long peduncles, which are a good source of fiber used in the textile industry,
while Sesquipedalis, the yard-long bean, has long, fleshy, and pendulous pods. The
yard-long bean whose pods can be as long as 90 cm or more is consumed as a veg-
etable especially in Asia. Yard-long bean with long pods may have evolved from
regular cowpea due to selection pressure exerted in Asia, where its consumption as
a vegetable is very popular. Despite the length of the pods, the number of seeds per
pod is usually not more than is found in cowpea which belongs to cultivar group
cv.-gr. Melanophthalmus. The cv.-gr. Unguiculata/Melanophthalmus comprises the
cultivated cowpea with most number of accessions. The protein-rich grains are the
most economically important part of the crop hence seeds are large and crowded in
the pods. This probably explains why cowpea is also referred to as crowder bean
in some communities. Dual purpose varieties are noted for their grain and fodder
yield and should be attractive to people in East Africa who consume cowpea leaves
as vegetables as well as livestock farmers in the dry savanna regions of SSA. Many
cowpea farmers in the dry savanna areas of SSA get almost the same amount of
income from sales of fodder as from grains.

4  Genetic Resources: Conservation and Utilization

The collection, conservation, characterization, documentation, and distribution of

genetic resources (germplasm) are important, and the diversity of germplasm gath-
ered in ex situ collections, or gene banks, is a key underpinning of current and future
breeding programs.
The most extensive collection of cowpea germplasm (15,371 accessions) is held
by the Genetic Resources Center (GRC) of the International Institute of Tropical
Agriculture (IITA), Ibadan, Nigeria. The IITA collection contains germplasm from
90 countries but around half of the collection (7912 accessions) is derived from
West Africa, the center of diversity for the crop. This collection, along with others
in the Consultative Group for International Agricultural Research (CGIAR) gene
banks, was designated by FAO as held “in trust” for the international community,
a status reinforced under Article 15 of the International Treaty on Plant Genetic
Resources for Food and Agriculture, which entered into force in 2004. Germplasm
from this collection is distributed to all those requesting it for research and breeding
for food, feed, and agriculture, free of charge under the standard material transfer
agreement. Cowpea seed is conserved under medium-term storage at 5 °C for the
224 O. Boukar et al.

active collection, from which seeds will be distributed, and in long-term storage at
−20 °C. Other significant collections are held in the USA with the US Department
of Agriculture (USDA), Griffin and with the University of California Riverside
(UCR), (around 10,000 and 6000 accessions, respectively). It should be noted that
IITA and these two USA collections have a combination of duplicate and unique
cowpea accessions.
Collection of genetic resources is accompanied by “passport data” giving details
of collecting site and other information recorded by collectors as well as accession
identifiers. Germplasm is characterized primarily by a number of morphological
“descriptors” of plant features which are relatively constant under different environ-
ments. A descriptor list developed by the International Board for Plant Genetic Re-
sources (IBPGR 1983) and modified by IITA and Bioversity International (Dumet
et al. 2010) is used for the characterization of cowpea germplasm; this includes veg-
etative and floral parts and seed. Progress in the development of improved cowpea
varieties that would be well adapted to different agroecologies in the tropics depend
largely on the genetic resources being conserved. Besides adaptation to different
agroecologies, many new varieties with specialty traits and consumer preferences
may also be developed using these resources (Table 7.1).
The core collection of a germplasm set is often developed to capture a high
proportion of the diversity in a number of accessions that can be more manage-
ably phenotyped, typically 5–10 % of the total collection. In many cases, both agro-
morphological characters and molecular markers are used to develop this. IITA has
developed a “core-collection” of 2062 accessions of cultivated cowpea using geo-
graphical, agronomic, and botanical descriptors (Mahalakshmi et al. 2007). In a fur-
ther refinement of this core collection concept, under the auspices of the Generation
Challenge Program (GCP) of the CGIAR (http://www.generationcp.org/research/
research-projects), a “mini-core” collection of cowpea was developed with 374 ac-
cessions. The continuing development and application of genomic tools, including
a draft cowpea genome sequence, will underpin new approaches to the characteriza-
tion of cowpea genetic resources and enhance their utility for breeders.
Hearne et al. (2012) studied a subset of the core collection comprising 86 acces-
sions plus 10 gene bank accessions, representing 84 countries of origin. Fourteen
SSR markers were used in the study, based on high polymorphism rates for two
alleles per marker and good technical resolution, to assess the levels of inbreeding
and heterogeneity within this group. The study revealed that inbreeding was not
as complete as previously assumed and that up to five plants per accession would
provide more accurate measures of diversity (Hearne et al. 2012).
A recent study of cowpea diversity analyzed the genetic relatedness of 433 cow-
pea landraces collected from 56 countries and 46 accessions of wild cowpeas us-
ing a set of > 1200 genome-wide single-nucleotide polymorphism (SNP) markers
(Huynh et al. 2013a). Among the landraces, 323 were from North, West, Central,
East, southeastern, and southern Africa, and 99 were representative of the rest of
the world. The wild cowpea accessions represented three countries in West Africa
and five countries in eastern and southern Africa. The genotyping was conducted
using the 1536-SNP cowpea Illumina GoldenGate assay (Muchero et al. 2009a).
7 Cowpea 225

Table 7.1   Cowpea germplasm accessions with desirable traits

Resistant/tolerant Germplasm accessions References
Diseases
Fusarium wilt TVu 109-2, TVu 347, TVu 984, TVu 1000 Singh et al. (1983)
Scab TVu 853, TVu 1404, TVu 1433. Singh et al. (1983)
Septoria TVu 456, TVu 483-2, TVu 486, TVu 1433, Singh et al. (2002)
TVu 11761, TVu 12349
Bacterial blight TVu 347, TVu 410, TVu 483-2, Danilla Singh et al. (1983)
(Nigerian landrace)
BICMV TVu 2480; TVu 2657, TVu 3433 Taiwo et al. (1982); Bashir
(1992)
CABMV TVu 401, TVu 1582 Bashir (1992)
CPMV TVu 227, TVu 345, TVu 612, TVu 2331 Patel (1982)
CPMoV TVu 3901 Allen et al. (1982)
Striga and Alectra B301; TVu 14676 Lane et al. (1997); Ouedraogo
et al. (2012)
Insect pests
Aphid TVu 36, TVu 62, TVu 408, TVu 410, TVu Singh et al. (1983)
801, TVu 2896, TVu 3000
Flower bud thrips TVu 1509, Sanzi (Ghanaian land race)
Leafhoppers TVu 59, TVu 123, TVu 662, Singh et al. (1983)
Bruchid TVu 2027, TVu 11952, TVu 11953 Singh et al. (1983)
Drought TVu 11979, TVu 14914, Danilla Watanabe et al. (1997), Agbi-
codo (2009)
TVu tropical Vigna unguiculata are germplasm lines available at the genetic resources center of
IITA
BICMV Blackeye cowpea mosaic virus, CABMV Cowpea aphid-borne mosaic virus, CPMV Cow-
pea mosaic virus, CPMoV Cowpea mottle virus

The diversity analysis using Bayesian inference identified two distinct cowpea gene
pools in Africa, one centered in western Africa and a second gene pool centered in
eastern Africa. Each gene pool was most closely related to the wild cowpeas col-
lected from the same geographic region. These results indicate that a process of di-
vergent domestication has occurred leading to the formation of the two gene pools.
Genetic variation was found to be slightly higher among the group of accessions
from non-African countries than among the African accessions, and accessions
from Asia and Europe were more related to those from western Africa while acces-
sions from the Americas were closer to the eastern Africa gene pool (Huynh et al.
2013a). The overlap in distribution and the chronological sequence of domestication
events is difficult to interpret precisely due to the lack of historical records of human
involvement in domestication and geographical movement of early cowpea forms.
However, these diversity studies are valuable in guiding introgression decisions in
breeding programs and for enhancing utilization of cowpea germplasm collections.
Availability of seed for distribution depends on number of seeds, their quality,
and health status. At IITA, the viability of seed under storage is maintained at 85 %
or above and regeneration of accessions is carried out when viability falls below
this level to ensure availability of sufficient seed of good quality for distribution.
226 O. Boukar et al.

An important aspect of the conservation and distribution of cowpea seed is health

testing, particularly virus indexing. Guidelines for the regeneration of cowpea were
developed by Dumet et al. (2008).
The cowpea breeding program at IITA makes extensive use of the cowpea core
collection from its GRC. In the last few years, accessions from the core collection
have been evaluated for a wide range of traits by the Cowpea Breeding Unit. These
include resistance to biotic stresses: Striga, aphids, bacterial blight, fusarium, smut,
brown blotch, and several viruses. Accessions were also evaluated for grain pro-
tein and mineral content (Boukar et al. 2011) and drought tolerance (Fatokun et al.
2012a). For a number of these traits, important sources of genes were identified, and
following crossing, lines were advanced for international trials in many countries
of SSA where lines with superior performance have been identified and released.
Cowpea has a number of related subspecies and species, which may be valuable
sources of important agronomic traits. The IITA’s GRC maintains about 2000 ac-
cessions of cowpea wild relatives. The genetic resources of these wild relatives are
not well represented in ex situ collections. The cultivated cowpea and some of its
wild relatives belong to the section Catiang of the genus Vigna. Previous activities
aimed at introgressing desirable genes through hybridization from some wild Vigna
species have shown that crosses are possible only among members of this section.
However, varying levels of compatibility have been observed when crosses were
made between cultivated cowpea and some of these wild relatives. Some wild Vigna
species such as V. vexillata have been found to show high levels of resistance to the
major insect pests (cowpea aphid, flower bud thrips, legume pod borer, pod-sucking
bugs, and cowpea bruchid) that cause immense grain-yield reduction in cultivated
cowpea (Singh et al. 1992). The efforts devoted to making interspecific crosses
between cowpea and vexillata did not yield any hybrid (Fatokun 2002). In addition,
experience has shown that seeds of wild cowpea relatives are very small, with hard
testa and unattractive color and texture. Breeders have tended to shy away from uti-
lizing the crop’s wild relatives. However, recent developments in the new genomic
tools for cowpea may change this attitude.
To avoid risk of loss of valuable germplasm it is good practice to “safety dupli-
cate” in another gene bank preferably in another country. The “safety backup” for
many important collections is the Svalbard global seed vault in Norway. The current
status of this vault was reviewed by Westengen et al. (2013) and 14,099 of the IITA
cowpea collection are currently held at Svalbard with the great majority of these
also safety duplicated in another gene bank in addition to that of IITA.
In 2008, the Global Crop Diversity Trust commissioned IITA to lead a survey
and expert consultation on the development of a strategy for cowpea conservation,
the results of which are summarized in Dumet et al. (2012). This highlighted the
need for capacity development of national systems, particularly in SSA.
The line B301 was collected from Botswana, and it has been a major source of
genes for resistance to Striga and Alectra. It has therefore been used extensively as
a donor for resistance to these two parasitic flowering plants. An improved cowpea
breeding line TVx 3236 with tolerance to flower bud thrips was selected from a seg-
regating population that resulted from a cross involving TVu1509 (Singh et al. 1992).
7 Cowpea 227

5  Major Breeding Achievements

The productivity of cowpea in SSA farmers’ fields is very low with mean grain yield
of less than 400 kg/ha, whereas in the USA yield is > 5000 kg/ha. Several factors,
notably an array of insect pests, diseases, and drought, militate against high grain
yield in SSA. That cowpea is grown in SSA by peasant farmers who are resource
poor and unable to procure the necessary pesticides to protect their crops in the field
and grain in storage, contributes to the low productivity of the crop. Most of the
breeding activities therefore focus on how to increase productivity by developing
improved varieties with high yield potential and resistance to the various abiotic
and biotic stresses. IITA has an active cowpea breeding program and very strong
collaboration with cowpea breeders in the various National Agricultural Research
Systems (NARS) of SSA. Besides, a number of advanced research institutions and
universities collaborate with IITA scientists in all aspects of cowpea research. The
cowpea germplasm lines available in the genetic resources center of IITA have con-
tinued to be the major source of genetic diversity upon which breeders depend for a
continuous generation of improved breeding lines.
Thus far, only cultivated cowpea germplasm lines have been exploited in the
development of improved varieties. Several improved cowpea breeding lines have
been developed, many of which have been evaluated across various agroecologies in
different countries and those with good performance and that are attractive to farm-
ers and consumers have been released as varieties in various countries (Table 7.2).
Through genetic improvement, the majority of cowpeas now adopted in SSA farm-
ers’ fields are varieties that are erect in growth habit and are day neutral. The grain
yield of traditional cowpea varieties is inherently low. In addition, because they
spread, farmers plant them at wide spacing thereby resulting in fewer plants per
hectare as compared to the erect or semierect improved varieties. Table 7.2 lists
cowpea varieties from IITA breeding nurseries that were released globally. Many of
the varieties combine resistance to diseases, Striga, Alectra, and flower bud thrips.
Generally, the key achievements in breeding have focused on introgression of
traits dealing with biotic and abiotic stresses to cowpea yield, combined with im-
proved agronomic qualities of enhanced grain size, grain quality, including seed-
coat color and texture, plant architecture, and time to maturity. Examples of ge-
netic improvements in West African country breeding programs are provided by
new cowpea variety releases in Burkina Faso and Senegal, with support from the
US Agency for International Development (USAID) Bean/Cowpea and Dry Grain
Pulses Collaborative Research Support Program (CRSP) programs. Institut Seneg-
alais Recherches Agricoles (ISRA) of Senegal has released a series of varieties over
the past 20 years, which combines targeted biotic stress resistances with enhanced
yield and grain qualities preferred by consumers and shortened maturity times to
hedge against drought years. These include Melakh, Mouride, Yacine, Pakau, and
in 2013 three new lines with large white grain types. These varieties have increased
yields over the national average by up to about 20 % and typically combine re-
sistance or tolerance to one or more cowpea insect pests such as flower thrips or
Table 7.2   List of cowpea varieties released in different countries. (Adapted and updated from Singh et al. 2002)
228

Country Variety released Country Variety released

Angola TVx 3236 Argentina IT82D-716
Australia IT82E-18 (as Big Buff) Belize VITA-3, IT82D-889, IT82E-18
Benin Republic IT81D-1137 Bolivia IT82D-889, IT83D-442
IT84S-2246-4,IT95K-193-12
Botswana ER-7, TVx 3236 Brazil VITA-3, VITA-7, TVx 1836-013J
Burkina Faso TVx 3236, KN-1
IT99K-573-2-1, IT98K-205-8 Myanmar VITA-4 (Yezin-1)
Cameroon IT81D-985 (BR1) Central African Republic VITA-1, VITA-4, VITA-7, VITA-5
IT81D-994 (BR2) TVx 1948-01F, IT81D-1137,
IT83S-818
TVx 3236 IT82E-18, IT81D-994
IT88D-363 (GLM-92)
IT90K-277-2 (GLM-93) Colombia IT83S-841
Cote d’Ivoire IT88D-361, IT88D-363
Cuba IT84D-449 (Titan) Democratic Republic of Congo. IT89KD-349, IT89KD-389
IT84D-666(Cubinata-666) IT89KD-355
IT86D-314 (Mulatina-314)
IT86D-368 (IITA-Precoz)
IT86D-782 (Tropico-782)
IT86D-792 (Yarey-792)
IT88S-574-3(OR 574-3)
Equatorial Guinea IT87D-885 Ethiopia TVx 1977-01D, IT82E-16, IT82E-32
Egypt Tvu 21, IT82D-716 Fiji VITA-1, VITA-3
IT82D-709, IT82D-812
IT82E-16 Gambia IT84S-2049 (Sosokoyo), IT83S-728-13
O. Boukar et al.
Table 7.2  (continued)
Country Variety released Country Variety released
Ghana IT82E-16 (Asontem)
7 Cowpea

IT83S-728-13 (Ayiyi) Guinea Conakry IT81D-879, IT83D-340-5

IT83S-818 (Bengpla) IT82E-16, IT85F-867-5 (Poku Togboi),
TVx 1843-1C (Boafa) IT85F-2805, IT83S-990,
TVx 2724-01F (Soronko) IT87S-1463, IT84S-2246-4
IT87D-611-3 (Nhyira), IT87D-2075
(Tona)
Guinea Bissau IT82E-889, IT82D-889 Haiti VITA-4, IT87D-885
India VITA-4, TVx 1502 Jamaica VITA-3, ER-7, IT84S-2246-4
Lesotho IT82E-889, IT87D-885 IT84E-124
IT82E-16, IT82E-32
Liberia IT82D-889, TVx 3236, VITA-5
Malawi IT82D-889, IT82E-16 VITA-4, VITA-7
IT82E-25, IT99K-494-6
Mali TVx 3236, IT89KD-374 (Korobalen)
Mauritius TVx 3236 IT89KD-245 (Sangaraka), IT97K-499-
35 (Jiguiya), IT93K-876-30, IT82D-
812, IT83S-818, IT85F-2020
Mozambique IT82E-16
IT00K-1263, IT97K-1069-6
Nepal IT82D-752 (Aakash)
IT82-889 (Prakash)
Nigeria TVx 3236, IT81D-994, IT86D-719
229
Table 7.2  (continued)
230

Country Variety released Country Variety released

Niger IT89KD-374, IT90K-372-1-2 IT84S-2246-4, IT90K-76
IT97K-499-35, IT97K-499-38 IT86D-721, IT88D-867-11
IT98K-205-8, IT99K-573-1-1, IT82E-60, IT89KD-374, IT90K-277-2
IT96D-610
IT90K-82-2, IT89KD-
288,IT97K-499-35, IT89KD-391,
IT99K-573-1-1
IT99K-573-2-1
Paraguay IT86D-1010, IT87D-378-4
IT87D-697-2, IT87D-2075
Philippines IT82D-889
Sierra Leone TVx 1990-01E, IT86D-721 Senegal TVx 3236
IT86D-719, IT86D-1010
IT82E-32, TVx 3236, Tvu 1190 Somalia TVx 1502, IT82D-889
VITA-3 IT82E-32
Sudan IT84S-2163
South Africa IT90K-59 (Daha ElGoz = Gold from Sand)
IT82E-16 (Pannar 311)
Swaziland IT82D-889 (Umtilane), IT82E-18
Sri Lanka IT82D-789 (Wijaya) IT82E-27, IT82E-71
IT82D-889 (Waruni)
TVx 309-01EG, VITA-4 Thailand VITA-3, IT82D-889
TVx 930-01B (Lita) Uganda TVx 3236, IT82E-60
IT86D-1010
Suriname IT82D-889, IT82D-789 USA IT84S-2246-4, IT84S-2049 (for nema-
tode resistance)
O. Boukar et al.
Table 7.2  (continued)
Country Variety released Country Variety released
Tanzania TKx 9-11D (Tumaini)
7 Cowpea

TVx 1948-01F (Fahari)

IT82D-889 (Vuli-1), IT85F-2020 Yemen TVx 3236, IT82D-789, VITA-5
IT99K-7-21-2–2-1, IT99K-573-1-1
(Vuli AR1), (Vuli AR2) Venezuela VITA-3, IT81D-795
IT82D-504-4, TVx 1850-01E
Togo VITA-5, TVx 3236 –
IT81D-985 (VITOCO) Zambia TVx 456-01F, TVx 309-01G
IT82E-16 (Bubebe)
Zimbabwe IT82D-889
231
232 O. Boukar et al.

pathogens such as virus or bacterial blight with higher innate yield potential. In
Burkina Faso, the Institut de l'Environnement et de Recherches Agricoles (INERA)
has released a series of cowpea varieties with larger grain size and resistance to the
parasitic weed Striga plus resistance to aphids and viruses. Interestingly, the variety
Melakh, which was bred and is now widely grown in Senegal, was found to be
an excellent variety in areas of Burkina Faso with similar agroecologies, and this
variety has also been released by INERA in Burkina Faso. This example of a line
developed in Senegal being evaluated and released in Burkina Faso demonstrates
the advantages of collaborative breeding programs and material exchange between
scientists from different countries of the subregion.
In the USA, cowpeas are bred to meet markets for use as both a vegetable and as
a dry bean. In California, the breeding program has focused on developing improved
blackeye dry grain cowpea types. The focus for cowpea improvement at UCR has
been to introgress resistance to Fusarium wilt and root-knot nematodes into high-
yielding backgrounds with improved grain quality. A recent example is California
Blackeye No. 50 (CB50) released in 2009, which has improved grain size and qual-
ity (brighter white seed-coat color) combined with resistance to Fusarium wilt races
3 and 4 plus strong resistance to the root-knot nematodes Meloidogyne incognita
and M. javanica (Ehlers et al. 2009). An earlier variety of blackeye cowpea, Cali-
fornia Blackeye No. 27 (CB27), which was released in 1999, was bred to combine
Fusarium wilt and root-knot nematode resistance with heat tolerance (Ehlers et al.
2000). In the southern USA, a successful breeding focus has been to incorporate
the persistent green seed-coat trait into high-yielding cowpea varieties for canning
or as fresh-shelled peas for freezing. This breeding focus was described in Ehlers
et al. (2002) and is based on the green cotyledon and green testa traits which result
in a persistent green seed color. The green cotyledon trait is conditioned by a single
recessive gene, with the symbol gc (Fery and Dukes 1994), and several successful
varieties have been released including Bettergreen, Charleston Greenpack, Petite-n-
Green, and Green Dixie (Ehlers et al. 2002).

6  Specific Goals in Current Breeding

The goals of cowpea genetic improvement change with time and usually depend on
agreed priorities set by stakeholders. The stakeholders include farmers, extension
agents, NGOs and donor representatives, seed companies, consumers, and research-
ers. Current cowpea breeding goals also vary with the target production areas but
are based on enhancing yield and grain quality, largely through introgression of
biotic and abiotic stress tolerance and resistance. The low productivity of cowpea
in the subregion is of major concern and efforts are directed primarily at addressing
the factors that appear responsible. In recent times, nutrition and health conscious
individuals and organizations seek cowpea varieties with higher protein content
than the levels in many of the available varieties which is around 25 %. Breeders
7 Cowpea 233

aim to develop varieties that overcome some of the identified production constraints
as follows.

6.1  Resistance to Insect Pests

From the seedling stage to time of harvest and even seed storage there are major
insect pests that damage cowpea. Aphids (Aphis craccivora) attack cowpea plants
in the field and if not controlled they can kill the plants especially the seedlings.
They are particularly troublesome when there is a short spell of drought after seed-
ling emergence. Earlier, a single dominant gene conferred resistance to aphids but
those varieties have now succumbed to the insect. New races of the insect have
evolved, so new sources of genes for resistance are being sought among cultivated
and wild cross-compatible cowpea relatives. A few lines among wild cowpea have
been found to be aphid-resistant. Molecular markers will be deployed to better un-
derstand the resistance and to facilitate marker-assisted selection (MAS).
In California, emphasis is also on cowpea aphid resistance and tolerance to Lygus
bug using resistance and tolerance traits identified in African cowpea germplasm
lines from IITA, including IT97K-556-6 for aphid resistance and IT93K-2046-1.
The aphid resistance in IT97K-556-6 has been shown by quantitative trait loci
(QTL) mapping to be inherited by one minor and one major QTL on cowpea ­linkage
groups 1 and 7, respectively (Huynh et al. 2015). In California, Lygus bugs cause two
types of yield loss in cowpea: First, feeding on young floral buds causes these buds
to drop which drastically reduces pod set and grain yield, and second, feeding by
Lygus bugs during pod and grain development leads to pitted and discolored grains.
Conventional breeding of Lygus-tolerant blackeye pea is underway by pedigree se-
lection from crosses between the African donor line and California Blackeye variet-
ies, and field phenotyping for tolerance in insecticide protected and unprotected plot
designs to assess grain yield and quality under natural Lygus bug infestation. The
Lygus tolerance determinants have yet to be mapped and SNP-tagged within the
cowpea genome, currently precluding MAS approaches for breeding.
A significant challenge for breeders is to better define traits for flower thrips
resistance and resistance to pod-sucking bugs. Phenotypic screening efforts are un-
derway in West African cowpea programs to provide genetic mapping data for QTL
discovery for these traits. The critical yield losses caused by these insect groups
make them a priority focus in cowpea breeding. In the case of flower thrips, Omo-
Ikerodah et al. (2008) identified DNA markers associated with QTLs that have ef-
fects on resistance to flower bud thrips in a biparental mapping population derived
from a cross that had Sanzi, the land race from Ghana, as one of the parents. Many
other improved breeding lines with resistance to diseases, drought, Striga, and pests
were derived from crosses that involved above listed (Table 7.1) and other germ-
plasm lines.
234 O. Boukar et al.

6.2  Tolerance to Drought and Low Soil P

Cowpea is grown mainly in drought-prone areas of SSA. Compared to many other

crops, cowpea is regarded as relatively drought tolerant. This notwithstanding, de-
pending upon severity, drought can still cause yield reduction in cowpea and the
variation observed among germplasm lines indicates the present level of drought
tolerance in the crop can be enhanced (Fatokun et al. 2012a). Molecular markers
have been identified that are associated with QTLs for drought tolerance (Agbicodo
2009). A series of QTLs have been identified following the analysis of different
recombinant inbred lines (RIL) segregating for drought tolerance. Seedling-stage
drought induced delayed senescence traits were identified in cowpea genotype
IT93K-503-1 and others in both greenhouse and field phenotyping experiments,
and reproducible QTLs for this trait were mapped in the cowpea genome (Muchero
et al. 2008, 2009b). More recently, the staygreen phenomenon, a trait which enhanc-
es delayed senescence, biomass, and grain yield under drought stress, was charac-
terized in cowpea through genetic mapping using SNP genotyping, field and green-
house phenotyping, and linkage disequilibrium association mapping in conjunction
with biparental QTL mapping (Muchero et al. 2013). Seven loci were identified;
out of which five exhibited pleiotropy for delayed senescence, biomass, and grain
yield. In particular, three of these putative staygreen QTLs were resolved at 3.2 cM
or lower map distances and provide important targets for introgression through
marker-assisted selection (MAS). In addition, co-location of these QTLs with those
governing early vegetative delayed senescence provides a rapid screening approach
by phenotyping plants at the seedling stage for drought response (Muchero et al.
2013). These markers will be useful in marker-assisted recurrent selection (MARS)
for drought tolerance in cowpea.
Like most other legumes cowpea has a need for phosphorus to be able to form
nodules adequately and fix nitrogen. The soils in SSA are generally low in phos-
phorus and farmers who grow cowpea usually do not apply fertilizer to their crops.
Improved breeding and germplasm lines have been evaluated for tolerance to low
soil P and differences were detected among them which are indications that lines
with a need for low levels of soil P can be developed.

6.3 Heat

Heat stress in cowpea disrupts flowering and pod set. It is an abiotic stress for which
genes for tolerance are available as targets for molecular breeding approaches. A set
of five heat tolerance QTLs were identified through QTL mapping in a biparental
RIL population developed from the heat-tolerant variety CB27 as one of the parents
(Lucas et al. 2013a). These QTLs provide resources for incorporating heat tolerance
into other elite heat-sensitive cowpea varieties using MAS.
7 Cowpea 235

6.4  Dual Purpose

Although grain is the most economically important product of cowpea, in some

parts of SSA such as Kenya, Tanzania, and Mozambique, young green and succulent
leaves are relished as pot herbs while the haulms are a source of quality fodder for
livestock in the dry savannas of West Africa. The young leaves are known to contain
high levels of protein while many farmers derive income from selling dried cowpea
fodder. The development of dual purpose varieties for leafy vegetables as well as for
grain could meet the needs of many more people across the African region. Some
attention is being devoted to selecting lines with this dual attribute that will serve
as a source of grains and leaves for human food and animal feed. Improved dual
purpose breeding lines have been identified in the IITA cowpea breeding program
and shared with collaborators for evaluation in their countries for acceptability to
farmers and consumers.

6.5  High Protein Content in Grains

The protein content of the grain is a major reason why cowpea is popularly con-
sumed at home in several SSA communities. It is also why cowpea is commonly
referred to as poor man’s meat. In SSA, the cost of meat is prohibitive and not af-
fordable in the quantity that is needed for a balanced diet. Depending on the variety,
cowpea grains contain between 18 and 29 % protein with a potential for 35 % (Duke
1981). Among 79 cowpea varieties studied by Evans and Boulter (1974), protein
content ranged from 21 to 34 %. In another study involving 100 lines, Nielsen et al.
(1993) found that protein content ranged from 22.9 to 32.9 % with a mean of 28.6 %.
Boukar et al. (2011) identified the following germplasm lines as having high protein
content—TVu 408, TVu 526, TVu 1820, TVu 2356, TVu 2508, TVu 2723, TVu
2880, TVu 3638, TVu 8810-1—which could be used in the development of im-
proved breeding lines. The cowpea protein consists of 90 % salt-soluble globulins
and 10 % water-soluble albumins (Duke 1981). The anti-nutritional factors found
in cowpea grains such as hemagglutinins and trypsin inhibitors are heat labile and
can be inactivated easily by heating, thus making cowpea protein readily digested
and absorbed. This makes cowpea protein suitable for infants and formulations of
baby foods containing cowpea should be encouraged and commercialized in SSA.

6.6  Resistance to Diseases

Many diseases afflict cowpea plants in the field. There are fungal, bacterial, and
viral diseases that attack the plants. Since farmers do not apply chemicals to protect
their cowpea crops the diseases are best controlled by planting varieties that are
resistant. Among the most devastating of fungal diseases is ascochyta blight caused
236 O. Boukar et al.

by Ascochyta phaseolorum Sacc., which is seed-borne (Emechebe and Shoyinka

1985). The disease causes severe defoliation and lesions on stem and pods and can
lead to the death of susceptible plants. Line TVu 11761 was identified as a potential
source of resistance to this disease (Singh et al. 2002). Brown blotch is another ma-
jor fungal disease of cowpea in SSA. The causal organism is Colletotrichum capsici
(Emechebe and Florini 1997). All plant parts above soil level show symptoms of
the disease in susceptible lines. Such symptoms include failure of seeds to germi-
nate, damping off of seedlings, girdling of stem and branches, and flower abortion,
among others. Treating seeds with fungicides, such as benomyl or carbendazim,
before sowing helps reduce incidence of the disease. However, the development of
resistant varieties appears the most attractive option for most SSA farmers. Scab,
smut, and Septoria caused by Elsino phaseoli, Protomycopsis phaseoli, and Septo-
ria vignicola, respectively, are also important fungal diseases of cowpea and cause
yield reductions.
The most important bacterial disease of cowpea is bacterial blight caused by
Xanthomonas campestris pv. vignicola. It is a disease that has been reported on
cowpea in different parts of the world. Disease symptoms include large irregular
foliar lesions with yellow margins, stem cankers, and preemergence and postemer-
gence seedling mortality (Emechebe and Florini 1997). Some germplasm lines have
been found that are resistant to the disease and the genes conferring resistance have
been transferred to several improved varieties. However, in view of the high rate of
mutation in the bacterium, it is necessary to continue identifying additional sources
of resistance.
Many viruses attack cowpeas and these can only be controlled by sowing resis-
tant varieties. The cowpea aphid-borne mosaic virus (CABMV) and bean common
mosaic virus (BCMV-BIC), both of which are seed-borne and occur worldwide, are
two economically important cowpea pathogens (Huguenot et al. 1997). In addition
to these two viruses, Hampton et al. (1997) reported that cucumber mosaic cucu-
movirus (CMV), cowpea mosaic (CPMV) and cowpea severe mosaic (CPSMV)
comoviruses make up the most devastating viruses of cowpea. They are also seed-
borne. Worrisome in cowpea production is the occurrence of mixed infections of
these viruses. Mixed infections cause drastic disease symptoms and even death of
plants in the field. Since there are no chemicals to control these pathogens the de-
velopment of resistant varieties is the only option for their control. Sources of genes
for resistance to several of the viruses have been identified and many have been
incorporated in released varieties.
A recent review of the important biotic stress resistance traits with molecular
marker-based associations is provided in Huynh et al. (2013b). Genetic map posi-
tions in the cowpea genome and linked, flanking SNP markers have been identified
for resistance to root-knot nematodes, Fusarium wilt, Macrophomina phaseolina
(ashy stem blight or charcoal rot), bacterial blight, several cowpea viruses (cowpea
mosaic virus, cowpea severe mosaic virus, blackeye cowpea mosaic potyvirus),
foliar thrips, cowpea aphid, and parasitic weed Striga gesnerioides. These resources
have helped to better define the breeding targets in several US, African, and Asian
cowpea-breeding programs. In California, the focus remains on Fusarium wilt and
root-knot nematode resistance.
7 Cowpea 237

6.7  Large Seed Size

In recent times, consumers have shown a preference for cowpeas with large grain
size. Breeding activities have been initiated towards developing varieties that meet
this preference. Experience has, however, shown that small seed size in cowpea is
dominant to large seed size hence there is need to embark on backcrossing to trans-
fer the genes for large seed size to preferred varieties.

6.8  Adaptation to Intercropping

Most of the improved cowpea varieties that have been released have erect or semi-
erect growth habit. They are, therefore, well adapted to sole cropping. Many farm-
ers in SSA still prefer to intercrop cowpea with sorghum, millets, and other cereals.
This habit is difficult to change because each farmer has access to a small land
area where he plants all the crops that provide the family with food and some in-
come. When intercropped, cowpea plants are readily shaded by the taller cereals.
The traditional varieties which farmers grow under this cropping system spread on
the ground and remain there until the cereals are harvested when they now receive
more sunlight, flower, and set pods. New varieties can be developed that can adapt
to intercrop conditions such that their flowering and podding are not adversely af-
fected by shading.

6.9  Striga Resistance

In Africa, most programs are targeting Striga resistance, in combination with

drought-tolerance traits as well as virus and insect resistance. For example, in
Burkina Faso, any new variety released must contain Striga resistance (Drabo per-
sonal communication 2014).

7  Breeding Methods and Specific Techniques

Cowpea breeding methods are similar to those of other self-pollinated crops such as
peanut, soybean, wheat, and barley. Cowpea breeders depend upon the germplasm
available in the different collections described earlier. The IITA GRC is a source of
genes of interest in both domesticated and wild cross-compatible cowpea relatives.
Several decades of breeding effort by different institutions also provide the chance
to build on the numerous improved breeding lines. The IITA cowpea breeding nurs-
ery distributes many breeding lines annually for testing, adoption as released vari-
eties or as parents to be used in the importing countries’ breeding programs. Most
238 O. Boukar et al.

cowpea breeding activities are focused on development of improved varieties with

farmers’ and consumers’ preferred traits. Breeding programs include the develop-
ment of populations segregating for desirable traits and from which selections are
made for both simple and complex characteristics (e.g., disease resistance, drought
tolerance, grain yield). Additional activities include determination of inheritance,
creation, and evaluation of new genetic variability and production of specific geno-
types.
Cowpea variety development programs follow the conventional breeding steps
for self-pollinated crops (pure-line breeding, pedigree breeding, single-seed de-
scent, etc.). Parents characterized by important traits of interest are chosen and
used to generate genetically variable populations through artificial hybridization
followed by selection. Appropriate selection pressure that favors identification of
desired traits such as imposition of disease pathogens, insect pests, drought, heat,
low phosphorus, etc. is exerted on the segregating populations. In addition, the lines
to be selected are assessed for agronomic and quality characteristics. As segregat-
ing populations advance, homozygosity also increases. The lines selected at this
stage on the basis of good performance are homozygous and ready for replicated
performance trials across multiple locations and cropping seasons. Seeds of the
best one to five percent of lines with superior agronomic performance or quality
characteristics are multiplied under controlled conditions for variety release and to
maintain their genetic purity.
As in all breeding programs, the exact techniques used in cowpea cultivar devel-
opment vary widely. In the case of simply inherited traits such as disease resistance,
the backcross method is used to introgress the associated gene(s) into existing cul-
tivars that are lacking the trait. When several traits are being moved from two or
more parents, hybrids from single, double, three-way, or other complex crosses are
advanced through any of several methods that support the acceleration of homozy-
gosity. Commonly used procedures include bulk population, the pedigree method,
single-seed descent and modifications of these methods as necessary. The ultimate
product of all the methods is a group of homozygous lines, which only vary in the
time frames during which selection pressures are applied. Cowpea breeders have
relied primarily on pedigree breeding to combine favorable traits from two parents
and by recurrent backcrossing to introgress a major trait from a donor line into an
elite recurrent parent which is usually a preferred current variety. Considerable suc-
cess has been achieved by both approaches to improve cowpea grain quality and
grain and biomass yield by the combining of traits determining grain size, texture
and color, drought and heat tolerance, and resistance to a range of pests and diseases.
Cowpea being primarily self-pollinated, hybridization between parents usually
involves emasculation (removal of anthers) from flowers of one parent (female or
seed parent) and artificial transfer of pollen from the alternate (male) parent. Cow-
peas are easier to cross than many other grain legumes due to the large size of the
flowers and to the fact that the keel is straight, beaked, and not twisted. Cowpea
flowers have few floral nodes per raceme and tend to have a lower rate of abortion
than many other species. Rapid and effective methods of hand emasculating and
crossing cowpeas were described by Myers (1996).
7 Cowpea 239

Although emasculation and pollination can be carried out all day, hybridization
of cowpea is less effective when the temperature is high. Night temperatures greater
than 20 °C reduce microsporogenesis leading to formation of indehiscent anthers
and pollen with low viability (Warrag and Hall 1983; Ahmed et al. 1992). Thus,
moderate temperature and increased humidity appear to increase the percentage of
pod set following hand-emasculated crosses. In general, the rate of such pod set-
ting varies enormously with environmental conditions, genotype, and manipulative
techniques.
In IITA, crossing activities are conducted in mesh houses or greenhouses to al-
low: (1) good control of insect pollen vectors, major pests, and diseases; (2) good
plant development (water, fertilizer, etc.); and (3) easy manipulation of plants dur-
ing crosses. When planted in the screenhouse, most cowpea lines tend to climb and
stakes are, therefore, needed which also help position the flowers at a height that is
comfortable for the person making the crosses. In the planning of crossing activi-
ties, photosensitivity and number of days to flowering of the parental genotypes are
always considered to avoid asynchronous flowering. Use of different planting dates,
removal of developing flowers and fruits, using black polythene to cover plants
from afternoon to next morning for a number of days (to reduce length of days) and
the use of cuttings are some of the techniques to ensure synchronous flowering by
the parental lines.
A hybrid plant reproduces to form a segregating population (segregation and
recombination of genes). Development of a new variety usually involves inbreed-
ing of a segregating cowpea population for three to seven generations, during which
selection is applied and individuals in the population become increasingly homo-
zygous (true breeding). Narrow crosses between closely related parents normally
require fewer generations of inbreeding than wide crosses (very different parents)
to become true breeding.
In IITA, screenhouses are used for rapid advancement of cowpea breeding popu-
lations with the possibility of 3–4 generations per year. When photoperiod-sensitive
parents are involved in the crosses, breeding programs can only have 2–3 genera-
tions per year. Generally, a high level of homozygosity is attained from F5 to F7 since
variation within rows derived from single selected plants will be relatively small.
Bulked F6/F7 populations are grouped according to maturity, plant type, seed quality,
and resistance to major pests and diseases. Farmers’ involvement through participa-
tory variety selection is encouraged. Homozygous materials are evaluated in initial
evaluation trials (IET) at 2–3 locations without replication. Selected lines from IET
are tested in preliminary variety trials and advanced variety trials (AVT) in 3 replica-
tions across 4 locations representing different agroecological zones: (a) Ibadan (7 °
25ʹ N, 3 ° 37ʹ E) derived savanna with bimodal rainfall (1500 mm); (b) Samaru (11 °
10ʹ N, 7 ° 38ʹ E) in the northern Guinea savanna (1000 mm rainfall); (c) Minjibir
(12 ° 08ʹ N, 8 ° 40ʹ E) in Sudan savanna with about 700 mm rainfall; and (d) Toumnia
(13 ° 58ʹ N, 9 ° 01ʹ E) or Malamadori representing the Sahel with about 350 mm rain-
fall. High-performing lines from AVT are compiled into cowpea international trials
which are sent to collaborators for testing in their local environments. The best lines
are released as varieties or used in their breeding programs for further improvement.
240 O. Boukar et al.

In some cases, this traditional approach to cowpea breeding which is based on

phenotypic selection of progenies carrying the desired traits is still continuing in
most programs. This is particularly the case where molecular markers for geno-
typing are unavailable, or where the marker-trait locus associations have not been
identified. The advent of new marker technologies for genotyping, explained in
detail in the next few paragraphs, is expected to facilitate a more efficient breed-
ing approach than the previous reliance on phenotypic selection which is time-
consuming. Thus, marker-assisted pedigree breeding (MAPB) and marker-assisted
backcrossing (MABC) have been incorporated into several cowpea breeding pro-
grams by taking advantage of the new marker-driven selection tools. For MAPB,
the design of an “ideotype” as the breeding goal is a useful first step in targeting the
genotype of the combined set of favorable alleles to be donated by the two parents.
Ehlers et al. (2012) provided a detailed example of this approach based on experi-
ence with two African cowpea populations derived from “elite” × “elite” crosses
produced by ISRA, Senegal (Mouride × IT84S-2246-4) and INERA, Burkina Faso
(IT93K-503-1 × IT84S-2246-4). The ideotype design is the full combination of the
target QTLs all in the homozygous condition for the favorable alleles. Molecular
breeding software programs (see next section) can then be used to analyze the ge-
notypic data to identify and rank families or individual plants in progenies with the
appropriate molecular scores (ideotype = maximum score or 100 %).
In MABC, the marker genotyping application has two components, namely
“foreground” selection for the presence of the target trait QTL using linked flanking
markers at the trait QTL, and “background” selection using genome-wide markers
to select for individual plants with the highest recurrent parent genotype profile. In
the California Blackeye cowpea-breeding program, transferring aphid, nematode,
and Fusarium wilt resistances into improved versions of the current varieties CB27,
CB46, and CB50 is being achieved with a MABC approach. For example, the QTLs
identified by Pottorff et al. (2012, 2014) for resistance to Fusarium wilt races 3 and
4 are being transferred through MABC into new lines. CB46 is race 3 resistant but
lacks race 4 and aphid resistances, so markers for the resistance loci can be used
to select the donated favorable alleles for resistances to aphid and Fusarium race
4 and also confirm the presence in the background of the favorable haplotypes for
Fusarium race 3 and root-knot nematode resistances. In Africa, several breeders
have started using MABC to add Striga and aphid resistances into elite local variet-
ies through collaborations with advanced research institutions.
A third breeding approach is MARS, which is being tested in four African cow-
pea breeding programs at INERA, Eduardo Mondlane University, IITA, and ISRA
in partnership with UCR. MARS has been used with success in some cereal breed-
ing programs (Charmet et al. 2001). The goal of MARS is to combine multiple
favorable traits from two parents in a complementary manner, in which early gen-
eration progenies with partial combinations of the full set of traits are intercrossed
for as many as three cycles of recombination to maximize the pyramiding of QTLs
for multiple traits into advanced breeding lines (Charmet et al. 2001). This ap-
proach overcomes the limitations which occur in pedigree breeding with manage-
able progeny sizes, especially when the favorable alleles at several QTLs need to be
7 Cowpea 241

combined. The geometric increase of a target locus becoming homozygous for the
unfavorable allele with each generation, renders individual homozygous positives
for all target QTLs rare or absent (Ehlers et al. 2012).
A series of MARS populations were developed from crossing elite parents with
complementary trait sets (Suvita 2 × IT97K-499-35, IT84S-2246-4 × IT98K-1111-1,
CB27 × IT97K-499-35, IT93K-503-1 × Mouride) relevant to each target environ-
ment in the four SSA cowpea breeding programs, with a focus on drought toler-
ance, seed size, and color, yield gain and biotic stress resistance (Striga and root-
knot nematode). About 300 F2 seeds were derived from each biparental cross fol-
lowed by selfing to produce about 300 F2:3 or F2:4 families. Each of the F2:3 or F2:4
populations was phenotyped in different field sites for yield and other agronomic
traits. Leaf samples of F2 individuals or F2:3/F2:4 bulks from the field were geno-
typed by KBioscience (www.lgcgenomics.com) using a customized list of poly-
morphic SNPs generated by SNP Selector (www.breedit.org) based on distance in
cM between genome-wide markers, and between markers at known trait positions.
QTLs were discovered at F2:3 or F2:4 generations. For recombination cycles, QTL
indices were computed by OptiMAS (http://moulon.inra.fr/optimas/), and members
of highest QTL-index families were then genotyped, selected, and intercrossed to
recombine favorable alleles. The outcome of this MARS breeding plan is that ad-
vanced lines have been produced that are homozygous for the favorable alleles of
the target QTLs for yield, seed size, heat tolerance, staygreen, and resistance to
root-knot nematodes and Striga. Currently the 20–30 advanced lines with the QTLs
confirmed by SNP-genotyping are in a 2-year performance testing phase in pro-
duction field trials, from which new variety releases are expected, and which will
provide elite lines for use as parents in further cowpea improvement.

8 Integration of New Biotechnologies in Breeding

Programs

High levels of resistance to several insects and diseases exist in wild Vigna spe-
cies, but cross incompatibility with cultivated lines is the biggest bottleneck limit-
ing their exploitation for cowpea improvement through conventional breeding.
Biotechnological approaches were suggested as ways to overcome these limita-
tions. If useful genes can be isolated from wild Vigna species, a genetic transfor-
mation system is a prerequisite for their deployment in cultivated cowpea. Initial
genetic transformation efforts using Agrobacterium tumefaciens as the gene vec-
tor were conducted by Garcia et al. (1986, 1987). This was followed by embryo
imbibition with or without subsequent electroporation (Akella and Lurquin 1993;
Penza et al. 1992).
In all these cases, the development of transgenic cowpea calli or chimeric plant-
lets from leaf discs, axilliary buds, or embryos were obtained but no mature trans-
genic plants could be generated. Microprojectile bombardment (biolistics) was also
used by several researchers to achieve the introduction of foreign DNA into cowpea
242 O. Boukar et al.

leaf tissues and embryos and to obtain high levels of transient expression of the
ß-glucuronidase transgene, but regeneration of plantlets from the transformed cells
was not possible (Kononowicz et al. 1997). The development of transformation
systems using either microprojectile bombardment or Agrobacterium cocultivation
gave some promising results with the coculturing of de-embryonated cotyledons
with A. tumefaciens resulting in selection of four plants on hygromycin (Kononow-
icz et al. 1997). This last approach helped in the development of a system that was
the first to be reproducible and that obeys Mendelian rules of inheritance (Popelka
et al. 2006). Critical features of this system include suitable explants from cotyle-
donary nodes or embryonic axes and a tissue-culture regime without auxins, but
which includes a cytokinin at low levels during shoot initiation. There are now
several reports showing experimental evidence for reproducible gene transfer to
cowpea including genes for resistance to pod borer (Higgins et al. 2012) and cow-
pea weevil (Solleti et al. 2008) as well as for weed control (Citadin et al. 2013) and
a range of model genes to evaluate the technology (Citadin et al. 2011).
The development of cowpea with a Bt gene was carried out successfully in the
Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Aus-
tralia. Field testing of these lines has been carried out in Nigeria, Burkina Faso and
Ghana in a Pod Borer-Resistant (PBR) Cowpea Project led by the African Agricul-
tural Technology Foundation (AATF) and supported by USAID. A selected Bt cow-
pea line with near complete resistance to Maruca pod borer is being used to intro-
gress the Bt gene into farmer preferred varieties. Selection using molecular markers
will expedite the rapid development of cowpea varieties with resistance to Maruca
and incorporating other traits preferred by farmers. Encouraging results have been
obtained by the relevant breeding programs in SSA, and the AATF is working to-
wards commercializing and making the PBR cowpea available to farmers in SSA.
In addition to genetic transformation, molecular breeding for cowpea is also well
advanced but requires a genotyping capability that is cost effective and efficient so
that genotyping results can be generated and interpreted quickly enough to make
breeding selection decisions for crossing or targeted phenotyping. The genomics
revolution has had important positive impacts on modern cowpea breeding. SNP
genotyping platforms were developed based on genic SNPs developed from ex-
pressed sequence tags. They have high rates of polymorphism among the primary
cowpea germplasm sources in the target breeding programs. Muchero et al. (2009a)
developed a 1536-SNP Illumina GoldenGate assay which included about 1100 ge-
netically mapped SNPs. This platform was used for extensive QTL discovery and to
develop a consensus genetic map for cowpea constructed from six RIL populations
(Muchero et al. 2009a).
The cowpea consensus map has been improved several times (Diop et al. 2012;
Lucas et al. 2011) and the current version constructed from 11 RIL populations and
two breeding populations is available online via HarvEST:Cowpea (Close and Wa-
namaker 2001). The 1536-SNP platform and the genetic maps were used to identify
the genomic positions of the many QTLs for important cowpea traits described
earlier. The QTL discovery has been based on extensive phenotyping for agronomic
as well as abiotic and biotic stress resistance traits in the genotyped biparental RIL
7 Cowpea 243

populations and cowpea diversity panels (Huynh et al. 2013b; Muchero et al. 2013;
Lucas et al. 2013b; Pottorff et al. 2014). The SNP calls associated with the favorable
alleles at each QTL provide the marker haplotypes needed for positive trait selec-
tion for use in foreground selection. The genome-wide markers across the 680 cM
genetic map, spaced on average, every 0.6 cM, provided the resource for back-
ground selection across the genome in the MABC breeding efforts.
Application of the SNP marker resource for cowpea breeders was further ad-
vanced by converting the mapped SNP markers to a flexible genotyping platform,
using the Kompetitive Allele Specific PCR (KASP) technology of LGC Genomics
(formerly KBiosciences). This platform enables choice in which and how many SNP
markers the breeder would like to use in a given breeding project and flexibility in
the number of DNA samples per genotyping run. This translates into a more cost-
efficient genotyping capability than the fixed GoldenGate platform. A new 60,000
SNP genotyping platform has been developed using the Illumina Infinium iSelect
technology (Close et al. 2015). The cowpea breeding programs with a strong focus
on molecular breeding have taken advantage of outsourcing the genotyping work.
In the programs at UCR, INERA, SARI, Eduardo Mondlane University, IITA, and
ISRA, the molecular breeding is underpinned by the outsourcing operation (Ehlers
et al. 2012). Leaf samples (leaf punches placed in 96-well plates and dried with
silica gel for preservation) are collected in the greenhouse or field, then express-
shipped to the genotyping facility (LGC Genomics in the UK or the USA for the
KASP platform), where DNA is extracted and genotyped with a preselected subset
of informative SNP markers. The data are made available usually within 4 weeks,
which can then be interpreted to make breeding selection decisions for crossing or
progeny selections.
Improvements in the workflow for molecular breeding have included develop-
ment of some in-house software programs for SNP selection and data analysis.
They are used in conjunction with the CGIAR IBP (Integrated Breeding Platform)
Breeding Management System software programs for analyzing the genotype and
phenotype data for QTL tracking and in the MARS, MAPB, and MABC breeding
schemes.

9  Seed Production

Improved seeds constitute one of the most important farm inputs needed for in-
creasing agricultural production. High quality seeds of improved varieties should,
therefore, be available to farmers to ensure sustainable crop production. However,
it has been observed that there is not much enthusiasm on the part of large seed
companies to engage in grain legume seed enterprises because of low margin of
profit, as farmers could recycle their own saved seed for up to 5 years (Abate et al.
2012). These authors reported that more than 70 % of farmers use their own saved
seed across the thirteen countries where the Tropical Legumes II (TL II) project is
being implemented. Kenya is the only exception where saved seed supplied just
244 O. Boukar et al.

over 34 % of farmers’ needs. In Mozambique, only 12 % of adopters of improved

cowpea varieties bought improved seed from agro-dealers, with the rest using their
own recycled seed (Fatokun et al. 2012b).
Strategies to ensure some level of production of good seeds include strengthen-
ing community-based and farmer-level seed production systems. Generally, the Na-
tional Agricultural Research Institutes are responsible for the production of breeder
and foundation seeds. Individual farmers and farmers’ groups, agricultural universi-
ties, and small private seed companies also produce foundation seeds. The private
sector and farmers’ groups are generally responsible for certified seed production.
In some countries, small-scale farmers and the public sector with the use of contract
farmers also produce certified seeds. Other quality seeds are also produced by farm-
ers’ associations generally supported by NGOs. In Nigeria for example, breeder and
foundation seeds of cowpea are produced mainly by research institutes such as IITA
and the Institute for Agricultural Research while certified seeds are produced by
seed companies (e.g., Maina Seeds, Alheri Seeds, Jikur Seed), Agricultural Devel-
opment Projects, some NGOs, out-growers, and National Program for Food Secu-
rity community seed growers.
Access to quality seed is a crucial factor in the adoption of improved technolo-
gies by farmers. Use of improved, modern varieties was generally low across some
SSA countries following baseline studies conducted at the beginning of phase I
of the TL II project (Abate et al. 2012). It was also reported by these authors that
unavailability of improved seed and, in some cases, lack of access to credit were
major bottlenecks for improved variety adoption. Fatokun et al. (2012b) noted that
in Mozambique over 70 % of non-adopters of improved cowpea indicated lack of
access to improved seeds as the major constraint. In Nigeria, 71 % of male-headed
households complained about lack of cash availability to purchase seeds and other
inputs. In their investigations of the cowpea seed subsector in Nigeria, they found
that access to quality, and affordability, of improved seeds was of concern. Most
cowpea producers (60 % in Kano and 86 % in Borno States of Nigeria) get informa-
tion on availability of seeds of improved varieties through Ministries of Agriculture
extension agents. Few producers get information from seed companies, research
institutes’ staff, fellow farmers, and NGOs in Kano State. These observations have
implications for the adoption of new varieties.
The cowpea seed system receives very little attention from the formal seed in-
dustry consisting of public sector research institutions, seed companies, and orga-
nizations (the National Agricultural Seed Council) in almost all the countries of
SSA. A larger proportion of the smallholder farmers’ seed needs are therefore met
by the informal sector. With the low level, or even absence, of the involvement of
large-scale seed companies, it is important to strengthen the informal sector and use
it as a means of providing resource-poor farmers with quality seeds of improved
varieties of crops at affordable prices. Concerted efforts are being made to promote
the ­dissemination of seeds of improved cowpea varieties in many SSA countries.
Farmer-to-farmer seed diffusion was jointly promoted by IITA, IAR and Kano State
Agricultural and Rural Development Authority to disseminate new cowpea variet-
ies (IT90K-277-2 and IT93K-452-1) in the late 1990s. About 8 kg of cowpea seeds
7 Cowpea 245

were given to each primary farmer selected to establish a 0.4 ha of seed farm. The
300 kg of seeds produced by each of the primary farmers (foundation seeds step
1 = FS 1) were distributed/sold to 12 secondary farmers. Each secondary farmer in
turn established a 0.4 ha seed farm (FS 2) and the 300 kg produced by each farmer
gave a total of 3600 kg which was enough to plant 1444 × 0.4 ha of commercial crop
(Utoh and Ajeigbe 2009).
This strategy was found to be faster and cheaper for seed dissemination than
previously used methods. In Nigeria, community seed production was promoted
by the National Program for Food Security. Farmers were trained in seed produc-
tion strategies and linked to seed companies and research institutions for renewal
of seed stocks. The role of extension agents is very important in seed production
and adoption of new improved varieties. In southern Borno State, the Promoting
Sustainable Agriculture in Borno State program implemented by IITA and national
partners selected and trained seed producers and assisted them with establishing
community-based seed multiplication schemes in 30 communities that covered
three agroecological zones (Fatokun et al. 2012b). The TL II project helped to estab-
lish an awareness creation system for improved varieties through field days, dem-
onstrations, seed fairs, agricultural shows, dealing with farmers’ research groups/
farmer field schools, and distribution of small packs of seed samples. The small
seed pack strategy, developed in partnership with the private sector, was helpful in
getting seeds of improved varieties to many more farmers. Marketing seed in small
quantities of 1- or 2-kg packs that are within the reach of smallholder farmers was
found to be both profitable to a small private seed company and attractive to farmers
(Fatokun et al. 2012b). Over 12,000 farmers were reached with this method over
a 3-year period (2010–2012) and this further popularized some improved cowpea
varieties.
Improved market linkages have encouraged seed producers to increase seed pro-
duction to supply a growing market. Market development for cowpea seed resulted
in increased production and sales of cowpea, making significant contributions to
improving livelihood and poverty reduction. Over 188 MT of seed was sold by seed
producers in Nigeria, 31.5 MT in Mali, and 93.7 MT sold in Niger, within the first
phase of TL II project (Fatokun et al. 2012b). This market is now established and
paying good prices for seeds, a situation likely to be sustained.
Based on our experience with the TL II project, strengthening of community-
based organizations, in particular the farmers’ groups and associations, through
training and support for quality certified and foundation seed production, reinforced
with postharvest processing, storage, distribution, and marketing will ensure that
quality the seed of newly developed and released varieties will be available to a
majority of farmers.
Another major constraint to seed trade in cowpea is the susceptibility of the
seeds to the bruchid weevil ( Callosobruchus maculatus). This insect is capable of
destroying all seed stored by farmers within 6 months. No cowpea variety has re-
sistance to this insect pest. In order to protect the seeds from damage by the in-
sect, farmers use a number of methods which are mostly not effective. In recent
times, ­researchers at Purdue University, USA have come up with a simple and cheap
246 O. Boukar et al.

chemical-free method for storing cowpea seeds. The technology is commonly re-
ferred to as Purdue Improved Cowpea Storage (PICS). With this method farmers
and seed retailers are able to store cowpea seeds longer than hitherto and this should
encourage them to produce and keep seeds for planting in the following cropping
season.

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Chapter 8
Grass Pea

Nuno Felipe Almeida, Diego Rubiales and Maria Carlota Vaz Patto

1 Introduction

Grass pea ( Lathyrus sativus L.) is a multipurpose robust grain legume crop. It can
grow in both drought- and flooding-prone environments and poor soils due to its
hardy and penetrating root systems (Campbell 1997; Vaz Patto et al. 2006b). It has
a high nutritional value (protein content ranging from 25 to 30 %), being important
both for human food and animal feed. In what concerns human consumption, it can
be consumed uncooked as a green snack, cooked in a stew, milled into flour or by
roasting the seed (Peña-Chocarro and Peña 1999). In addition to its uses as food
and feed, symbiosis with rhizobia allows an efficient nitrogen fixation in the soil,
lowering the inputs needed in crop rotation and making them suitable to be used as
green manure in sustainable farming systems (Hanbury et al. 2000). As an example
of its versatility, grass pea is easily introduced in intercropping systems, rotations or
used along with paddy rice in relay cropping systems (Abd El Moneim et al. 2001;
Campbell et al. 1994; Hillocks and Maruthi 2012).
There is great potential for the expansion in the utilization of grass pea in dry
areas or zones which are becoming more drought prone, with increased salinity or
increased tendency to suffer from biotic stresses. However, the crop is unpopular

N. F. Almeida ()
Instituto de Tecnologia Química e Biológica António Xavier (ITQB),
Av. da República, EAN, Apartado 127, 2781-901 Oeiras, Portugal
e-mail: [email protected]
D. Rubiales
Institute for Sustainable Agriculture, CSIC, Avenida Menendez Pidal s/n,
14004 Córdoba, Spain
e-mail: [email protected]
M. C. Vaz Patto
Instituto de Tecnologia Química e Biológica António Xavier (ITQB),
Av. da República, EAN, Apartado 127, 2781-901 Oeiras, Portugal
e-mail: [email protected]
© Springer Science+Business Media New York 2015 251
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_8
252 N. F. Almeida et al.

with governments and donors because the plant contains small amounts of a toxin,
β-N-ozalyl-l-α, β-diaminopropanoic acid (ODAP). Although this toxin can cause a
neuronal disorder, known as “lathyrism”, the condition develops in humans with a
6 % chance only when grass pea is consumed in large quantities, unaccompanied by
other foodstuffs in an unbalanced diet and during a long period of time (Lambein
et al. 2009). Also, seeds can be partly detoxified by various processing methods
(Kumar et al. 2011; Kuo et al. 2000).
Even though this robust crop is rightly considered as a model crop for sustain-
able agriculture and despite the lathyrism stigma, the development of new breed-
ing technologies and the growing interest in its use in Mediterranean-type environ-
ments, all over the world, will provide a bright future to this crop (Vaz Patto et al.
2006b; Vaz Patto and Rubiales 2014).

2  Origin and Systematics

Grass pea belongs to genus Lathyrus, within the Fabaceae family (syn. Legumino-
sae), subfamily Faboideae (syn. Papilionoideae), tribe Fabeae (syn. Vicieae), along
with genera Pisum, Vicia, Lens and Vavilovia (Kenicer et al. 2005; Schaefer et al.
2012; Smýkal et al. 2011; Wojciechowski et al. 2004).
Natural distribution of grass pea has been completely obscured by human cul-
tivation. Its use for food, feed and forage makes it difficult to distinguish between
wild and domesticated populations, toughening the task to precisely locate its cen-
tre of origin (Kumar et al. 2013). The most probable grass pea centre of origin
is believed to have been in the eastern Mediterranean or Fertile Crescent, around
6000 before present (BP). This has been supported by archaeobotanical and recent
phylogenetic reports (Kislev 1989; Schaefer et al. 2012), refuting the hypothesis by
Smartt (1984) that the centre of origin was located in Southwest or Central Asia.
Domestication of grass pea seems to have occurred alongside with other pulses, be-
ing normally found with early domesticates of pea ( Pisum sativum L.), lentils ( Lens
culinaris Medik.) and bitter vetch ( Vicia ervilia (L.) Willd.; Erskine et al. 1994).
Hopf (1986) hypothesized that L. sativus is a derivative from Lathyrus cicera,
its genetically nearest wild species. In addition, in what concerns domestication
in Southern Europe (France and Iberian peninsula), evidences of cultivation of L.
cicera were found, dating from 4000 or 3000 BP, suggesting that expansion of L.
sativus farming may have also led to the domestication of the local L. cicera (Camp-
bell 1997).
Within the economically important legume crops and model species, P. sativum
is reported as the closest relation to grass pea, followed by lentil, faba bean (Vi-
cia faba L.), barrel medic ( Medicago truncatula Gaertn.), chickpea ( Cicer arieti-
num L.) and Lotus corniculatus L. (Asmussen and Liston 1998; Ellison et al. 2006;
Wojciechowski et al. 2004).
The infrageneric classification of Lathyrus genus has been revised several times,
the one reported by Kupicha (1983) being the most accepted one. In this treatment,
8  Grass Pea 253

the genus is organized in 13 clades (Orobus, Lathyrostylis, Lathyrus, Orobon, Pra-

tensis, Aphaca, Clymenum, Orobastrum, Viciopsis, Linearicarpus, Nissolia, Neu-
rolobus and Notolathyrus). This morphological-based classification has been re-
cently supported by molecular phylogenetic studies using sequence data from the
internal transcribed spacer (ITS) region and from cpDNA (Kenicer et al. 2005,
2009). Schaefer et al. (2012), using nuclear and chloroplast phylogenetic data, fur-
ther suggested that the genus Lathyrus is not monophyletic and recommended that
a more natural classification would be to transfer Pisum and Vavilovia to a then
monophyletic Lathyrus genus.

3  Varietal Groups

Great morphological variation is reported in grass pea, especially in vegetative

characters such as leaf length, while, for instance, its floral characters are much
less variable, showing a clear grouping in flower colour (Fig. 8.1; Jackson and Yu-
nus 1984), as well as its seed and yield traits (Hanbury et al. 1999). Several stud-
ies divided grass pea accessions broadly into two groups: those from the Indian
subcontinent and those from the Mediterranean region. Jackson and Yunus (1984)
reported that all blue-flowered accessions came from Southwest and South Asia,
while the white and mixed-coloured accessions had a more western distribution,
from the Canary Isles to the western republics of the Soviet Union. These authors
also pointed out that white-flowered accessions only had white seeds with no sec-
ondary markings on the seed coat. In accordance with this, Hanbury et al. (1999)
reported that Mediterranean accessions were characterized by larger and whiter
seeds, selected for human consumption, with higher yield potential than the Indian
accessions. Grass pea small-seeded accessions are considered more primitive types

Fig. 8.1   Blue-flowered

Lathyrus sativus genotype
254 N. F. Almeida et al.

and normally associated with hardened seeds like what happens in other Old World
grain legumes such as pea, chickpea or lentil (Chowdhury and Slinkard 2000).
A particular case is the germplasm selected for forage, in the Mediterranean
region, with landraces with broad leaves and pods, but low seed yield (Chowdhury
and Slinkard 2000; Kumar et al. 2013).

4  Genetic Resources and Utilization

Conservation of Lathyrus genetic resources has recently attracted more attention

because of the potential role of this species under the climate change scenario (Ku-
mar et al. 2013).
Grass pea is mentioned in two conservation programmes for major food legumes.
One is the International Treaty on Plant Genetic Resources for Food and Agriculture
(ITPGRFA; FAO 2009), which aims at guaranteeing food security through conser-
vation of biodiversity, fair exchange and sustainable use of plant genetic resources.
This is being accomplished by establishing a global system to provide farmers,
plant breeders and scientists access to plant genetic materials, ensuring that recipi-
ents share benefits with the countries where they have been originated and by rec-
ognizing the contribution of farmers to the diversity of crops used as food.
The other, a more specific programme developed by the Global Crop Diversity
Trust (CGDT) in collaboration with the International Center for Agriculture Re-
search in the Dry Areas (ICARDA), aims for a long-term conservation strategy of
L. sativus, L. cicera and L. ochrus (GCDT 2009). This programme is detailing the
current status of national collections and identifying gaps in collections of these
three species from areas of diversity. Their strategy recommends that documenta-
tion on collections should be upgraded and that more work should be carried out
on characterizing and evaluating collections for key traits, making this data widely
available (Gurung and Pang 2011).
Several ex situ and a few in situ conservation examples exist for grass pea germ-
plasm. The largest Lathyrus ex situ collections are maintained at the Conservatoire
Botanique National des Pyrénées et de Midi-Pyrénées in France (4.477 accessions;
previously at Pau University), by the ICARDA comprising 3.239 accessions and by
the National Bureau of Plant Genetic Resources (NBPGR) in India (2.619 acces-
sions). Smaller, but still relevant, collections are maintained by other banks such as
the Germplasm Resource Information Network (GRIN) from the US Department
of Agriculture (USDA) in the USA, the Leibniz Institute of Plant Genetics and
Crop Plant Research (IPK) in Germany and the Centro de Recursos Fitogenéticos
(CRF) from the Instituto Nacional de Investigación y Tecnología Agraria y Alimen-
taria (INIA) in Spain. Backups from 2.134 grass pea accessions, from 44 countries,
are deposited at the Svalbard Global Seed Vault (http://www.nordgen.org/sgsv/,
accessed June 2014). In what concerns in situ conservation, five genetic reserves
for Lathyrus diversity conservation have been proposed in Syria and Turkey (Hey-
wood et al. 2007). These authors also stressed the importance of increasing public
8  Grass Pea 255

awareness for the significance of crop wild relatives in agricultural development

and the need for their simultaneous conservation.
This conserved germplasm represents a valuable reservoir of diversity, providing
access to sources of a wide range of interesting agromorphological traits such as
earliness, plant architectural traits, disease and pest tolerance, as well as low ODAP
content. Characterization of this diversity through phenotyping and genotyping
studies will unveil novel alleles that can be used to improve this crop. Diversity
characterization in Lathyrus germplasm has focused, for example, on ODAP con-
tent (Fikre et al. 2008; Grela et al. 2012; Kumar et al. 2011), phenology and yield
(Grela et al 2012; Mera 2010), parasitic weed resistance (Fernández-Aparicio et al.
2012), disease resistance (Gurung et al. 2002; Vaz Patto et al. 2006a; Vaz Patto and
Rubiales 2009) or quality traits (Granati et al. 2003). Some of these characterization
studies have represented the first step of selection programmes.

5  Major Breeding Achievements

Conventional grass pea-breeding programmes have been established in several

countries, including Australia (Hanbury et al. 1995), Bangladesh (Malek 1998),
Canada (Campbell and Briggs 1987), China (Yang and Zhang 2005), Chile (Mera
et al. 2003), Ethiopia (Tadesse and Bekele 2003), India (Lal et al. 1986; Pandey
et al. 1996), Nepal (Yadav 1996) and Syria (Abd El-Moneim et al. 2000). Some
of these breeding programmes are still active, but most are small in comparison to
other legume crops (Vaz Patto et al. 2011).
Due to the occurrence of lathyrism in humans, major breeding programmes are
essentially aimed for low ODAP content, besides productivity and adaptability. This
has resulted at present in several L. sativus or L. cicera breeding lines or released
varieties with reduced ODAP content (from 0.5 to 1.5 %, down to 0.01 % or less;
Kumar et al. 2011). For instance, low ODAP cultivars have been released in several
countries, such as ‘Wasie’ in Ethiopia, ‘Ali-Bar’ in Kazakhstan and ‘Gurbuz 1’
in Turkey (ICARDA 2006, 2007). Similarly, low ODAP, high-yielding cultivars
have been released in India such as ‘Pusa 24’, ‘Prateek’, ‘Ratan’ and ‘Mahateora’
(ICAR 2009). In Bangladesh, examples of the low ODAP and high-yielding varieties
are ‘BARI Khesari 1’, ‘BARI Khesari 2’ and ‘BARI Khesari 3’ (Malek 1998) or
the ‘BINA Khesari 1’ (Kumar et al. 2011). In Canada, high yield and low ODAP
(0.03 %) ‘LS8246’ was released for feed and fodder (Campbell and Briggs 1987),
and in addition, a high N-fixation variety, ‘AC Greenfix’ was released specially as
green manure (Krause and Krause 2003). In Chile, ‘Luanco-INIA’, a large-seeded,
high-yielding grass pea variety was released, used locally as feed and for export,
especially for some European markets where larger seed size is desirable for human
consumption (Mera et al. 2003). Finally, in Australia, the variety ‘Ceora’ was bred
to be used as forage, hay or as a green manure crop (Siddique et al. 2006). Also in
Australia, a L. cicera cultivar, ‘Chalus’, was selected for high yields and low ODAP
levels (Hanbury and Siddique 2000).
256 N. F. Almeida et al.

6  Specific Goals in Current Breeding

Low ODAP content is still one important goal of many of the current grass pea-
breeding programmes. Nevertheless, other traits have always been associated with
this.
Increased yield has been a selection criterion for most crop improvement
programmes. However, some of the yield components that affect yield such as
double podding or increased seeds per pod have received insufficient attention. Also
the biomass yield of L. sativus has started to receive more attention during the past
few years (Campbell 1997; Abd El Moneim et al. 2001; Vaz Patto et al. 2006b). This
is a very important area due to the large potential of this crop for forage and straw
in the North African and South Asian regions (Campbell 1997). Additionally, unde-
sirable traits such as prostrate plant habit, indeterminate growth, late maturity and
pod shattering (Rybinski 2003) are being handled by several breeding programmes.
The concentrated effort on reducing ODAP content resulted in many other ar-
eas of evaluation and crop improvement, such as resistance to biotic and abiotic
stresses, being neglected. However, with the release of these low ODAP lines, the
development of varieties with increased resistance to prevalent pests and diseases
has gained new strength. This crop is usually grown by poor farmers and under
poor management, where it is difficult to adopt chemical control for diseases and
pests. Therefore, the development of varieties having resistance to prevalent biotic
stresses is essential, and more efforts are required in this area of improvement of
this very hardy pulse crop (Vaz Patto et al. 2006b).

7  Breeding Methods and Specific Techniques

Collection and evaluation of germplasm, local or introduced, is the cornerstone in

any breeding programme. Subsequent hybridization and selection of the resulting
progeny using different strategies will allow incorporating interesting traits into
a more adapted background. This may include backcrossing, recurrent selection,
single-seed descent and pedigree/bulk breeding methods. All of these methods can
be applied on grass pea improvement.
Grass pea is predominantly a self-pollinated crop, although outcrossing up to
30 % has been reported (Ben Brahim et al. 2001; Chowdhury and Slinkard 1997;
Rahman et al. 1995). Large size of flower, bright colour of petals, flower density
and nectar production are reported to influence the outcrossing in Lathyrus species
(Kiyoshi et al. 1985). Entomophilic pollination in grass pea is due especially to
bees and bumblebees (Kumar et al. 2011). Due to this observed outcrossing level, in
most grass pea-breeding programmes, crosses are done under controlled conditions,
in greenhouse or under insect-proof coverings (Vaz Patto et al. 2011).
Conventional grass pea breeding focussed essentially on hybridization of se-
lected accessions, with the screening and evaluation of the resulting progeny. In
8  Grass Pea 257

the particular case of breeding to reduce ODAP content, low ODAP accessions are
crossed with high-yield material with good agronomic potential (Campbell 1997).
Intergeneric hybridization, although difficult, is possible with L. amphicarpos
and L. cicera (Yunus and Jackson 1991). Crosses have also been made with other
species such as L. chrysanthus, L. gorgoni, L. marmoratus and L. pseudocicera
(Heywood et al. 2007), but only ovules were produced.
Also with the objective of reducing ODAP content, grass pea has been subjected
to induced mutagenesis by physical and/or chemical mutagens. Other traits have
been affected by mutagenesis such as plant habit, maturity, branching, stem shape,
leaf size, stipule shape, flower colour and structure, pod size, seed size and colour
and NaCl tolerance (Biswas 2007; Nerkar 1972, 1976; Rybinski 2003; Talukdar
2009a, b, 2011). In vitro culture was also employed, inducing somaclonal variation
(Ochatt et al. 2002a; Roy et al. 1993; Zambre et al. 2002). Induced mutagenesis
and somaclonal variation created new diversity, allowing the selection of lines with
interesting traits.
Ochatt et al. (2002b) developed an in vitro system coupled with in vitro stages
in order to shorten regeneration cycles, obtaining up to almost four cycles per year.
However, this approach is only applicable when few seeds/plant are intended, as in
single-seed descendant breeding schemes.
The advent of various molecular-marker techniques and the ability to transfer
genes across different organisms, using transgene technology, have begun to have
an impact on plant genome research and breeding. These techniques offer new ap-
proaches for improving important agronomic traits in Lathyrus species and break-
ing down transfer barriers to related legume species (Vaz Patto et al. 2006b). This
would allow exploring the variability existing in other Lathyrus genepools and
hopefully transfer the interesting grass pea traits to related legume species.
Genetic transformation of grass pea was attempted with only one successful re-
port obtaining stable transformed plants (Barik et al. 2005). Given that regeneration
protocols for grass pea are often genotype specific, it may be necessary either to
develop more generally applicable protocols or to adapt the protocol after transfor-
mation (Ochatt et al. 2013).

8 Integration of New Biotechnologies in Breeding

Programmes

In order to be able to perform marker-assisted selection (MAS), it is necessary to

identify molecular markers that are closely linked to the trait of interest. Once a trait
is associated with a marker (or more), plants can be selected early on its growth
stage, allowing a faster and more efficient breeding process.
Until now, only two linkage maps using molecular markers were developed for
L. sativus. One developed by Chowdhury and Slinkard (1999) used 11 random am-
plified polymorphic DNA (RAPD) markers, 1 isozyme marker and 1 morphological
trait (flower colour). The other linkage map was constructed by Skiba et al. (2004),
258 N. F. Almeida et al.

using 47 RAPDs, 7 cross-amplified pea microsatellite simple sequence repeats

(SSR) markers and 13 cleaved amplified polymorphic sequence (CAPS) markers
and was used to study the genetic basis of resistance to Ascochyta blight. Never-
theless, these maps were not informative enough to allow bridging that mapping
information between them, as reviewed by Vaz Patto et al. (2006b).
Compared to other grain legumes such as pea, faba bean or chickpea, genomic
recourses for grass pea are still scarce. In July 2014, the National Center for Bio-
technology Information (NCBI) database had made available the information of
178 EST sequences from a cDNA library of one L. sativus accession inoculated with
Mycosphaerella pinodes (Skiba et al. 2005), 89 nucleotide sequences mainly from
the Bowman–Birk (BBI) inhibitor coding sequences (41 accessions), chloroplast
sequences (21 accessions) and 216 protein sequences (44 amino acid sequences
from BBI inhibitors, 150 sequences from chloroplast proteins).
Specific molecular markers have been developed or adapted for grass pea in
order to assist diversity studies and further develop linkage maps. Almeida et al.
(2014a) studied the transferability of molecular markers from M. truncatula, P.
sativum, L. culinaris, Lupinus spp. and V. faba to Lathyrus spp. and their appli-
cation in mapping and diversity studies. Cross-genera amplification of molecular
markers provided an alternative for the development of new molecular markers
on understudied genus, allowing also performing comparative mapping between
the sequence donor and the target species. This survey for similar genetic regions
among closely related species will contribute to the potential future exchange of
interesting traits among them.
Earlier molecular markers, specific or cross amplification studies in grass pea,
included the work of Shiferaw et al. (2011) that successfully amplified nine ex-
pressed sequence tag-simple sequence repeats (EST-SSRs) developed from the EST
sequences of Skiba et al. (2005) and 12 EST-SSRs from M. truncatula, which have
been previously proven to be transferable to other legume species by Gutierrez et al.
(2005).
Lioi et al. (2011) were able to genotype in a grass pea diversity study, ten SSRs
developed from nucleotide sequences stored at public databases, being nine from L.
sativus sequences and one from a L. japonicus sequence.
More recently, Yang et al. (2014) employed next-generation sequencing (NGS)
to develop 144 specific grass pea SSRs, from which, 74 where polymorphic and
therefore useful for diversity studies and genetic mapping.
The first grass pea expression analysis was performed by Skiba et al. (2005),
identifying 29 potential defence-related genes differentially expressed in response
to M. pinodes inoculation. These included genes associated with pathogen recogni-
tion, the phenylpropanoid pathway, hypersensitivity, pathogenesis-related and dis-
ease resistance response proteins.
In addition, expression analysis using RNA-sequencing was also employed in
grass pea to tackle the molecular mechanisms underlying prehaustorial rust resis-
tance (Almeida et al. 2014b). These authors identified several pathogenesis-related
proteins as possibly involved in grass pea resistance to rust, that included some
regulated by the well-studied mildew resistance locus O (MLO) gene. In this study,
8  Grass Pea 259

several potential rust effectors were also identified. These could be used as probes to
identify target grass pea host proteins, as a first step in the development of effector-
driven legume breeding, maximizing the durability of resistance against this quickly
evolving pathogen (Vleeshouwers et al. 2011). Finally this RNA-sequencing study
also identified several polymorphic single-nucleotide polymorphism (SNPs) and
EST-SSRs between parental lines of existing grass pea segregating recombinant
inbred lines (RILs), allowing its use for linkage mapping.
Expression analysis of the response to infection with Ascochyta lathyri in a resis-
tant grass pea accession was performed using deepSuperSAGE. This approach has
identified several differentially expressed genes (Almeida et al. 2015), opening the
way to a powerful route of identification of candidate resistance genes and more de-
tailed study of resistance gene networks in L. sativus (Vaz Patto and Rubiales 2014).

9  Future Prospects

The present paradigm change towards the study of crop species instead of focus-
ing on model species will aid in the development of plant species that have been
neglected. Lowering costs in high-throughput sequencing and the development of
high-throughput phenotyping have encouraged the development of new molecular
tools to boost the genetic characterization and utilization of the rich Lathyrus germ-
plasm.
Grass pea research was tied to the persecution of an ODAP-free variety for sev-
eral decades. This has hampered progress in this crop for the improvement of other
traits. As an example and despite its importance, ODAP-related research should
not block the understanding of the reasons behind the success of grass pea when
dealing with biotic and abiotic stresses, and for which it is considered a survival
crop. In an alternative to low ODAP varieties, an option might be improving quality
traits that can lower ODAP’s negative effects. These include increasing the content
in homoarginine, cysteine or methionine. Although this is an old objective, it is
still unachieved, due to the presence of technical barriers in the regeneration of
transformed tissues (Girma and Korbu 2012) or the high influence of genotype ×
environment in those traits (Piergiovanni et al. 2011; Piergiovanni and Damascelli
2011).
For quicker progress on these and other quantitative traits improvement via
MAS, it would also be useful to have a saturated linkage map, including cross-
transferable markers to other related species such as pea, faba bean or the model
M. truncatula, to apply in quantitative trait loci (QTL) mapping. In this way, com-
parative mapping would also be possible to other closely related legume species,
assisting knowledge transfer among these species and facilitating candidate gene
discovery for the detected potential QTL regions.
With the development of high-throughput and dense genotyping, assessment of
the correlation between phenotype and genotype, needed for the development of
MAS approaches, has shifted from focusing on two parental lines, differing strong-
260 N. F. Almeida et al.

ly in phenotype, to the analysis of populations of unrelated individuals. Association

mapping panels by sampling more genetic diversity can take advantage of many
more generations of recombination and avoid the time-consuming generations of
crosses (Morrell et al. 2012). High-throughput genotyping associated with a core
collection evaluation will facilitate trait dissection and gene discovery through as-
sociation mapping as well as characterization of the collection genetic structure
(Cobb et al. 2013). That is why Vaz Patto and Rubiales (2014) supported the idea
of concentrating international evaluation efforts on to a grass pea core collection,
representative of all the existing diversity, but of a manageable size. For adaptive
traits, core and mini-core collections may not capture the needed diversity (Gepts
2006). As an alternative, the Focused Identification of Germplasm Strategy (FIGS)
approach, which is a trait-based approach with high probability of identification of
desired genetic material (Khazaei et al. 2013) is being applied at ICARDA to the
Lathyrus germplasm collection to develop subset collections.
In terms of grass pea plant resources for functional genomics studies, various
mapping populations including RILs, near isogenic lines (NILs) and targeting in-
duced local lesions in genomes (TILLING) populations are critically needed for
trait–marker association and gene inactivation/deletion studies (Kumar et al. 2013).

10  Seed Production

Several grass pea improved varieties have been originated from various breeding
programmes as already described in the section “Major Breeding Achievements”
section. As in any plant species with outcrossing frequency rate up to 30 %, special
efforts are needed for cultivar conservation. Strategies like an isolation distance or
the use of a buffer crop between cultivars when producing seeds are essential to
maintain genetic purity and phenological features of the developed cultivars.
Nevertheless, the most common seed available is from landraces or farmers’
varieties, inherently heterogeneous. These farm-saved seeds are obtained and traded
within an informal seed system where seeds are exchanged among farmers that
mainly do not sell the product of the seed, but use it for self-consumption.
In conclusion, presently available genetic resources, established breeding
achievements and recent biotechnological progress, associated with a growing in-
ternational interest on grass pea cultivation, will definitely provide a bright future
to this highly potential crop.

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Chapter 9
The Legume–Rhizobia Symbiosis

Jean-Jacques Drevon, Nora Alkama, Adnane Bargaz, A. Paula Rodiño,

Kiriya Sungthongwises and Mainassara Zaman-Allah

1 Introduction

Rhizobial symbiosis is the term for symbiosis between nitrogen-fixing soil bacteria
commonly called rhizobia, and roots of legumes like soya beans, broad beans and
stems in some species like Sesbania sp. However, nonleguminous plant can also
form symbiosis with nitrogen-fixing bacteria, such as actinomycetes, of the genus
Frankia (Tate 1995). Hellriegel and Wilfarth (1888) isolated the first rhizobia able
to induce the nodulation in a controlled experimental environment, thus establish-
ing the basis of the symbiotic relationship between legumes and rhizobial species.

J.-J. Drevon ()
Eco&Sols, INRA, 1 Place Viala, 34000 Montpellier, France
e-mail: [email protected]
N. Alkama
Department des Sciences Agronomiques, Universite de Mouloud Mammeri,
Tizi Ouzou, Algeria
e-mail: [email protected]
A. Bargaz
Biosystems and Technology, Swedish University of Agricultural Sciences,
Sundsvägen 16, 230 53 Alnarp, Sweden
e-mail: [email protected]
A. P. Rodiño
Biology of Agrosystems, Misión Biológica de Galicia (MBG),
Spanish National Research Council (CSIC), El Palacio-Salcedo, 36143 Pontevedra, Spain
e-mail: [email protected]
K. Sungthongwises
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
e-mail: [email protected]
M. Zaman-Allah
CIMMYT, Southern Africa Regional Office, Harare, Zimbabwe
e-mail: [email protected]
© Springer Science+Business Media New York 2015 267
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_9
268 J.-J. Drevon et al.

This symbiosis is readily observable since the nodules are numerous and usually
1–3 mm in size. During symbiotic life, rhizobia differentiate into bacteroids encap-
sulated within the peribacteroid membrane (PBM) inside the infected cells consti-
tuting an organite called the symbiosome (Young and Haukka 1996; Sy et al. 2001).
Bacteroids are able to alter metabolic and molecular processes in the cells and cause
cellular enlargement.
The nodules formed by most of these bacteria are benign and mutually useful
structures, conducting the N2 fixation for the host plants. Generally, symbiosis is
characterized by a more or less distinct host preference or even host specificity.
Poor nodulation may occur even if good seed inoculation practices were used (Zah-
ran 1999). The large differences of the combination between rhizobial strains and
legume genotypes may offer the potential to increase N2 fixation by both rhizobial
and legume selection (Peoples and Craswell 1992). In addition, genetic diversity of
legumes and rhizobia strongly affect SNF as a function of the environment (Poehl-
man 1991). The rhizobia–legume symbiosis is the primary source of biologically
fixed nitrogen for agricultural system (Vance 1997; Kinzig and Socolow 1994).
Some 40–60 million metric tons (Mt) of N2 are fixed by agriculturally important
legumes annually, with another 3–5 million Mt fixed by legumes in natural eco-
systems (Smil 1999). The following revision of the literature focuses on symbiotic
nitrogen fixation (SNF) of legumes.

2  Nodule Formation

Many legumes can nodulate with a diversity of rhizobia strains. Thus, most legume
species have a specific rhizobia strain that maximizes N2 fixation. However, some
rhizobia strains are effective and some are ineffective for SNF. Ineffective strains
will form many small nodules on the legume root but fix little or no nitrogen. Ef-
fective rhizobia strains that fix high rates of nitrogen form fewer but larger nodules
that have dark pink centres. In soil, rhizobia are mobile and rely on chemical signals
like phenolic compounds secreted by the roots, for example, luteolin by Medicago
truncatula, within the rhizosphere of legumes (Peters et al. 1986).
The interaction between a particular strain of rhizobia and the “appropriate” le-
gume is mediated by a Nod factor (NF), a lipochitin oligosaccharide secreted by the
rhizobia and recognized by transmembrane receptors on the root-hair cells of the
legume. NF are produced by rhizobia and act as the main morphogenic molecules
regulating nodule organogenesis (Ferguson and Mathesius 2003). They can be rec-
ognized by receptors for chitin oligosaccharides (Stacey and Shibuya 1997). Mol-
ecules which are related to NF may have a more general role in plant development
(Spaink et al. 1993; van der Holst et al. 2001). Benhamou and Asselin (1989) and
Spaink et al. (1993) have identified within plants, molecules related in their struc-
ture to chitin oligosaccharides which are known to play a role in animal develop-
ment. More particularly, chitin oligosaccharides are substrates for chitinases, which
have been shown to play a role in different aspects of plant development (Collinge
9  The Legume–Rhizobia Symbiosis 269

et al. 1993). Modifying chitin structures by expression of the bacterial nodA and
nodB genes, which modify NF in rhizobia, led to changes in plant development
(Schmidt et al. 1993). If the combination is correct, the rhizobia enter the root-hair
cell or an epithelial cell if the infection proceeds by root-hair curling or lateral-
root cracking, then multiply within the infection thread progressing extracellularly
within the cortex (Crespi and Galvez 2000; Stougaard 2001; Kistner and Parniske
2002). This infection thread is constructed by the root cells and is formed only in
response to the infection (Fernández-Pascual et al. 2007). When the infection thread
reaches a cell in the developing cortex, the rhizobia are engulfed by endocytosis into
endosomes. At this time, the cell goes through several rounds of mitosis without
cytokinesis so the cell becomes polyploid. The cortex cells then begin to divide
rapidly forming a nodule. This organogenesis of nodule starts earlier as a response
to the NF and progresses during the progression of the infection thread (Fernández-
Pascual et al. 2007). Each nodule becomes connected by the xylem and phloem to
the vascular system of the plant.
Thus, the development of nodules, while dependent on rhizobia, is a well-coor-
dinated developmental process of the plant. This response is driven by the translo-
cation of cytokinins from epidermal cells to the cells of the cortex (Sturtevant and
Taller 1989) with subsequent nodule formation in other regions of the root (Suther-
land et al. 1990). Several pieces of evidence suggest that rhizobia induce changes
in the cytokinin balance of the root, through cytokinin synthesis, turnover or sen-
sitivity in the roots during nodule initiation (Ferguson and Mathesius 2003). The
reactivation of the cell cycle was demonstrated to initiate nodule primordium for-
mation (Foucher and Kondorosi 2000; Goormachtig et al. 1997; Yang et al. 1994).
In pea, Newcomb et al. (1976) showed that nodule cytokinin levels were related to
the maturity of the nodule. The application of cytokinins induced the formation of
pseudo-nodule structures on legumes and nonlegumes, such as Nicotiana tabacum
(tobacco; Arora et al. 1959), Alnus glutinosa (Rodriguez-Barrueco and Bermudez
de Castro 1973), Pisum sativum (Libbenga and Harkes 1973), Macroptilium at-
ropurpureum (siratro; Relic et al. 1994) and Medicago sativa (Cooper and Long
1994; Bauer et al. 1996). Moreover, since cytokinins can induce starch formation,
they probably also play a role in setting up a carbohydrate sink for the developing
nodule (Bauer et al. 1996).
The rhizobia may go through a period of rapid multiplication within the nodule.
The term symbiosome refers to the organelle-like structure composed of the PBM,
the peribacteroid space which it encloses and the bacteroids within that space.
Symbiosomes are formed when rhizobia are released from the infection thread into
the infected cells of the root cortex. This occurs by endocytosis of the infection
thread membrane and results in encapsulation of the bacteria in a plant membrane.
Only a few bacteria infect any one cortical cell, and after endocytosis, the rhizobia
proliferate resulting in the cytoplasm of mature infected cells being packed with
symbiosomes. The symbiosomes are intercalated with a honeycomb-like network
of actin filaments, which may play a role in the organization of the symbiosomes
and metabolite movement between them (Whitehead et al. 1998). In the nodules of
temperate legumes such as pea, all symbiosomes contain only single bacteria. In
270 J.-J. Drevon et al.

tropical associations, such as soya bean, the symbiosomes become multi-bacteroid.

Infected root cells may contain up to 20,000 bacteroids.

3  Nodule Function

Generally, effective root nodules are big and have a pink colour by contrast with in-
effective root nodules that remain small and with a white colour (Poehlman 1991).
N2 fixation starts between 10 and 21 days after infection (Marschner 1995). Nodules
occur at 9 days after sowing (DAS), and N2 fixation activity occurs at 11 DAS in
soya beans and 14–25 DAS in mung beans. The number of nodules will increase un-
til flowering stage but the size and weight, leghaemoglobin and nitrogenase activity
will be maximized at 30 DAS. Pawar and Ghulghule (1980) found that nodulation
of cowpea occurs at 7 DAS, then the number of nodules and the weight will be max-
imized at 21 DAS and decrease after 28 DAS. Bushby (1991) studied nodulation
pattern of the mung bean variety Satin and found that this variety produces 40 % of
the nodule weight before flowering stage which will reach 60 % at flowering stage.
SNF with legumes host is catalyzed by the nitrogenase enzyme with the follow-
ing reaction:

N 2 +8e- +8 H + +16 ATP → 2NH3 + H 2 +16 ADP +16 Pi

The overall activity of the whole nitrogenase complex decomposes in two steps:
first step consists in ferredoxin transfer of electron to Fe in component II of nitroge-
nase (dinitrogenase reductase); in second step, component II will transfer electron
to component I (dinitrogenase). Two molecules of ATP are used for transferring
of each one electron. The steps repeat will load electrons in component II until it
reaches a redox potential that will make it possible to reduce N2 and H + into NH3
and H2 (Fig. 9.1). Electron source to reduce N2 comes from leaf photosynthates.
Three major factors are involved in the large potential of nodulated legumes for N2
fixation, namely photosynthate supply, O2 concentration, fixed-N export.
The first factor is the direct supply of photosynthates to the N2-fixing nodules.
About 30 % of carbohydrates from photosynthesis are transferred to roots in order
to support symbiosis (Söderström and Read 1987; Söderström 1992; Jones and Dar-
rah 1996; Farrar and Jones 2000).
During the vegetative growth stage of the legume, the nodules may consume as
much as 20 % of the photosynthates in a legume like cowpea (Pate and Herridge
1978) and half of these photosynthates are respired as CO2. Although, between up to
30 % of the respired CO2 can be reassimilated by the nodules via phosphoenolpyru-
vate (PEP) carboxylase providing up to 25 % of the carbon needed for the synthesis
of malate and aspartate (Fig. 9.2) for the assimilation of NH3 and export to the host
plant (Deroche and Carrayol 1988). Consequently, SNF will decrease or stop if
nodulated-roots lack carbohydrates supply.
9  The Legume–Rhizobia Symbiosis 271

Fig. 9.1   Nodule conductance to O2 diffusion

Fig. 9.2   Infection process in the cells of the nodule. ATP adenosine triphosphate
272 J.-J. Drevon et al.

The second factor is effective maintenance of O2 concentrations in the interior

of the nodules for protection of the nitrogenase against inactivating O2. The low O2
concentrations within nodules are determined by two mechanisms: The existence
of an O2 diffusion barrier in the cortex of the nodules; the high respiration rates of
the bacteroids in an O2-limited environment. Indeed, a short-term decrease in the
nodule conductance to oxygen diffusion appears to be linked with the inhibition of
nitrogenase activity by water deficiency (Durand et al. 1987; Weisz et al. 1985) or
by exposure to acetylene (Witty et al. 1984; Drevon and Hartwig 1997). It has been
suggested that the variable component of the nodule conductance involves changes
in the distribution of air spaces within the nodule cortex. These changes have been
supposed to result from an occlusion of intercellular spaces (Sinclair and Goudriaan
1981; Sheehy et al. 1985; Davis et al. 1987; Witty et al. 1987; Hunt et al. 1988) and/
or changes in the volume of some of its cells (Walsh et al. 1989; Parsons and Day
1990; Vance and Heichel 1991). Short-term decreases in Lupinus albus conduc-
tance, associated with lowering temperature, detopping or darkening plants, or root
exposure to nitrate, were correlated with the occlusion of cortical intercellular spac-
es with a glycoprotein (Iannetta et al. 1993). In the inner cortex, both the “boundary
layer” and the more internal “distributing zone” may be implicated in long- and/
or short-term changes in the nodule conductance to oxygen diffusion (Parsons and
Day 1990). We reported that the inhibition of soya bean nodule nitrogenase activ-
ity and/or respiration by a short-term exposure to supra optimal pO2 or NaCl was
linked with a rapid decrease in the nodule O2 conductance caused by cell collapse
in the inner cortex (Drevon et al. 1988; Serraj et al. 1994). This structural variation
would involve a change in the size and water content of intercellular spaces thus
modifying the nodule conductance.
In addition, the high O2 consumption in nodules for provision of energy as ATP
for nitrogenase creates a great potential for production of toxic oxygen species.
Leghaemoglobin is probably the most important of these sources of reactive O2
because this protein is extremely abundant in nodules and is subject to autoxidation
in which O2−, H2O2 and OH− are produced (Puppo et al. 1981; Puppo and Halliwell
1988). Oxidative damage in nodules is especially important during nodule senes-
cence when free radical production and lipid peroxidation are enhanced by elevated
levels of free iron (Becana and Klucas 1992). For protection against this toxicity,
a defence mechanism by the enzymes of the ascorbate–glutathione cycle rapidly
responds to changes in O2 concentrations in legume nodules (Dalton et al. 1991).
Controlling nutrient transport and oxygen permeability at nodular cortex are par-
ticularly important aspects for the effective function of the nodule, and negatively
influenced by nutrient deficiency, salinity and drought stress (Serraj 2002).
Finally, the third factor is the rapid export of the fixed nitrogen (Vance and Gantt
1992). NH3 produced from SNF is thought to move through PBM to cytoplasm of
the host cell. Then NH3 is incorporated into amino acids by glutamate synthase-
glutamine oxoglutarate aminotransferase (GS-GOGAT), and partly transformed
into ureides such as allantoin and allantoic acid (Pate et al. 1980). These products
are then transported to the above part of the plant in order to produce amino acid,
protein and other N compounds for the growth of the plants.
9  The Legume–Rhizobia Symbiosis 273

4  Environmental Factors Limiting Nitrogen Fixation

Among environmental factors that influence the quantity of nitrogen fixed, the tem-
perature is essential for nodule formation. In temperate zones, the development of
nodules in legumes is maximized between 17 and 22 °C, while in the tropical zone
nodules best develop between 19 and 35 °C. When the temperature reaches these
extrema, the size of nodules is reduced and only a low bacteroids population can
develop. Moreover, temperature extrema also reduce nitrogenase activity signifi-
cantly. In addition, the competitiveness of the rhizobia in forming nodules and the
effectiveness of the rhizobia species–host plant symbiosis to fix N2 are controlled
by a series of edaphic, chemical and biophysical factors (Wangnai 1998).

4.1  Nitrate Inhibition of the Rhizobial Symbiosis

Nitrate (NO3−), ammonium (NH4 + ) and urea around 15–20 kg N ha−1 stimulate
leguminous plants growth at the beginning of the vegetative growth. Nitrogen sup-
ply is needed during the initial phase of nodulation, when the plant cannot rely on
symbiotic N2 fixation (Sousanna and Hartwig 1996). The nitrate nutrition of the le-
gume can however inhibit SNF (Arrese-Igor et al. 1997). It has been suggested that
there are multiple effects of nitrate inhibition. In some studies, nitrate nutrition was
responsible for stopping nodulation and decreasing the number of nodules as well
as their weight. A decrease in the activity of nitrogenase (Fujikake et al. 2003) and
N2 fixation activity was moreover recorded (Arrese-Igor et al. 1998). Other studies
suggested that the regulation of N2 fixation has a direct effect on nitrogenase activ-
ity in the nodules (Ribet and Drevon 1995b; Drevon and Hartwig 1997; Almeida
et al. 2000). This inhibition occurs in areas of legume cultivation, especially in
temperate regions where it prevents the maximum exploitation of SNF.
In addition, Gordon and James (1997) have reported that sucrose synthase activ-
ity and gene transcription in legume nodules are reduced by nitrate absorption. A
decrease in nitrogenase activity inside the nodules was observed within 18 h after
exposing soya bean plants to nitrate application and coupled with the disappearance
of sucrose synthase mRNA within 24 h. These observations suggest that nitrate
feeding may produce a plant-to-nodule signal which could affect both the O2 diffu-
sion barrier and the expression of the sucrose synthase gene. The existence of mu-
tants that hypernodulate even in the presence of nitrate also support the hypothesis
that nitrate is not the inhibiting factor itself, but that it leads to secondary signals
that suppress nodulation (Carroll et al. 1985). Although mechanisms of signals re-
main unclear, Neo and Layzell (1997) have suggested that glutamine and/or as-
paragine could act as signals. It is possible that, in legumes, at least some of the ef-
fects of nitrate are also mediated by auxin. According to the auxin burst hypothesis
(Gresshoff 1993), high auxin levels inhibit nodule formation. It is hypothesized that
nitrate increases the sensitivity of the root to auxin, thus reducing nodule formation.
An effect of nitrate on the auxin response pathway has been found in Arabidopsis
274 J.-J. Drevon et al.

thaliana (Zhang et al. 1999). The nodular NO3− reduction is suggested to be the
major cause of this inhibition.
The nodular reduction of NO3− could also inhibit bacteroidal nitrogenase activity
via NO2− generation and/or reducing power competition. In addition, the nitroge-
nase activity of ex planta bacteroids (Stephens and Neyra 1983) and of free-living
rhizobia (Keister and Evans 1976) is inhibited by NO3− in strains having nitrate re-
ductase (NR) capacity, this inhibition being alleviated in NR mutants (Stephens and
Neyra 1983). The mechanism of this ex planta inhibition could be related to nitrite,
the first product of NO3− reduction. Indeed this ion accumulates in isolated bacte-
roids subjected to NO3− in microaerobiotic conditions (Rigaud et al. 1973); it is also
detected in bacteroids extracted from nodules of symbiosis receiving NO3− (Becana
et al. 1985). It inhibits nitrogenase at concentrations as low as 0.1 mM (Trinchant
and Rigaud 1982). Finally, the local effect of nitrate inhibition on nodulation and
N2 fixation may be related to the fact that high accumulation of nitrate is restricted
in the root parts in direct contact with nitrate (Ohyama et al. 1993). The intensity of
inhibition of nodule growth by NO3− is linked to the treatment period as well as the
tolerance of legume species (Fujikake et al. 2003).

4.2  Salinity and Drought

Abiotic constraints such as salinity and drought (González et al. 2001) strongly
reduce SNF. Several hypotheses have been advanced to explain the negative effect
of salt on SNF in plant legumes: diminished photosynthate supply to the nodule
(Vessey and Waterer 1992; Georgiev and Atkins 1993); reduced supply of respira-
tory substrates to the bacteroids (Delgado et al. 1994); alterations in the oxygen
diffusion barrier which reduces oxygen flux into the nodule and avoids nitrogenase
damage (Serraj et al. 1994). The provision of substrates by the legume host in order
to support N2 in the nodules is an important facet of effective symbiosis, which
has been studied more extensively in ureide-exporting nodules (Day and Copeland
1991; Gordon and James 1997). Salinity can seriously change the photosynthetic
carbon metabolism, leaf-chlorophyll content, as well as photosynthetic efficiency
(Seeman and Critchley 1985; Sharkey et al. 1985). However, salinity is known to
boost the nodular carbohydrate content, and sucrose is the predominant carbohy-
drate in legume root nodules (Fougère et al. 1991; Gordon et al. 1993).
Eventually, there is genetic variability among legumes regarding their tolerance
to salinity. Some legumes such as Vicia faba, Phaseolus vulgaris and Glycine max
are more salt tolerant than others as Pisum sativum. It has been reported that some
Vicia faba tolerant lines sustained SNF under saline conditions (Cordovilla et al.
1995). Other legumes, such as Prosopis spp. (Fagg and Stewart 1994), Acacia spp.
(Zhang et al. 1991) and Medicago sativa (Abdel-Wahab and Zahran 1983) are salt
tolerant. However, these legume hosts are less tolerant to salt than are their rhizobia.
Salt inhibits the initial steps of rhizobia–legume symbioses. Indeed, soil microbial
populations are also negatively affected by increasing salt concentrations as a result
9  The Legume–Rhizobia Symbiosis 275

of direct toxicity as well as through osmotic constraint (Tate 1995).The hypothesis

that the oxygen diffusion barrier is solely responsible for the control of nodule N2
fixation during osmotic constraint has been challenged (Guérin et al. 1990; Diaz del
Castillo et al. 1994; Diaz del Castillo and Layzell 1995) because nodule metabolic
potential is also impaired in response to drought. Under drought conditions, only
the activity of sucrose synthase, the key sucrose hydrolytic enzyme, declined. It is
possible, therefore, that the reduced potential to metabolize sucrose may be an im-
portant factor contributing to the overall response of soya bean nodules to drought.
Sucrose synthase activity and transcript levels also declined significantly and rap-
idly in nodules when soya bean plants were subjected to dark-induced constraint
(Gordon et al. 1993). Therefore, it is possible that the sensitivity of sucrose synthase
gene expression and the in vivo activity of sucrose synthase may be a common fea-
ture of the response of soya bean nodules to environmental constraints.
In drought conditions, the reduced import of photosynthates may cause less car-
bohydrate to be available for nodule function (Durand et al. 1987). Another pos-
sibility is that export of carbohydrates from nodules could be blocked because of
a lack of water (Walsh 1990). The resulting accumulation of N products some-
how causes feedback inhibition of nitrogenase activity or ammonia assimilation. In
previous studies, an accumulation of ureides under drought was observed in both
shoots (Serraj and Sinclair 1996) and nodules (Serraj et al. 1999; Vadez et al. 2000).
Serraj et al. (2001) suggested that ureides accumulation within the plant could re-
sult in the inhibition of nodulation either as a result of a direct feedback within the
nodule or an indirect feedback originated from shoots. Moreover, supply of external
ureides increased ureide concentration in leaves and inhibited nitrogenase activity
(Serraj et al. 1999; Vadez et al. 2000). Albrecht et al. (1994) reported that nodule
initiation, growth and activity are all more sensitive to water deficit than are general
root and shoot metabolism implying that soil moisture deficiency has a pronounced
effect on N2 fixation.
The response of nodulation and N2 fixation to water deficit depends on the growth
stage of the plants. Pena-Cabriales and Castellanos (1993) pointed out that water
deficit imposed during vegetative growth was more detrimental to nodulation and
SNF than that imposed during the reproduction stage. Sellstedt et al. (1993) found
that water deficiency decreased the amount of N derived from N2 fixation by about
26 % when measured by the acetylene reduction assay. Moreover, during the harvest
period of soya bean in symbiosis with rhizobia, nodule P concentrations and P use
efficiency declined linearly with soil and root water content (Franson et al. 1991).

4.3  Soil Acidity

It is also well documented that legume productivity is limited by soil acidity which
is a significant problem for agricultural production in many areas of the world (Gra-
ham 1992; Clarke et al. 1993; Bordeleau and Prevost 1994; Correa and Barneix
1997). Most leguminous plants require a neutral or slightly acidic soil for growth,
276 J.-J. Drevon et al.

especially when they depend on SNF (Brockwell et al. 1991; Bordeleau and Prevost
1994). Poehlman (1991) showed that the suitable soil pH for rhizobia activity was
6.5. However, legumes and their rhizobia exhibit varied responses to acidity. Some
species, like alfalfa ( Medicago sativa) are extremely sensitive to acidity, while oth-
ers, such as Lotus tenuis, tolerate relatively low soil pH (Correa and Barneix 1997).
The failure of legumes to nodulate under acid soil conditions is common, especially
in soils of pH less than 5.0. The inability of some rhizobia to persist under such con-
ditions is one cause of nodulation failure (Carter et al 1994; Bayoumi et al. 1995).
However, poor nodulation can occur even when a viable rhizobial population can
be demonstrated (Graham 1992; Graham et al. 1994). Taylor et al. (1991) reported
that acidity had more severe effects on rhizobial multiplication than did Al stress
and low P conditions. Low pH reduced the number of Rizhobium leguminosarum
bv. trifolii cells in soils, which resulted in no or ineffective nodulation by clover
plants (Ibekwe et al. 1997). The number of nodules, the nitrogenase activity, the
nodule ultrastructure and the fresh and dry weights of nodules were affected to a
greater extent at a low–medium pH (4.5; Vassileva et al. 1997). Similar results were
obtained with Bradyrhizobium japonicum in symbiosis with soya bean.
Legume species differ greatly in their response to low pH with regard to growth
and nodulation (Tang and Thomson 1996). Recently, it has been found that the
amount of N2 fixed by forage legumes on low-fertility acidic soil is dependent on
legume growth and persistence (Thomas et al. 1997). Recent reports indicated the
destructive effects of acidic soils on rhizobia–legume symbiosis and N2 fixation.
Low pH reduced the number of R. leguminosarum bv. trifolii cells in soils, which
resulted in no or ineffective nodulation by clover plants (Ibekwe et al. 1997). The
number of nodules, the nitrogenase activity, the nodule ultrastructure and the fresh
and dry weights of nodules were affected to a greater extent at a low medium pH
(4.5) (Vassileva et al. 1997). In acidic soils with pH of 5.0, where heavy metal activ-
ity is relevant, the presence of available aluminium inhibits nodulation (Bordeleau
and Prevost 1994). Graham (1992) reported that Al is more detrimental to nodula-
tion than it is to plant growth in legumes. At pH 4.5 and with 0.5 mM Ca, nodula-
tion in cowpea was delayed by 13 mM Al and nodule number and dry weight were
severely depressed (Alva et al. 1990).
The pH and calcium concentration strongly affect root exudation and the adsorp-
tion of the rhizobia at the root surface (Richardson et al. 1988; Carter et al. 1994).
Availability of Ca in acidic soils with a high Al content appears very important for
nodulation. At the concentration between 0 and 1.5 mM, the adsorption of Sino-
rhizobium meliloti on Medicago sativa is linearly related to the concentrations of
calcium and magnesium. Higher calcium concentrations are required to compensate
for the negative effect of low pH (i.e. high H + concentrations) on the adsorption of
rhizobia (Caetano-Anollés et al. 1989). Thus, the negative effects of low pH and
low calcium concentrations on nodulation of alfalfa are reflected at the first step
of nodulation. In addition, similar observations indicated that the nodule number,
nitrogenase activity and nodule ultrastructure of the common bean, Phaseolus vul-
garis, were greatly affected at low Ca concentration (0.13 mM) in acidic soils with
a pH of 4.5 units (Vassileva et al. 1997). Calcium moreover is essential in several
9  The Legume–Rhizobia Symbiosis 277

mechanisms of energy storage and transfer being indispensable in the reduction

process of N2 to NH4 (Vidor et al. 1983). The critical Ca level for nodule forma-
tion in pigeon pea ( Cajanus cajan) and guar is more than 50 mM, whereas peanut
and cowpea nodulated very well in solution culture with 2 mM calcium (Bell et al.
1989). Nodulation and nodule development in cowpea were strongly depressed at
low pH (4.5–5.5) and low calcium concentration (0.05–2.5 mM; Alva et al. 1990).

4.4  Phosphorus Requirement of the Symbiosis

Acid-weathered soils of the tropics and subtropics are particularly prone to P defi-
ciency. Worldwide, phosphorus is considered as the principal yield-limiting nutrient
along with nitrogen (Zahran 1999). Phosphorus deficiency is a primary constraint
to plant growth in many terrestrial ecosystems (Bonser et al. 1996). Under low soil
pH, phosphate is adsorbed by clay minerals. Other factors such as low soil moisture
affect on the availability of phosphorus (Karmakar et al. 1997; Raychaudhury et al.
2003). P deficiency has two main causes: (i) the low content in total P of some
soils poor in organic matter or highly lessivated and (ii) the complexation of P with
cations such as Ca, Al or Fe, under the form of insoluble oxides which are unavail-
able for the plants, like in acid soils. Nowadays, vast areas of potentially good land
are still agriculturally poor because of P deficiency (Basu et al. 2008). The avail-
ability of P in the soil for plants and microorganisms can moreover be limited by
the competition with microorganisms in the rhizosphere and geochemical sinks. In
addition, P deficiency in the soils can also be caused by low phosphorus contents in
the parent material.
Sanginga et al. (1996) reported that legumes introduced in cereal-based cropping
systems with P applications showed an enhanced ability to establish, nodulate and
grow. Under P-deficient conditions, P fertilization will usually result in enhanced
nodule number and mass and greater N2 fixation per plant and per gram of nodules
(Serraj and Adu-Gyamfi 2004). Besides increasing nodulation and N2 fixation, P
fertilizer may increase grain yields (Mugwira et al. 1997). The increasing com-
mon bean grain yields up to 900 kg ha−1 were obtained by applied doses of P as
high as 352 kg P ha−1. Previous observations in the northern Guinea savanna have
shown that legumes require about 30 kg P ha−1 for optimal growth and N2 fixation
(Fig. 9.3; Weber et al., personal communication).
Bell et al. (1990) reported that suitable phosphorus concentration for groundnut
is around 0.3–0.4 % P of dry weight. Shu-Jie et al. (2007) showed that P fertiliza-
tion stimulates the growth of nodulated plants and N2 fixation. This hypothesis has
been confirmed with the isotopic method 15N for soya bean at the end of its cycle
(Pongsakul and Jensen 1991) and for the pea for which an optimal fertilization in
P stimulates tenfolds more the accumulation of N than the growth. Cassman et al.
(1993) has shown that the application of P fertilizer increased number of nodules of
soya bean grown in acid soils. Moreover, the application of P fertilizer at the correct
place and time has positive effects on growth and N2 fixation of soya bean in acid
278 J.-J. Drevon et al.

Fig. 9.3   Growth of nodule (a) and shoot (b) for different P concentration

soils. This may increase the availability of P in the vicinity of the soya bean seed-
lings and has a positive effect on the survival of introduced rhizobia which stimulate
root growth and then promote infection of the plants.
Phosphate is essential for SNF of legumes (Waluyo et al. 2004). The P require-
ments for N2 fixation have been investigated in various legume crops such as cow-
pea, pea, soya bean and Acacia mangium (Serraj and Adu-Gyamfi 2004). First et al.
(1987) demonstrated that leguminous species differ in their phosphorus requirement
for growth from 0.8 to 3.0 mM. Regarding the phosphorus use efficiency (PUE)
concept, it was initially defined by Siddiqi et al. (1991) and discussed by Gourley
et al. (1994), and it has gained scientific interest (Cure et al. 1988). According to
Cassman et al. (1981), efficient P utilization in N2-fixing symbioses may be closely
related to an adequate P partitioning between shoot and nodulated root, and between
root and nodules.
ATP is an essential energy provider molecule for the metabolism of organic com-
pounds containing P such as sugar phosphates, phospholipids, nucleic acids, nucleo-
tides and coenzymes which are key molecules for biological metabolisms (Schacht-
man et al. 1998). Plants dependent on SNF have therefore high ATP requirements
9  The Legume–Rhizobia Symbiosis 279

for nodule development and function (Ribet and Drevon 1996), and need additional
P for signal transduction and membrane biosynthesis. Phosphorus is also particu-
larly important for leguminous plants because of its influence on the activity of the
rhizobia species. P supplies in the soil are essential to N2-fixing soya bean plants
compared to N resources in the soil (Serraj and Adu-Gyamfi 2004). Wan Othman
et al. (1991) reported that nodulation of cowpea ( Vigna unguiculata) was impaired
by a very low P status of the soil. Therefore, maximum benefits from N2 fixation
depend on soil P availability (Kennedy and Cocking 1997).
The symbiosis between legumes and rhizobia requires additional P uptake for
the initial growth of the plant and to establish functioning nodules (Serraj and Adu-
Gyamfi 2004). Shu-Jie et al. (2007) suggested that the P deficiency specifically
inhibited the nodule development and thereby the total N2 fixation. Several physi-
ological characteristics of the nodule such as N2-dependent growth, nodule respi-
ration and control of oxygen diffusion affect SNF under phosphorus deficiency.
Al-Niemi et al. (1997) suggest that bacteroids can be P limited even when plants
have received otherwise adequate P levels. Cassman et al. (1980) showed that for
soya bean, the P requirement for the nodules appeared higher than for root and shoot
growth. Phosphorus concentrations in the nodule are often significantly higher than
those in shoot or root tissue (Serraj and Adu-Gyamfi 2004). Hart (1989) suggested
that nodules are strong sinks for P and range in P content from 0.7 to 1.2 % of dry
matter. Vadez et al. (1997) concluded that the P concentration in nodules can be
threefold higher than in the other organs confirming the sink ability of nodules for
P in nodulated legumes.
However, P requirement for N2 fixation has been shown to vary among geno-
types in pigeon pea and mung beans or Casuarina–Frankia symbioses (Serraj and
Adu-Gyamfi 2004). Differences in N2 fixation related to the efficiency of utilization
of P were also found among soya bean genotype (Guanawardena et al. 1993) and
A. mangium populations (Vadez et al. 1995). In addition, the growth rate of most
rhizobia strains is reduced by low levels of P (Al-Niemi et al. 1997). However,
strains of rhizobia differ markedly in tolerance to phosphorus deficiency (Beck and
Munns 1985). This P-deficiency response occurred when the medium P concentra-
tion decreased below 1 mM. Nodulation and N2 fixation and survival of rhizobia
in soil are particularly affected under low P and acid soil conditions (Graham and
Vance 2003).
Nodule biomass has been proven to be highly correlated to the availability of
P for the plant. P deficiency decreases the number of nodules per plant (Singleton
et al. 1985) and/or the individual mass of nodules (Gates 1974; Jacobsen 1985; Isra-
el 1987; Guanawardena et al. 1992), the mass of the bacteroid in soya bean (Sa and
Israel 1991) as well as the specific nitrogenase activity of nodules (Jacobsen 1985;
Singleton et al. 1985; Israel 1987; Ribet and Drevon 1995b; Vadez et al. 1996;
Drevon and Hartwig 1997; Qiao et al. 2007). Additional effects of P deficiency have
been reported in common bean, soya bean, lupin and alfalfa, such as to increase the
absorption surface and density of the roots resulting in more exploration of soil vol-
ume (Vance 2001), and to acidify the rhizosphere by root exudates (Neumann and
Römheld 1999) and H + efflux (Tang et al. 2001a, b, 2004). Finally, P may increase
280 J.-J. Drevon et al.

the nodulation and stimulate the nitrogenase activity by improving the plant growth
(Gentili and Huss-Danell 2003; Yang 1995; Reddell et al. 1997).

5 The Diversity of Rhizobial Symbioses in Their N2

Fixation Under Low P Soil

Previous studies have proven that although N2 fixation increases P demand of the
plant for Pisum sativum (Jacobsen 1985) and for G. max at the end of its devel-
opment cycle (Cassman et al. 1981; Israel 1987), it does not increase P demand
for Stylosanthes (Gates 1974), Trifolium (Robson et al. 1981), Vigna unguiculata
(Cassman et al. 1981) and Aracia mangium (Sun et al. 1992).
Among legumes, cowpea is more tolerant to phosphorus deficiency than others
like soya bean and common bean (Cassman et al. 1981; Gómez et al. 2002). More-
over, SNF in common beans is affected by P deficiency more than in other legumes
(Israel 1987; Ribet and Drevon 1995a, 1996; Vadez et al. 1996, 1997; Drevon and
Hartwig 1997). Consequently, SNF is considered to be less efficient in common
beans than in other legumes (Pereira and Bliss 1987; Isoi and Yoshida 1991). In
works reviewed by Bliss (1993), high SNF of common bean under P deficiency was
reported to be related to nodule number (Jebara et al. 2005), nodule mass (Kipe-
Nolt and Giller 1993; Kipe-Nolt et al. 1993), late nodule senescence (Hungria and
Franco 1988), early nodulation (Chaverra and Graham 1992) and secondary nodu-
lation (Wolyn et al. 1989). Regarding common bean Phaseolus vulgaris, there is
evidence of genotypic variability for SNF at different levels of available P which
show a possibility of selecting bean cultivars able to support biological N2 fixation
under low P soils (Pereira and Bliss 1989; García et al. 1996; Vadez et al. 1999).
Sanginga et al. (2000) studied PUE and N balance of cowpea on 94 lines in a
low P soil of the derived savanna zone in West Africa. The cowpea lines fixed on
average 22 kg N ha−1, which was 70 % of the plant total N. The N balance based on
the difference between the amount of N2 fixed and N exported through the harvest,
ranged between − 10.6 kg N ha−1 and  + 7.7 kg N ha−1. Based on its adaptability to
grow in low P soils and overall positive N balance, the cowpea line IT81D-715
should be recommended for cultivation when P is the limiting factor.
Most studies of low P tolerance in beans have been undertaken in hydroponics or
sand–alumina culture systems in order to optimize the control of P supply (Whita-
ker et al. 1976; Pereira and Bliss 1987; Vadez et al. 1997, 1999). In these conditions,
Vadez et al. (1999) identified tolerant lines with threefold greater N fixed per P sup-
plied than sensitive lines, and higher P use efficiency for SNF, the ratio of N fixed
per nodule P concentration. They also found that P deficiency delayed the onset of
nitrogenase activity, measured as acetylene reduction activity (ARA) and acceler-
ated the ARA decline during pod filling. They concluded, in agreement with Pe-
na-Cabriales and Castellanos (1993) and Vikman and Vessey (1992), that the time
course of ARA may be a valuable trait for screening SNF tolerance to P deficiency.
9  The Legume–Rhizobia Symbiosis 281

In addition, the best-adapted genotypes increased the soil P availability by about

50 % after a culture cycle (Ankomah et al. 1995; Rajput and Singh 1996). The later
was associated with an increase in SNF covering 89 % of the plant N requirement
and an accumulation of 200 kg ha−1 N in the soil (Sanginga 2003). Attempts have
been made to increase bean productivity by selecting lines able to fix adequate N2
under conditions of low P availability (Pereira and Bliss 1987; Vadez et al. 1999).
Conducting a screening procedure through 600 lines of common bean, Vadez et al.
(1999) found a large genotypic variability for SNF tolerance to P deficiency. They
reported that SNF tolerance to P deficiency was mostly found among late flowering
varieties. The SNF tolerance to P deficiency was correlated with (i) low nodule P
concentration, (ii) intense and early nodulation and (iii) large root dry weight and
high nodule nitrogenase activity.
The lower values of the ratio SNF/nodule dry weight for tolerant lines than for
sensitive lines indicate that the tolerant lines were essentially early N2-fixing sym-
bioses. The negative correlation between SNF and nodule P concentration suggests
higher PUE for N2 fixation (Fig. 9.4). This conclusion that the tolerance was mostly
due to efficient utilization of P for SNF is also suggested by the lower correlation
between SNF and P concentration in other organs than in nodules (Vadez et al.

Fig. 9.4   Efficiency in use of the rhizobial symbiosis. PUE phosphorus use efficiency, SNF symbi-
otic nitrogen fixation, DW dry weight, EURS efficiency in use of rhizobial symbiosis
282 J.-J. Drevon et al.

1999). The correlations between SNF and P concentrations in shoot or root would
be a consequence of the effect of nodule PUE on the N-determined growth of root
and shoot, and the subsequent dilution effect on P partitioning in these organs. The
lower P concentration in nodules of tolerant lines compared to sensitive lines may
also explain the higher nodule mass of tolerant lines as a result of a lower immobili-
zation of P in nodule structures, allowing more nodules to develop. This observation
may also explain the higher nodule persistence in tolerant lines during reproductive
stages when P is remobilized for pod and seed development. However, it does not
exclude that some tolerant lines have relatively higher nodule efficiency (Vadez
et al. 1999).
The lower difference in growth than in N fixed or ARA per tolerant versus sensi-
tive line, indicates limited benefit in growth subsequently to SNF tolerance to P de-
ficiency, though the SNF tolerance to P deficiency is associated with higher PUE for
plant growth. Vadez et al. (1999) concluded that the improvement of SNF for low
P soils might be feasible through an extension of vegetative growth using climbing
indeterminate progenitors in common bean. Although, it may be possible to breed
among early flowering lines for enhanced SNF and tolerance to P deficiency with
the ratio SNF per plant P concentration in glasshouse hydroponic culture.

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Chapter 10
Nutritional Value

Francesca Sparvoli, Roberto Bollini and Eleonora Cominelli

1 Introduction

Consumption of legumes is associated with physiological and health benefits, such

as prevention of cardiovascular disease, obesity, diabetes mellitus and cancer, as
indicated by an increasing number of studies. The growing body of research on
these health benefits has stimulated interest in developing innovative technologies
to expand the use of pulses in food products. Nevertheless, growing global food se-
curity challenges and protein malnutrition remain critical in many countries around
the world.
Currently, nearly 870 million people are suffering from chronic undernourish-
ment. Moreover, 100 million children under five are underweight, and this child-
hood malnutrition is a cause of death for more than 2.5 million children every year.
Most hungry and undernourished people live on diets based on very high amounts
of staple foods but few micronutrient-rich foods such as fruits, vegetables and ani-
mal and fish products. As a consequence, they lack the required protein, fat, vitamin
A, iodine, zinc and iron. A particular type of malnutrition is the so-called hidden
hunger, a chronic lack of vitamins and minerals as a consequence of poor dietary
quality, which negatively impacts on health, cognition, function, survival and eco-
nomic development. Hidden hunger mainly affects women and children from the
lower-income groups in developing countries; however, it also claims victims in the
developed world, where people apparently do not look hungry. Indeed, obesity or
being overweight can be a sign that bodies are still hungry for crucial micronutri-
ents. The impact of hidden hunger is serious: Globally stunted growth and anaemia

F. Sparvoli () · R. Bollini · E. Cominelli

CNR Institute of Agricultural Biology and Biotechnology, Via Bassini15, 20133 Milan, Italy
e-mail: [email protected]
R. Bollini
e-mail: [email protected]
E. Cominelli
e-mail: [email protected]
© Springer Science+Business Media New York 2015 291
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_10
292 F. Sparvoli et al.

in children become a major cause of health problems in later life, particularly the
increasing prevalence of overweight/obesity and chronic diseases. This results in
a double burden for the health systems, with associated massive health costs and
negative impact on economic productivity.
Grain legumes (pulses) are considered an essential source of nutrients and are
also recognized as a poor man’s meat, showing their importance for people of de-
veloping countries, where the consumption of animal protein is limited by non-
availability or is self-imposed because of religious or cultural habits. Furthermore,
legume seeds contain many bioactive and/or antinutritional compounds, such as
phytate, oligosaccharides, phenolic compounds, nonprotein amino acids, lectins
and enzyme inhibitors that play metabolic roles in humans or animals that frequent-
ly assume these seeds. These effects may be regarded as positive, negative or both
(Champ 2002).
Considerable genetic variation has been reported in the chemical composition of
legume seeds, both among and within species. In addition, chemical composition
can be modified by environmental factors during plant development, since many of
the bioactive compounds are secondary metabolites produced during seed develop-
ment and maturation. Existing data show that the balance between deleterious and
beneficial effects of these compounds depends on their chemical structure, concen-
tration, time of exposure and interaction with other dietary components. Therefore,
it is important to know not only the amounts but also the types of compounds in
the food and how they affect human body. The scientific understanding of how
these bioactive molecules act on organisms is an important challenge for the future
research and a special attention should be paid to the potential synergistic effects be-
tween/among the different classes of bioactive compounds (Rochfort and Panozzo
2007).
In this chapter, main classes of bioactive compounds, together with some spe-
cies-specific ones, are described in relation to their biological activities, abundance
in legume crops and role in nutrition and health.

2 Major Seed Proteins: Storage Proteins

and Enzyme Inhibitors

Legume seeds contain large amounts of proteins, mostly with a storage role, rang-
ing from about 16 % (dry weight) in cowpea, pigeon pea and chickpea to as much
as about 50 % in lupin and soybean, according to species, genotypes within spe-
cies and environments (Table 10.1). Storage proteins are synthesized during seed
development, stored in specific subcellular compartments, the storage vacuoles or
protein bodies and then hydrolyzed during germination to provide nitrogen and car-
bon skeletons for the developing seedling. The major storage proteins of legume
seeds are oligomeric globulins and albumins, which usually account for about 70
and 20 % of the total protein, respectively.
10  Nutritional Value 293

Table 10.1   Range of variation (% of seed weight) of principal constituents of grain legume seeds
Carbohydrates
Species Protein Total Fibres Starch Oil
Common bean 20.9–30.1a 54–64a 10c 41.5c 1.3–2.5a
Pea 21.9–31a 52–62a 5.9–12.7b 18.6–54.5b 1.3–3a
Chickpea 16–28a 54–66a 2.7–9b 42–54.9b 3.1–7a
Faba bean 24.3–32.2a 57–60a 7.5–13.1b 37–51.5b 1.1–4
Cowpea 16–36b 56–68a 6.3b 46.84–53.63e 1–1.3a
Lentil 20.6–32a 54–58a 12–14.7b 46–49.7b 1–2.1a
Pigeon pea 15.9–24.1 b
57.3–58.7 a
10 c
44.3 c
1.2–1.6b
Mung bean 23.3–27.7 a
61–62 a
7–12.9 b
45 c
0.7–2.4
Lupinus spp. 28–47c 26–47c 3d 0.4b 4–15.5c
Soybean 26.5–55.2 b
30.2–35 c
20 c
1.5 c
6.5–28.7b
a
Chibbar et al. 2010
b
Burstin et al. 2011
c
Hedley 2001
d
Reddy et al. 1984
e
Sreerama et al. 2012

2.1 Globulins

Globulins are classified as 7S and 11S proteins, according to their sedimentation

coefficients (S), and are collectively named vicilins and legumins, respectively.
Legumins are compact hexamers of about 350–400 kDa, and each monomer is
made up of two disulphide-bonded subunits derived from posttranslational prote-
olysis of a single precursor polypeptide. Vicilins are typically trimeric proteins of
150–190 kDa that lack cysteine residues and hence cannot form disulphide bonds.
Their subunit compositions vary considerably, mainly because of differences in the
extent of posttranslational processing (proteolysis and glycosylation). Vicilins can
be divided into two groups: In the first one, the precursor polypeptides are exten-
sively fragmented to give rise to mature subunits in the range of 12–34 kDa. Con-
versely, the precursor polypeptides belonging to the second group undergo little or
no posttranslational cleavage, and mature polypeptides are about 40–76 kDa. Pea
vicilin is a typical example of group one vicilins, while soybean β-conglycinin,
and common bean (Phaseolus vulgaris L.) are among the best characterized of the
second group. Although legumins and vicilins are both present in most legumes,
their relative abundance is highly variable, and some species are virtually devoid
of either one or the other. Vicilins are usually less abundant then legumins, but a
remarkable exception exists in P. vulgaris and P. lunatus, in which normally vicilin
is the most abundant storage protein. In P. coccineus, the ratio between vicilin and
legumin is highly variable, and seeds virtually depleted of legumin or phaseolin
have been reported (Durante et al. 1989).
From a nutritional point of view, the amino acid profile of legume storage pro-
teins reveals low amounts of the essential sulphur-containing amino acids (i.e. me-
thionine and cysteine) and tryptophan, while lysine, another essential amino acid,
294 F. Sparvoli et al.

is quite abundant. Legume proteins complement very well those of cereals, which
are normally rich in sulphur amino acids and poor in lysine and threonine. Besides
the composition in essential amino acids, the nutritional quality of seed proteins
is also largely determined by their digestibility. In fact, amino acids composition
only represents the potential nutritional quality of a protein, being their bioavail-
ability critical for the supply of amino acids in the diet. A number of data obtained
with experimental approaches devised to assess the bioavailability of amino acids in
foods concurrently demonstrated that seed proteins have a lower overall nutritional
quality than animal proteins. This can be related to their low content in sulphur
amino acids, the compact proteolysis-resistant structure of the native protein and the
presence of antinutritional compounds in the seed, which may affect digestibility
of proteins themselves as well as of other components. For example, the nutritive
value of common bean phaseolin, the 7S globulin of this species which normally
accounts for about 40–50 % of total seed proteins, is limited by a low content in
sulphur amino acids and by resistance to enzymatic hydrolysis, even after heat treat-
ment (Montoya et al. 2006). The three-dimensional structures of 7S vicilins and 11S
legumins have been resolved and confirmed that the two globulins are structurally
related (Lawrence et al. 1994). Vicilin trimers are arranged in a disk-shaped fash-
ion with each monomer arranged around a threefold symmetry axis and are rich of
β-sheet and β-turn structures which have been proposed to be responsible of the low
digestibility of globulins (Carbonaro et al. 2012; Lawrence et al. 1994).
Although the nutritional value and digestibility of globulins are not optimal,
there are data showing that soybean β-conglycinin, the 7S globulin, is responsible
for cholesterol/triglyceride-lowering activity, and it seems that the N-terminal ex-
tension domain of the α′ chain is responsible for inducing this biological response
(Consonni et al. 2011). A similar positive role in reduction of hypercholesterolemia
(and prevention of cardiovascular risk) has been shown for lupin proteins, the ma-
jor role being played by γ-conglutin, an unusual basic 7S-type globulin specific of
white lupin (Sirtori et al. 2004). Interestingly, γ-conglutin is also able to counteract
the plasma glucose increase and improve insulin sensitivity when administered to
rats, thus suggesting a potential use in the control of glycaemia in type 2 diabetes
(Lovati et al. 2012).
Different approaches have been undertaken to improve the nutritional quality
of legume storage proteins, and many have been directed towards increasing the
content of sulphur amino acids and/or change in relative abundance/type of storage
proteins. An example strategy, applied in common bean, consisted in the selection
and breeding for highly digestible phaseolin types (Montoya et al. 2010). In fact,
comparison of the degree of hydrolysis of 43 different phaseolin types showed vari-
ability ranging from 11 to 27 % for uncooked phaseolin and from 57 to 96 % for
heat-treated one (Montoya et al. 2008). An alternative approach is the manipulation
of seed protein compositions by decreasing the percentage of those types with low
or limiting amino acid content. Using three common bean lines differing for major
storage proteins content (devoid of phaseolin and/or major lectins), Taylor et al.
(2008) evaluated the impact of storage protein deficiency on protein accumulation
and amino acid composition, especially those containing sulphur, in mature seeds.
10  Nutritional Value 295

They found that deficiency of phaseolin and major lectins was associated with a
progressive and compensatory increase of the content of other proteins, mainly le-
gumin, α-amylase inhibitor (αAI) and mannose lectin FLT-3 receptor interacting
lectin (FRIL; Marsolais et al. 2010). However, the most interesting finding was that
the deficiency of some classes of storage proteins caused a modulation of sulphur
amino acid content. The deficient lines showed a decrease of S-methyl-Cys and
γ-Glu-S-methyl-Cys (both nonprotein amino acids) that were compensated with an
increase of the Cys (70 %) and Met (10 %) pools, and the combined content raised
from 18.9 to 26.8 mg/g protein, a value slightly above Food and Agriculture Orga-
nization (FAO) guidelines of 25 mg/g protein for human nutrition.
Albumins are the second most abundant class of legume storage proteins. They
are water-soluble proteins comprising most of the bioactive polypeptides, such as
lectins, protease inhibitors and αAI. The abundance of these bioactive molecules
is quite variable in the different legumes. Lectins are widespread in many legume
seeds, while αAI activity has been detected only in few legume species. Remark-
ably, the majority of these proteins have evolved within the seed as a protective
mechanism against insects, fungi, predators and a number of stress conditions
(Chrispeeels and Raikhel 1991). On the other hand, very often, the biological activ-
ity of these proteins is also responsible for the nutraceutical and health properties
of legumes, thus the interest for the potential uses of these molecules has increased
in recent years.

2.2 Lectins

Lectins are a family of highly homologous glycoproteins that exhibit specific and
reversible carbohydrate-binding properties. As a result, lectins can bind to specific
sugars and glycoproteins on the surface of cells in the gut wall, thereby interfering
with nutrient breakdown and absorption.
Many lectins are able to agglutinate red blood cells, thus their presence is tra-
ditionally measured by their haemagglutinating activity (HA). Lectins’ abundance
and their biological activity in legume seeds vary among species as well as among
genotypes of the same species (Table 10.2). Null or very-low lectin activity/pres-
ence has been reported for chickpea, lupin and Vigna genus, on the contrary, seeds
of Phaseolus species have the highest content, although common bean genotypes
devoid of lectins have been identified (Campion et al. 2009a; Confalonieri et al.
1992).
Growth suppression, diarrhoea and bloating are the most common effects of
raw lectin ingestion by humans and livestock (Vasconcelos and Oliveira 2004).
The toxicity of lectins is very often due to their high resistance to proteolysis and
stability over a large range of pH. Even though some lectins are heat sensitive,
they are not always completely destroyed by cooking because of the use of gentle
cooking methods, such as dry heat and short cooking times. Lectin activity can, to
various degrees, be removed from foods by different technological processes. For
296 F. Sparvoli et al.

Table 10.2   Comparison of haemagglutinating activity of different legume seed protein extracts
towards rabbit or human erythrocytes. One unit of haemagglutinating activity (HU) is defined as
the amount of seed extract per ml (range from 12,500 to 0.1 µg/ml) in the last serial dilution giving
50 % of agglutination (the lowest the HU value, the highest is the haemagglutinating activity of the
sample). (Data adapted from Grant et al. 1983)
Haemagglutinating activity range
Species Rabbit erythrocytes Human erythrocytes (AB
type)
Phaseolus vulgaris (white kidney beans) 6–24 12–390
Phaseolus vulgaris (pinto beans) 6250–12,500 12,500
Phaseolus coccineus 1.5–12 98–390
Phaseolus acutifolius 1.5–12 24
Phaseolus lunatus 12,500 98
Lentil 49–780 1560–6250
Pea 49–195 3120
Chickpea 12,500 12,500
Cowpea 12,500 12,500
Pigeon pea 12,500 12,500
Mung bean 12,500 12,500
Faba bean 49–3120 3120–12,500
Soybean 24–390 12,500

example, soaking, autoclaving and toasting completely destroyed the lectin in P. lu-
natus (Adeparusi 2001). Apart from common bean, microwave heating adequately
destroys haemagglutinins and trypsin inhibitors in legume seeds without affecting
protein quality, and irreversible lectin denaturation is achieved using boiling water
(Hernandez-Infante et al. 1998).
Active lectins, that survived cooking and/or passage in the gastrointestinal tract,
may induce changes in some or all of the digestive, absorptive, protective or secre-
tory functions of the whole digestive system and affect cellular proliferation and
turnover. For example, phytohaemagglutinin (PHA), the common bean lectin, binds
to the gastric mucosal and parietal cells inhibiting the gastric acid secretion in con-
scious rats (Kordas et al. 2001), while in pigs it causes an increase in stomach
weights and thickness (Radberg et al. 2001).
Although lectins are considered antinutrients, positive and beneficial roles for
human health and nutrition have also been reported. Studies have revealed that
oral administration of low doses of lectins can produce beneficial effects on the
digestive/absorptive efficiency of the gut, its immune system and bacterial ecology
and that, by modulating the secretion of gut hormones, some lectins can influence
the body’s endocrine system with beneficial consequences for general metabolism
(Pusztai and Bardocz 1996). Some lectins may play a key role in preventing certain
cancers (De Mejia and Prisecaru 2005) or can be used as therapeutic agents for
preventing or controlling obesity (Bardocz et al. 1996).
10  Nutritional Value 297

2.3  Protease Inhibitors

Many legume seeds also contain inhibitors of proteolytic enzymes. These are con-
sidered antinutritional molecules interfering with protein digestion, due to their
ability to irreversibly inhibit, if not properly inactivated, the action of digestive
enzymes, such as trypsin, chymotrypsin, carboxypeptidases and elastase. However,
once inactivated, protein inhibitors may even play a positive nutritional role, due to
their high content of sulphur-containing amino acids compared to the majority of
the seed proteins.
Most protease inhibitors belong to two major classes, the Kunitz trypsin inhibi-
tors, particularly abundant in soybean seeds, and the Bowman–Birk inhibitors, that
are widely found in the other legume seeds (Clemente et al. 2011; Oliva et al. 2011).
Kunitz-type inhibitors have a molecular mass of about 20 kDa, with two disulphide
bridges, and act specifically against trypsin. The Bowman–Birk inhibitors are dou-
ble-headed inhibitors of 8–9 kDa with a high proportion of disulphide bonds. They
usually comprise two distinct binding loops responsible for the inhibition of two
identical or different proteases (chymotrypsin and/or trypsin) per inhibitor mole-
cule. Differences in inhibitor concentrations and activity have been reported among
legume species as well as varieties (Guillamon et al. 2008) and may be affected by
environmental conditions during seed maturation (Piergiovanni and Pignone 2003).
Trypsin inhibition (measured as trypsin inhibitor units per mg, TIU/mg) can range
from negligible, as in the Lupinus spp., to very abundant in soybean (43–84 TIU/
mg) and common bean (17–51 TIU/mg). The TIU content of different Lathyrus
cultivars is in the range 19–30 TIU/mg sample, and this is higher than in chickpea
(15–19 TIU/mg) and pea (6–15 TIU/mg). Most lentil and faba bean cultivars have
lower values (3–8 and 5–10 TIU/mg sample, respectively) (Guillamon et al. 2008).
Kunitz and Bowman–Birk inhibitors’ antinutritional effects are not only a con-
sequence of inhibition of intestinal protein digestion for their presence in a diet
consisting of free amino acids still has adverse effects resulting in decreased growth
(Lajolo et al. 2004). It has been proposed that these inhibitors act suppressing the
negative feedback regulation of pancreatic secretions through the release of hor-
mone cholecystokinin from the intestinal mucosa (Liener 1994). The consequence
is the stimulation of pancreas enlargement and hypersecretion of digestive enzymes
(sulphur-rich proteins), causing a loss of sulphur-rich endogenous proteins. This
would depress growth, as legume seed proteins are deficient in sulphur amino acids.
On the other hand, the presence of trypsin inhibitors in the diet has been linked also
to health-promoting properties. Bowman–Birk inhibitors are effective in preventing
or suppressing carcinogen-induced transformation in vitro and carcinogenesis in
animal assays. Anti-inflammatory properties of protease inhibitors have also been
demonstrated (Ware et al. 1999). A number of patents on the use of Bowman–Birk
inhibitors to combat obesity (Defreitas et al. 2003), degenerative and autoimmune
diseases (Kennedy and Rostami 2004) and, in general, skeletal muscle atrophy
(Sweeney et al. 2005) have been released.
298 F. Sparvoli et al.

2.4  α-Amylase Inhibitors

Presence of αAI differs greatly among legume species and the best described and
most abundant are those found in Phaseolus species. Grant et al. (1995), analyzing
the αAI levels in seeds of a number of legumes found in Europe, detected the high-
est contents of αAI in Phaseolus species (2–4 g/kg). Much lower levels (0.1–0.2 g/
kg) were found for lima bean, cowpea, chickpea, faba bean and sweet lupin, while
no αAI activity was found in seeds of adzuki bean, lentil, mung bean, pea, soybean
and winged bean. In common bean, αAIs belong to a group of evolutionarily related
seed proteins, comprising lectins and arcelins, whose presence has been frequently
associated to resistance against phytophagous insects (Pueyo and Delgado Salinas
1997; Zaugg et al. 2013).
αAIs do not inhibit plant amylases, while they are active against a different type
of amylases, such as human (saliva and pancreatic), porcine, fungal and, most inter-
esting, insect amylases (Ishimoto et al. 1995). Hence, it is not surprising that most
of the studies looking for αAI presence in seeds have been motivated by the protec-
tive role of this molecule against predatory insects. The best characterized αAI is
that of common bean; it is a quite stable molecule being relatively heat resistant (it
is still active after 30 min at 80 °C). In native conditions, it can resist tryptic diges-
tion up to 24 h at 37 °C (Adeparusi 2001); however, proper thermal treatment, such
as heating for at least 10 min at 100 °C, is sufficient for complete inactivation and
loss of resistance to trypsin (Sparvoli et al. 1999). These properties could in part
explain the biological effects of αAI. In clinical studies, purified αAI inhibited in-
traduodenal amylase. High dietary intakes of αAI can cause a number of potentially
deleterious alterations in the metabolism of experimental animals. Starch digestion
in the rat small intestine was also inhibited, with occasional blockage of the caecum,
particularly at daily intakes of αAI higher than 20 mg, leading to losses of body
nitrogen, lipids and carbohydrates and growth depression (Pusztai et al. 1995). In
humans, αAI consumption decreases postprandial plasma glucose and insulin levels
(Jain et al. 1989; Layer et al. 1986), thus the starch-blocking properties of αAI have
prompted several studies to exploit the use of this molecule to control obesity as
well in the prevention and treatment of diabetes (Obiro et al. 2008).

3  Starch, Fibres and Oligosaccharides

Starch and fibres, along with carbohydrate derivatives, such as oligosaccharides,

constitute the major components of seed carbohydrates, making up to 30–40 % of
seed dry matter in those species with higher protein content, such as lupin and soy-
bean, and up to 50–65 % in less protein-rich legumes species (Table 10.1). There is
increasing evidence that the addition of fermentable fibre to the diet alters the func-
tion and structure of the gut, modifies the production of gut-derived hormones, and
is associated with improved whole-body glucose homeostasis even in the absence
of disease.
10  Nutritional Value 299

3.1 Starch

Generally, starch constitutes the largest part of carbohydrate fraction, accounting

for 35–45 % of seed dry weight, the only exceptions being soybean and lupin in
which oil replaces starch as the main energy storage source (Table 10.1). Amylose
and amylopectin are the two basic components of starch; amylose is a linear mole-
cule with molecular weight ranging between 70 and 200 kDa, whereas amylopectin
is a highly branched molecule consisting of main chains of (1–4)- α-d-glucose with
short chains of (1–6)- α-d-glucose-linked branches with molecular weight greater
than 2 × 104 kDa.
Starch composition is one of the determinants that define legume seed nutritional
value and health effects. On the basis of its susceptibility to amylases and conse-
quent digestibility profile, starch is classified as rapidly digestible starch (RDS),
slowly digestible starch (SDS) and resistant starch (RS). RDS is rapidly digested
and absorbed in the duodenum and proximal regions of the small intestine leading
to a rapid elevation of blood glucose and usually a subsequent episode of hyper-
glycaemia. SDS is slowly digested in the small intestine to provide sustained glu-
cose release with a low initial glycaemia and subsequently a slow and prolonged
release of glucose. On the contrary, RS is not hydrolyzed in the small intestine and
is fermented by the colonic microflora in the large intestine, producing short-chain
fatty acids (SCFA) that provide additional energy to the body along with butyrate
that is beneficial to colonic health (Aller et al. 2011; Fig. 10.1). Due to the differ-
ent method used to evaluate the content of starch fractions, it is difficult to make a
meaningful comparison of the levels of SDS, RDS and RS starches among legume
seeds. Factors such as amylose content, crystallinity and amylopectin structur have
been shown to influence SDS and RS levels (Hoover et al. 2010). Most starches
from grain legumes have a relatively high amylose content (30–40 %) compared to
those from cereals or tubers. These characteristics may lead to increase in RS con-
tent after processing, hence having important effects on human physiology (Guillon
and Champ 2002), such as promoting slow and moderate postprandial glucose and
insulin responses (Sievenpiper et al. 2009).

3.2  Dietary Fibre (DF)

DFs consist of chemically heterogeneous molecules such as cellulose, and non-

cellulosic polysaccharides like hemicellulose, pectins, oligosaccharides and lignin
derived from structural carbohydrates of the plant cell walls. DFs resist digestion
and absorption in the small intestine and are partially or completely fermented in
the large intestine, thus exerting various physiological effects with health implica-
tions (Tharanathan and Mahadevamma 2003). Depending on their water solubility,
total DF (TDF) are classified into insoluble DF (IDF) and soluble DF (SDF). The
first class (IDF) is made up by cellulose, hemicellulose and lignin, and its consump-
tion reduces intestinal transit time, thereby improving laxation. The second class
300 F. Sparvoli et al.

Fig. 10.1   Scheme representing starch classification and its main postprandial effects. SDS slowly
digestible starch, RDS rapidly digestible starch, RS resistant starch. (Adapted from Aller et al.
2011)

(SDF) consists of oligosaccharides, glucans, gums and pectins, and its action is
mainly in helping lowering blood cholesterol and regulating glucose. The composi-
tion and concentration of DF in legume seeds vary depending on their localization:
the seed coat or the cotyledons, with the first having higher DF concentrations. DFs
from seed coats contain large amounts of cellulose (35–57 %) and less amounts of
hemicellulose and pectins, while the major polysaccharides in cotyledons are pectin
compounds (about 55 %), cellulose (9 %) and non-starchy glycans (ranging between
6 and 12 %; Guillon and Champ 2002). Cellulose has been found to be the major
constituent of crude fibre in pea and common bean, while hemicellulose is more
abundant in lentil, faba bean, pigeon pea and mung bean (Reddy et al. 1984).
10  Nutritional Value 301

3.3 Oligosaccharides

The most common oligosaccharides in legume seeds are α-galactosides, which are
soluble low-molecular-weight sugars mostly represented by the raffinose family
oligosaccharides. They are α-(1→6)-galactosides linked to carbon C-6 of the glu-
cose moiety of sucrose and include raffinose (trisaccharide), stachyose (tetrasac-
charide) and verbascose (pentasaccharide). Their relative and total abundance vary
among species and cultivars. Appreciable levels of these oligosaccharides, rang-
ing between 0.4 and 16.1 % of dry matter, are accumulated in the seeds of lentil,
chickpea, lupin, pea and faba bean. Lupins contain higher levels of stachyose and
raffinose compared to peas and faba beans, while verbascose is more abundant in
peas and faba beans compared to lupin, chickpea and common bean (Muzquiz et al.
2012); stachyose is the most abundant α-galactoside in common bean (Diaz-Batalla
et al. 2006). Interestingly, in chickpea there is a marked difference in oligosac-
charides content between desi-type and kabuli-type chickpeas with the last having
16.8 % higher content than the former (Saini and Knights 1984).
From a nutritional point of view, α-galactosides are considered antinutritional
factors as they are not hydrolyzed by mucosal enzymes in the small intestine of
monogastric animals and are then fermented in the lower gut by resident anaerobic
bacteria with the consequent production of carbon dioxide and hydrogen gases that
are responsible for digestive discomfort (Rochfort and Panozzo 2007). On the other
hand, α-galactosides have also proven to exert prebiotic effects by promoting the
beneficial activity of specific members of the intestinal microflora, thus improving
gut health by suppressing intestinal putrefaction, reducing constipation and diar-
rhoea, stimulating the immune system and increasing resistance to infection (Bud-
dington et al. 2002).

4  Minerals and Phytic Acid

Legume seeds are an excellent source of essential minerals, particularly iron, zinc
and calcium (Campos-Vega et al. 2010). The highest levels of iron can be found in
seeds of common bean, faba bean, mung bean and lentil. High zinc contents have
been reported for Lupinus spp., lentil and chickpea, while the highest calcium con-
tent is found in seeds of common bean, lupin, faba bean and chickpea (Table 10.3).
The increasing concerns about food security, together with a widespread “hidden
hunger”, have stimulated research on crop biofortification, that, in many cases, has
been translated into wide screenings of natural variability to identify donor geno-
types with high mineral content. These genetic materials have been further used for
breeding high Fe and high Zn varieties as well as for the identification of useful
molecular markers to assist breeding (Amarakoon et al. 2012; Blair et al. 2013;
DellaValle et al. 2013; Nair et al. 2013).
 302

Table 10.3   Range of variation of essential minerals (µg/g seed dry weight) and phytic acid content (mg/g seed dry weight) in different legume species
Species Fe Zn Ca PA Reference
Mung bean 44.5–107 23.3–48 273 1.8–5.8 Sompong et al. 2010; Taunk et al. 2012
Pea 46–73 39–63 622–1219 3.1–7.1 Amarakoon et al. 2012; Muzquiz et al. 2012; Trinidad
et al. 2010
Lentil 64.6–90 44–73.1 480–1280 2.5–12.2 Cabrera et al. 2003; Karakoy et al. 2012; Muzquiz et al.
2012; Thavarajah et al. 2009
Chickpea 46–77 37–74 517–1974 2.8–13.6 Grant et al. 2003; Konietzny and Greiner 2003; Muzquiz
et al. 2012; Thavarajah and Thavarajah 2012; Wang et al.
2003
Pigeon pea 54 61 514 3.5–17.5 Chitra et al. 1995; Sompong et al. 2010; Trinidad et al.
2010
Lupinus spp. 24–108 29–176 1350–2225 6–8.9 Muzquiz et al. 2012; Porres et al. 2007; Trugo et al. 1993
Cowpea 106 65 209 5.4 Chitra et al. 1996; Grant et al. 2003; Konietzny and
Greiner 2003
Common bean 62–280 10–42 562–4065 3.4–28.7 Cabrera et al. 2003; Doria et al. 2012; Grant et al. 2003;
Guzman-Maldonado et al. 2000; Konietzny and Greiner
2003; Muzquiz et al. 2012; Trinidad et al. 2010
Faba bean 55–110 20.5–58 610–1973 5.9–15 Cabrera et al. 2003; Campos-Vega et al. 2010; Konietzny
and Greiner 2003; Muzquiz et al. 2012; Oomah et al.
2011; Uzun et al. 2011
Soybean 161 66 1502 4.8–20.1 Muzquiz et al. 2012; Trinidad et al. 2010
F. Sparvoli et al.
10  Nutritional Value 303

Although legume seeds have a good content in essential minerals, they also ac-
cumulate significant amounts of compounds that lower their nutritional value by
lowering nutrient bioavailability. Phytic acid ( myo-inositol-1,2,3,4,5,6-hexa-kis-
phosphate, InsP6, PA) and its lower phosphorylated derivatives (InsP5 and InsP4)
are some of such compounds. Phytic acid is the main phosphorous storage form in
seed and is stored as a mineral complex (phytate salts) in specific subcellular struc-
tures, called globoids, in the protein vacuole of embryo and cotyledonary cells. It
accounts for an average of 75 % of total seed P and constitutes 1–3 % of dry weight.
However, PA and its less abundant derivatives, InsP5 and InsP4, are well recog-
nized antinutrients, as, during gastrointestinal passage, they bind trace elements
(e.g. Fe, Zn, Ca and Mg) and reduce their absorption leading, under certain dietary
circumstances, to mineral (mostly Fe, Zn, Ca) deficiencies (Gibson et al. 2006).
Phytic acid also interferes with other nutrient absorption: Its ability to complex with
proteins decreases their solubility, therefore, impacting on digestive enzyme activ-
ity (Urbano et al. 2000). Recent studies have also identified PA as an antioxidant
and have demonstrated that it possesses anticarcinogenic/antineoplastic properties,
can reduce or prevent kidney stone formation, and plays important roles in many
physiological processes (Raboy 2003; Vucenik and Shamsuddin 2006). The amount
of PA accumulated in seeds varies among species, varieties and soil P availability;
however, accumulation of very low PA levels has been detected only in induced mu-
tants. Among legumes, mung bean, pea, lentil and chickpea have relatively lower
levels of PA compared to common bean, faba bean and soybean (Table 10.3).
Mutations that reduce the level of seed PA (low phytic acid, lpa) have been iden-
tified in major crops such as maize, rice, wheat, common bean, pea and soybean
(Campion et al. 2009b; Rasmussen et al. 2010; Warkentin et al. 2012; Wilcox et al.
2000). Decreased accumulation of seed PA varies depending on the type of muta-
tion and generally ranges between 30 and 90 %. The highest reductions are found
associated to mutations affecting a multidrug resistance-associated protein (MRP)-
type adenosine triphosphate (ATP)-binding cassette (ABC) transporter, which is a
high-affinity InsP6 transporter (ABCC5) necessary for PA vacuolar storage (Maroof
et al. 2009; Panzeri et al. 2011). Since PA and inositol phosphate derivatives play
key roles in plant and cell functions, reduction in plant agronomic performance and
fitness has been reported for a number of lpa mutants. Moreover, the extent of the
negative pleiotropic effects of the mutation appears to correlate to the level of PA
reduction (Panzeri et al. 2011). Despite this, there are indications that in some cases
low PA levels in the seed are compatible with good plant performance and seed vi-
ability, as shown in common bean (Campion et al. 2013), or there is a potential for
breeding to obtain acceptable performances, as shown for other crops (Israel et al.
2007). From a nutritional point of view, recent papers demonstrated that lpa mutants
are effectively biofortified, being able to provide more micronutrients to humans
than their wild type (WT) counterparts (Petry et al. 2013).
304 F. Sparvoli et al.

5  Phenolic Compounds: Tannins and Anthocyanins

Phenolic compounds are mainly represented by tannins and flavonoids and are
mostly accumulated in the seed coats where they contribute to the determination of
the color. Total phenolic compounds vary in composition and contents across differ-
ent species, tissues, stages of development and in response to environmental factors
(Caldas and Blair 2009; Diaz-Batalla et al. 2006; Marles et al. 2010; Oomah et al.
2011). A survey on phenolic compounds content in legume species can be found in
the US Department of Agriculture (USDA) flavonoid, isoflavone and proanthocy-
anidin databases (USDA, 2013).
In nutritional terms, the major effect of tannins is the reduction of protein digest-
ibility by inhibition of proteolytic activity and/or formation of indigestible com-
plexes with dietary protein. Tannins also form complexes with polysaccharides and
iron in the gastrointestinal lumen; therefore, they reduce the efficiency of carbohy-
drate absorption and the bioavailability of the minerals in the grain. Despite these
concerns, tannins may function as anticarcinogenic compounds and antioxidants
(Serrano et al. 2009), thus the balance between health benefits and antinutritional
effects is important when planning breeding work for this trait. Moreover, since
some legume seeds, such as faba bean and peas, are also used as a protein source
for feeding monogastric animals, tannin-free varieties are considered superior to
tannin-containing ones (Crepon et al. 2010).
Flavonoids have been shown to exert many beneficial roles on human health
since they possess diverse biological activities such as antioxidation, antiageing,
anticancer, antiinflammation, antiatherosclerosis, cardiovascular protection, im-
provement of endothelial function, as well as inhibition of angiogenesis and cell
proliferation activities. In legumes, the highest polyphenolic contents are found in
dark, highly pigmented seed varieties, mostly belonging to Phaseolus and Vigna
species. Xu and Chang (2007) made a comparative analysis of phenolic composi-
tion in a number of widely cultivated legume species and showed that lentil, black
and red varieties of common bean and black soybean have the highest total phenolic
content (TPC), total flavonoid content (TFC) and condensed tannins content (CTC).
These high phenolic contents correlate with the highest antioxidant activities, as
assessed with different evaluation methods (22,2-diphenyl-1-picrylhydrazyl radi-
cal scavenging assay DPPH; ferric reducing antioxidant power, FRAP; and oxygen
radical absorbance capacity, ORAC; Table 10.4). In a recent work, black chickpea
genotypes with high TPC and TFC content as well as FRAP levels have been de-
scribed (Segev et al. 2010).
Common beans, and in general Phaseolus species, exhibit a wide variety of seed
coat colours and patterns, thus they have been the subject of most of the published
studies regarding composition and abundance of the different classes of phenolic
compounds in legume species. Extensive genetic analyses have identified specific
loci, controlling seed coat colour ( P, C, R, J, D, G, B, V and Rk, that regulate flavo-
nol and anthocyanin synthesis) and pattern ( T, Z, L, J, Bip and Ana), and 12 quan-
titative trait loci (QTL) controlling condensed tannin concentration. Among them,
 10  Nutritional Value

Table 10.4   Phenolic contents in seeds of different legume species. (Adapted from Xu et al. 2007)
Species Market class TPC TFC CTC DPPH FRAP ORAC
(mg GAE/g) (mg CAE/g) (mg GAE/g) (µmol Trolox eq/g) (mmol Fe2+eq/100 g) (µmol Trolox eq/g)
Pea Yellow pea 0.86–1.14 0.09–0.17 0.22–0.58 0.57–2.65 0.62–0.82 3.26–12.8
Green pea 0.65–0.99 0.05–0.15 0.23–0.61 0.98–2.25 0.43–0.86 1.73–9.95
Lentil 4.86–9.6 3.04–4.54 3.73–10.2 19.07–19.87 8.75–12.44 59.55–95.19
Common bean Black bean 3.37–6.99 2.51–3.3 4.09–5.73 14.49–18.95 6.05–9.70 48.91–92.73
Navy bean 0.57 0.92 0.47 1.48 1.27 13.3
Small red bean 5.76 4.24 5.16 17.9 4.53 70.58
Soybean Black soybean 5.57 4.04 1.96 18.44 9.43 131.34
Yellow soybean 1.74–1.82 1.06–1.24 0.37–0.79 0.92–1.83 1.09–1.49 35.1–44.23
Chickpea 0.98 0.72 0.52 1.26 0.8 9.26
TPC total phenolic content, TFC total flavonoid content, CTC condensed tannins content, GAE gallic acid equivalent, CAE catechin equivalents, DPPH
2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, FRAP ferric reducing antioxidant power, ORAC oxygen radical absorbance capacity
305
306 F. Sparvoli et al.

the P gene plays a key role in the regulation of seed colour, since it is epistatic on the
expression of C, D and J, and it is considered the controlling factor for the presence
or absence of flavonoids in the seed coat (Caldas and Blair 2009). An example of
the variability of phenolic compounds in common bean has been reported by Diaz-
Batalla et al. (2006) who quantified the concentrations of flavonoids (kaempferol,
quercitin) and phenolic acids (p-hydoxybenzoic acid, vanillic acid, p-coumaric acid
and ferulic acid) in a collection of ten cultivated and four wild varieties of Mexican
bean seeds. A similar analysis was performed on a collection of Italian common
bean landraces (Doria et al. 2012) and the content of genistein and daidzein isofla-
vones was also assessed. Only some genotypes contained this class of compounds
and the highest values were 101 μg/g for daidzein (average value 36.7 μg/g) and
21.6 μg/g for genistein (average value 9.14 μg/g). However, these values are very
far from those reported in soybean, which is a well-known good font of daidzein
(470 μg/g) and genistein (740 g/g; Rochfort and Panozzo 2007).

6 Saponins

Saponins are naturally occurring compounds widely distributed and particularly

abundant in legume seeds. Saponins consist of a lipid-soluble nucleus, having either
a steroid or a triterpenoid aglycone structure, with one or more side chains of water-
soluble carbohydrates. Based on their aglycone structure, saponins are generally
categorized into three main groups as groups A, B and E. The main saponin compo-
nents in legumes are the group B saponins, which contain the aglycone, soyasapo-
genol B. Many saponins are bitter and reduce the palatability of livestock feeds and
have long been considered antinutrients due to toxicity and their haemolytic activity
(Khalil and El-Adawy 1994). However, only a few are toxic since an enormous
structural diversity within this chemical class exists, depending on the aglycone
structure, the attachment of the glycosidic moieties and the nature of the glycosides.
Saponins are attracting considerable interest as a result of their diverse properties.
Clinical studies have suggested that they are health-promoting components that af-
fect the immune system in ways that help to protect the human body against cancer,
and also lower cholesterol levels. Saponins decrease blood lipids, lower cancer risks
and lower blood glucose response. A high saponin diet can be used in the inhibi-
tion of dental caries and platelet aggregation, in the treatment of hypercalciuria in
humans, and as an antidote against acute lead poisoning. In epidemiological stud-
ies, saponins have been shown to have an inverse relationship with the incidence of
renal stones (Shi et al. 2004).
Presence of saponins has been reported in many edible legumes such as lupin,
lentil, chickpea, faba bean, as well as soybean, bean and pea. Contents vary from
10 μg/g in pea up to 5000–6000 μg/g in soybean and chickpea (Campos-Vega et al.
2010). A study on a group of Italian landraces of common bean has reported an
average soyasapogenol B content of 304 μg/g, and values were ranging from 105 to
454 μg/g (Doria et al. 2012). Another study on Spanish varieties showed a variation
10  Nutritional Value 307

from 890 up to 2050 μg/g and a third study on navy bean seeds reported even higher
values up to 7620 μg/g (Burbano et al. 1999; Shi et al. 2009).

7 Other Minor Antinutritional/Bioactive Compounds:

Vicine, Convicine, l-DOPA and ODAP

The nutritional quality of some legume seeds may be affected also by other more
species-specific molecules of different chemical origin. The seeds of faba bean ac-
cumulate vicine and convicine, two pyrimidine glycosides, whose aglycone forms,
divicine and isouramil, respectively, are the causative agent of favism. This is a
haemolytic anaemia that affects male individuals carrying a specific genetic defect
in the gene coding for erythrocyte-located glucose-6-phosphate dehydrogenase.
Vicine and convicine have antinutritional effects also in the diet of monogastric
animals, and several efforts, related to breeding as well as to processing treatments,
have been undertaken to reduce their amounts in seeds (Crepon et al. 2010).
Faba bean, together with Mucuna pruriens (a tropical legume also known as
velvet bean), is one of the best plant sources of l-3,4-dihydroxyphenylalanine (l-
DOPA), a naturally occurring nonprotein isomer of the amino acid 3,4-dihydroxy-
phenylalanine, which is potentially toxic if ingested in large amounts. l-DOPA has
been reported to cause serious hallucinations in addition to gastrointestinal distur-
bances, such as nausea, vomiting and anorexia. Despite this, a lot of interest exists
for this compound, since it is the major ingredient in medicines used to treat Par-
kinson disease (PD) patients. In fact, l-DOPA is a substrate of l-DOPA decarbox-
ylase, which converts l-DOPA to the biologically active catecholamine dopamine,
a compound that is depleted in the brain of people affected by PD. In faba bean,
l-DOPA accumulates mostly in embryo axis of germinating seeds, and levels of
around 75 mg/g dry weight have been detected after 9 days of germination, while
much lower amounts (0.34 mg/g dry weight) have been found in the seed (Goyoaga
et al. 2008). On the contrary, l-DOPA levels in the seeds of Mucuna species are
around 3.1–6.7 % dry weight, and can reach up to 9 % (Pras et al. 1993).
Another nonprotein amino acid, the neuroexcitatory, β-N-oxalyl-l-α,
β-diaminopropionic acid (ODAP), is found in Lathyrus sativus seeds. It is respon-
sible for neurolathyrism, a disease associated to prolonged overconsumption of this
protein-rich seed in a monotonous diet and consisting in the degeneration of up-
per motor neurons and the irreversible paralyzing of the legs in up to 6 % of the
affected individuals. Assessment of ODAP content in L. sativus has shown that
germplasm from South Asia contained relatively high amounts of ODAP (0.7–2.4 %
dry weight), whereas those from North Africa, Syria, Turkey and Cyprus had sig-
nificantly lower quantities of ODAP (0.02–1.2 %). No accessions were found to be
free of the toxin (Hillocks and Maruthi 2012)
308 F. Sparvoli et al.

8  Health Benefits of Grain Legumes

Legumes have been consumed for thousands of years for their nutritional qualities,
but only during the past few decades the potential impact of pulses on human health
has been revived. Many different studies have reported that the consumption of
pulses have beneficial physiological effects in the prevention and control of a broad
range of chronic and degenerative diseases such as obesity, cardiovascular diseases
(CVD), diabetes and cancer which are typical of industrialized societies (Bazzano
et al. 2011). Pulses could potentially be considered as “functional foods” in addition
to their accepted role of providing proteins and fibres. The consumption of pulses is
in fact recommended as part of healthy eating by governments and health organiza-
tions globally. A high consumption of pulses is also one of the eight components
of the highly lauded Mediterranean diet. However, according to FAO, the current
consumption ratio of cereal grains to pulses in the diet is 8:1, which is considerably
different from the ideal consumption ratio of 2:1. Particularly, the consumption of
pulses in the Western world remains quite low, at less than 3.5 kg/capita per year,
while in other parts of the world annual pulse consumption can range from 10 kg/
capita (South America and India) to 40 kg/capita (Burundi). The role of pulses in the
prevention and control of different pathologies is summarized below.

8.1  Metabolic Syndrome

The metabolic syndrome includes different risk factors of chronic diseases such
as CVDs and diabetes, abdominal obesity, atherogenic dyslipidemia (high level
of serum triglycerides and LDL cholesterol and low blood concentrations of HDL
cholesterol), raised blood pressure, insulin resistance, proinflammatory state and
prothrombotic state.
Results of two meta-analyses showed the long-term benefits of pulse consump-
tion (2–5 cups per week for 3–12 weeks) on risk factors of the metabolic syndrome
(Bazzano et al. 2011; Sievenpiper et al. 2009). Another study demonstrated that
frequent consumption (5 cups/week over 8 weeks) of different legumes (yellow
peas, chickpeas, navy beans and lentils) in an ad libitum diet reduced risk factors
of metabolic syndrome in overweight and obese adults. These effects were similar
or even stronger, depending on the different parameters analyzed, to the ones ob-
tained with an energy-restricted diet (by 2093 kJ/day, corresponding to 500 kcal/
day) implemented by counselling (Mollard et al. 2012).
The effects of consumption of grain legumes in the reduction of CVDs, obesity
and diabetes mellitus are very strictly correlated and dependent on the different
nutritional and nutraceutical components present in pulses, as recently reviewed
(Hayat et al. 2014) and shown in the model reported in Fig. 10.2. Particularly, due
to their slow release of carbohydrates, pulses are considered as low glycemic index
foods (Atkinson et al. 2008), contributing to a reduction in the insulinemic respons-
es. On the other hand, the consumption of pulses, through different mechanisms,
reduces serum total cholesterol and LDL cholesterol (Anderson and Major 2002).
10  Nutritional Value 309

Fig. 10.2   A simplified model representing how different pulse compounds can protect from dis-
eases associated with metabolic syndrome. PUFA polyunsaturated fatty acids, SCFA short-chain
fatty acid, T2DM type 2 diabetes mellitus, CVD cardiovascular disease (Adapted from Hayat et al.
2014)

8.2  Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder, resulting from

insulin resistance, a condition in which cells fail to use insulin properly. This dis-
order leads to several secondary complications, including cardiovascular disease,
chronic renal failure, diabetic retinopathy, hypertension, atherosclerosis, coronary
artery disease and hyperlipidaemia. Approximately 150 million people worldwide
are affected by T2DM, with a projection of 300 million people being affected by
2025. Diabetes has become a serious public health problem, particularly in devel-
oped countries.
The glycemic index (GI) of a food is defined as the incremental area under the
blood glucose curve following ingestion of a test food, expressed as a percentage
of the corresponding area following an equivalent load of a reference carbohydrate,
either glucose or white wheat bread. After consumption of high GI foods, there is
a large, rapid increase in blood sugar levels (glycemic response) and in response a
rapid increase in insulin levels, while the consumption of low GI foods is correlated
to a reduction in postprandial glucose and insulin elevations. Pulses are foods with
low GI (Atkinson et al. 2008). Epidemiological studies have suggested that the
consumption of foods with low GI protects against the development of T2DM and
is useful also in the management of T2DM patients (Campos-Vega et al. 2010; Venn
310 F. Sparvoli et al.

and Mann 2004). Particularly, people consuming about three portions per week of
whole grain foods have lower probability (risk reduction of 20–30 %) to develop
T2DM than low consumers (< 3 servings per week; Venn and Mann 2004). Differ-
ent short-term studies have shown that consumption of seeds of common beans and
other pulses typically reduces postprandial glucose elevations in nondiabetic and
diabetic individuals compared with most starchy foods. Moreover, pulses combined
with a high GI food produce a glycaemic response that is intermediate between the
high- and low GI foods, but it is not clear if the nature of the effect is additive or lin-
ear (Tappy et al. 1986; Thompson et al. 2012). A clear association between a higher
intake of legumes and a reduced risk of T2DM was particularly evident from results
of a large, prospective, population-based study of middle-aged Chinese women. In
this study, 64,227 women with no history of T2DM, cancer or cardiovascular dis-
ease at study recruitment, were followed up for an average of 4.6 years. An inverse
association between quintiles of total legume intake of three mutually exclusive
legume groups (peanuts, soybeans and other legumes) and T2DM incidence was
observed (Villegas et al. 2008).
Different mechanisms of action have been proposed to explain the low glycemic
response to legumes consumption (Hutchins et al. 2012). The high content of vis-
cous fibres in pulses contributes to the low glycemic response, as they form a gel-
like substance along the digestive tract, slowing down absorption rate of nutrients.
The inclusion of a viscous fibre with a test meal is able to reduce the blood glucose
response by an average of 44 % (Wolever and Jenkins 2001). However, the viscous
fibre component of legumes is not sufficient to determine the low glycemic and
insulin response to legumes, as the addition of bean fibre to a potato meal is sig-
nificantly less effective in lowering glucose and insulin response than a bean meal
alone (Tappy et al. 1986).
Legumes are particularly rich in amylose; its lower molecular weight, smaller
surface area and linear structure make it subject to slower digestion than amylo-
pectin. The presence of RS in legumes results in the lower availability of glucose
with the consequent slow entry of glucose in the bloodstream, the reduction of the
demand for insulin, the lowering of the GI and the insulinemic postprandial re-
sponse (Tappy et al. 1986). The protein fraction of pulses may also reduce starch
digestibility and consequently glycemic response by directly interacting with starch
(Alli and Baker 1980).
Moreover, proteins from pulses with a specific role in the prevention and
management of diabetes have been extensively studied, such as γ-conglutin and
α-amylase inhibitor isoform 1 (αAI-1; Barrett and Udani 2011; Lovati et al. 2012).
The use of seeds decoctions of white lupin as “antidiabetic” is well known in the old
pharmacopoeia. In the past years, γ-conglutin has been identified as the molecule
conferring hypoglycemic properties to lupin. It was demonstrated that it has a glu-
cose-lowering effect in normal rats upon glucose overload trials. This effect is very
similar to that of metformin, a well-known antidiabetic drug. Moreover, the chronic
oral γ-conglutin treatment in rats, in which hyperglycaemia had been induced, at-
tenuated the rise in plasma glucose and insulin (Lovati et al. 2012). γ-conglutin is
able to interact in vitro with mammalian insulin (Magni et al. 2004). To explain
10  Nutritional Value 311

its antidiabetic effect, it has been hypothesized that it acts as an insulin-like agent.
These data suggest the potential use of this protein in the control of glycaemia in pa-
tients with manifest or preclinical diabetes as well as for applications as functional
foods and dietary supplements (Lovati et al. 2012; Magni et al. 2004). Another
pulse protein with a well-characterized role in lowering the postprandial increases
in blood sugar level is αAI-1 from common bean, also referred to as phaseolamin
in starch blockers preparations (Barrett and Udani 2011). Starches are digested into
sugars by α-amylase secreted in the saliva and by the pancreas and consequently
absorbed in the small intestine. The use of purified forms of αAI-1 as a dietary
supplement reduces the postprandial spikes of glucose and insulin following a high-
GI meal (Obiro et al. 2008).
The glycemic response is influenced also by phytic acid. It was demonstrated
that the consumption of unleavened bread made from navy bean flour, containing
phytic acid, significantly reduced blood glucose area compared with that of bread
made with wheat flour, while the opposite effect was obtained removing phytic
acid. Moreover, phytic acid is also able to directly interact with starch and inhibit
in this way starch digestibility (Thompson et al. 1987). As phytic acid binds cations
such as Ca2+, its presence may also reduce the stability of α-amylase, dependent on
Ca2+ (Yoon et al. 1983).
Another possible mechanism of action responsible for the low glycemic response
to pulses consumption is independent on components but depends on the fact that
beans are commonly consumed in their whole form or minimally processed with
little or no grinding. The integrity of the cell wall is then maintained after eating.
Moreover, the cell wall of pulses is generally more resistant to digestion than the
cell wall of cereal grains. These aspects may contribute to slow digestion and con-
sequent low glycemic response (Noah et al. 1998).

8.3  Obesity and Overweight

Based on data from the World Health Organization (WHO), in 2008 approximately
1.4 billion adults worldwide were overweight and at least 500 million were obese.
Increased consumption of foods rich in DFs, such as pulses, is associated with a
lower body mass index (BMI), defined as the individual’s body mass divided by the
square of their height. Moreover, intake of foods with a high-fibre content helps in
reaching satiety faster, and this effect is maintained longer as fibre-rich foods re-
quire a longer time to chew and digest in the intestinal system (Marlett et al. 2002).
Different epidemiological studies demonstrated the efficacy of combined diets con-
taining wholegrains and pulses in conferring a lower average BMI, a smaller waist
circumference and demonstrated the negative correlation of this kind of diet with
waist-to-hip ratio (Koh-Banerjee and Rimm 2003). Only a few published studies
have specifically measured the effects of pulses consumption on body weight and
satiety. Papanikolaou and Fulgoni (2008) reported on the association of consump-
tion of beans with dietary quality and obesity risk in > 8000 adult participants in the
312 F. Sparvoli et al.

US National Health and Nutrition Examination Survey (1999–2002). Compared to

nonconsumers, bean consumers ( N = 1475) had a lower body weight and a smaller
waist size. Additionally, consumers of beans had a 23 % reduced risk of increased
waist size and a 22 % reduced risk of being obese (Papanikolaou and Fulgoni 2008).
Experimental studies in humans to evaluate the effects of pulse consumption on
weight loss were performed during intentional energy restriction or without energy
restriction. The first group of studies showed that, when pulse consumption is cou-
pled with energy restriction, there is a beneficial effect on weight loss and on other
parameters important to evaluate obesity risk. One of these trials was performed
on 30 overweight and obese participants consuming a reduced energy intake diet
(30 % energy restriction based on initial energy requirement) for 8 weeks eating
either four servings/week of pulses or none. Results showed significantly greater
decreases in BMI and body weight, expressed as percent of initial value, in the
pulse-consuming group compared with the control group (22.0 vs 20.9 kg/m2 and
27.8 vs 25.3 %, respectively). However, percentage body fat and waist circumfer-
ence decreases did not differ significantly between groups (Hermsdorff et al. 2011).
Similar results were obtained from other studies (Abete et al. 2009; Karlström et al.
1987). On the other hand, randomized controlled trials performed without energy
restriction did not support a beneficial effect of pulses on weight loss, as reviewed
by McCroryet al. (2010).
There is available evidence in support of pulse grains’ ability to induce satiety.
Subjects consuming at least 1200 g/week canned chickpeas for 12 weeks reported a
significant increase in satiety compared with subject consuming their habitual diet
(Murty et al. 2010). Similar effects on reducing appetite were described for navy
beans (Wong et al. 2009). In both cases, despite an increase in DF intake, no differ-
ences in total energy intake were observed.
RS and DF are mainly responsible of the pulse effects on the control of appe-
tite through increased satiety for their low digestibility. The fermentation of fibre
and RS by bacteria in the large intestine generates specific SCFA, mainly butyric
acid (Marinangeli and Jones 2012). This compound is the main product of indigest-
ible fractions of black beans, lentils and chickpea by human microbiota, as dem-
onstrated by in vitro fermentation (Hernández-Salazar et al. 2010). Rats fed high
diets containing 25 % adzuki beans or two varieties of common beans significantly
increased cecal butyric acid concentrations, compared with rats fed control corn-
starch diet (Han et al. 2003). Butyrate was shown to increase hepatic and muscle
expenditure as well as fat oxidation, mitochondrial oxidation and biogenesis when
supplemented to mice diet (Gao et al. 2009). Thus, increased production of SCFA
by fermentation of RS and fibres is an underlying reason for the protective benefits
by the consumption of pulses (Finley et al. 2007). Moreover, high-fibre foods are
believed to stimulate and prolong cholecystokinin secretion, a gastrointestinal pep-
tide acting as hunger suppressant (Holt et al. 1992). Therefore, it is reasonable to
hypothesize that appropriate dosages of pulse grain fibres can stimulate cholecysto-
kinin release (de Graaf et al. 2004).
Protein component of pulses also plays an important role in weight management.
Proteins, compared to carbohydrates, produce the highest thermic effect of food,
10  Nutritional Value 313

which depends on the energetic costs associated with dietary peptide catabolism,
protein synthesis and gluconeogenesis (Robinson et al. 1990). Amino acid composi-
tion could facilitate increase in energy expenditure (Marinangeli and Jones 2012).
Arginine and glutamine, present at high level in pulses, have been shown to possess
thermogenic properties (Iwashita et al. 2006; McKnight et al. 2010).
A specific antiobesity role for some proteins has also been described. Extracts
of the already-cited phaseolamin have an antiobesity effect, as shown by different
studies, although some conflicting results have been reported (Barrett and Udani
2011; Obiro et al. 2008). The αAI-1 inhibitory action results in the mobilisation of
body fat reserves, due to energy restriction. In different studies, the efficacy of a
commercial αAI-1 extract, referred to as Phase 2® (Pharmachem Laboratories, Inc.,
Kearny, NJ, USA), in reducing obesity was reported. Celleno et al. examined the ef-
fects of a dietary supplement containing 445 mg of Phase 2® on body composition
of overweight human subjects in a 30-day study. They found greater reduction of
body weight, BMI, fat mass, adipose tissue thickness and waist/hip/thigh circumfer-
ences, while maintaining lean body mass in subjects treated with Phase 2® com-
pared to subjects receiving placebo (Celleno et al. 2007). Similar results were ob-
tained in other studies, as reviewed by Barrett and Udani 2011. On the other hand,
other reports did not confirm the efficacy of this starch blocker (Chokshi 2006). The
effect of the extracts depends on a given manufacturer’s methods of extraction, as
regards the maintenance of high anti-amylase activity and purity (Obiro et al. 2008).
As raw beans contain the lectin PHA, a highly toxic protein if consumed in native
conditions, the protocol for the preparation of starch blockers requires a specialized
process to inactivate haemagglutinin activity. To overcome this problem, common
bean genotypes not able to accumulate PHA in the seed have been developed (Bol-
lini et al. 1999). On the other hand, it was suggested that PHA as a dietary adjunct or
therapeutic agent may be efficacious to stimulate gut function and ameliorate obe-
sity if a safe and effective dose range can be established. The effects of inclusion of
different levels of raw kidney bean of high lectin content (27 g/kg meal) in the diet
of obese Zucker rats and their lean littermates in comparison with pair-fed controls
were tested. It was shown that the growth of both obese and lean rats on bean diets
was retarded by the daily bean intake in a dose-dependent manner, and most of this
decrease was because bean-fed rats contained less body fat than the controls after
10 days (Pusztai et al. 1998). Consumption of bean-derived lectins was shown to
increase cholecystokinin secretion, compared with controls fed lactalbumin, con-
tributing to induce satiety (Herzig et al. 1997).

8.4  Cardiovascular Disease

CVDs are the number one cause of death, globally accounting for 30 % of all deaths,
and are projected to remain the single leading cause of death by 2030.
In general, increased consumption of soluble fibre from foods results in reduced
serum total cholesterol and LDL-cholesterol and has an inverse correlation with
314 F. Sparvoli et al.

coronary heart disease (CHD) mortality (Noakes et al. 1999). Legume consumption
has been associated with lower risks of CVD and CHD in observational epidemio-
logic studies (Bazzano et al. 2001; Kushi et al. 1999). For example, a study involv-
ing a total of 9632 men and women revealed a significant inverse relationship be-
tween legume intake and risk of CHD and CVD. In fact, legume consumption four
times or more per week, compared with less than once a week, was associated with
a 22 % lower risk of CHD and an 11 % lower risk of CVD (Bazzano et al. 2001).
Among the different controlled trials that have examined the potential hypo-
cholesterolaemic effects of a diet rich in non-soy legumes, such as peas, lentils,
different market classes of common beans, lima beans, chickpeas and faba beans,
the majority identified positive effects, particularly in some cases (Anderson et al.
1984; Nervi et al. 1989; Sowmya and Rajyalakshmi 1999), while in a very few stud-
ies no effect was identified (Cobiac et al. 1990; Mackay and Ball 1992; Winham
et al. 2007). A meta-analysis of randomized controlled trials was conducted to quan-
tify the direction and magnitude of the potential effect that consumption of non-soy
legumes may have on serum cholesterol concentrations (Bazzano et al. 2011). From
140 reports on the subject, the authors selected ten publications including studies
performed on a total of 268 participants, and in which a comparison between a non-
soy and a control diet was carried out for at least 3 weeks. This meta-analysis study
provided a strong evidence that non-soy legume consumption lowers serum total
cholesterol (Bazzano et al. 2011). Very recently, another study confirmed the effi-
cacy of a diet rich in pulses (two servings daily of beans, chickpeas, peas or lentils,
about 150 g/day) for reducing CVD risk factors in individuals 50 years or older, an
age group who are at increased risk of this disease and on which a few studies had
focused before (Abeysekara et al. 2012).
Low glycaemic index of pulses is important for lowering the risk of CVD (Du-
ranti 2006). Moreover, several components of pulses are likely to contribute to their
cholesterol-lowering effects. Soluble fibre is thought to bind to bile acids in the
intestines and prevent reabsorption into the body. Consequently, an increase in the
production of bile acids reduces the liver pool of cholesterol, triggering uptake of
serum cholesterol by the liver, thereby lowering circulating cholesterol in the blood
(Galisteo et al. 2008).
Another mechanism for the reduction of serum cholesterol depends on the activ-
ity of SCFA, particularly propionate, which alters metabolic pathways resulting in
reduced serum cholesterol (Venter et al. 1990).
Chickpea contains a higher amount of fat than other pulses, and it is a relatively
good source of nutritionally important polyunsaturated fatty acids (PUFAs), oleic
acid and linoleic acid, constituting almost about 50–60 % of chickpea fat (Jukanti
et al. 2012). It was shown that the intake of PUFA such as linoleic acid has a ben-
eficial effect on serum lipids, insulin sensitivity and haemostatic factors, thereby it
could be helpful in lowering the risk of CHD (Hu et al. 2001).
Recent evidence suggests that legume saponins, in addition to their anticancer
activity may also be beneficial for hyperlipidemia (Shi et al. 2004) and in reducing
the risk of heart diseases in humans (Geil and Anderson 1994).
10  Nutritional Value 315

8.5 Cancer

Cancer is a leading cause of death, mainly in industrialized countries, in the USA,

for example, it is second only to CVD. In many developing countries cancer inci-
dence appears much lower, but it is expected to raise due to increased control over
infectious diseases and control of childhood diseases, leading to rise in life expec-
tancy and in proportion of elderly people (Khatib and Aljurf 2008).
Different epidemiological analyses indicated a decreased risk of death associ-
ated with colon, breast and prostate cancer in countries with higher consumption
of pulses (Mathers 2002). Moreover, experiments performed on laboratory animals
have confirmed these results. One such study showed that feeding black or navy
beans to rats exposed to the carcinogen azoxymethane reduced both the incidence
and number of colon tumours by 50 % (Hangen and Bennink 2002). Most of these
studies were focused on common beans, while a few investigations have been per-
formed on other pulses. For example, a 64 % suppression of azoxymethane-induced
aberrant cryptic foci was shown in mice fed with 10 % chickpea flour (Murillo et al.
2004). It was reported a systematic comparative study on antiproliferation effects of
hydrophilic extracts from 13 commonly consumed food legumes (green pea, yel-
low pea, chickpea, lentil, yellow soybean, black soybean, pinto bean, black bean,
small red bean, red kidney bean, mung bean, adzuki bean and black-eyed pea), us-
ing nine in vitro cultured human cancer cell lines (Xu and Chang 2012). Among the
legume tested, lentil, the four common beans, mung bean and adzuki bean exhibited
dose-dependent inhibitory effects against cell proliferation of all cancer cell lines.
In particular, adzuki bean exhibited the strongest antiproliferative properties in a
dose-dependent manner against seven of the nine cancer cell lines. Moreover, other
legumes tested showed antiproliferative effects only against some cancer cell lines
(Xu and Chang 2012). These results indicate that commonly consumed legumes may
serve as an excellent dietary source for cancer prevention and further studies are
warranted to characterize the potentiality of different legumes in cancer protection.
There are several bioactive food components of pulses that could be responsible
for the cancer-preventive effect, as shown in the model reported in Fig. 10.3.
It is recognized that a major role in the anticancer effects of food is played by
phenolic components. Beneficial effects of isoflavonoids in preventing breast and
prostate cancers have been extensively studied (McCue and Shetty 2004). The al-
ready-mentioned paper from Xu and Chang (2012) showed that the total phenolic
content of 13 food legumes exhibited significant linear correlation with the overall
antioxidant activities. Coloured common beans, black soybean, lentil, adzuki bean
and mung bean exhibited stronger antioxidant capacities and cancer cell prolifera-
tion inhibitory effects on different human cancer cell lines, as compared to green
and yellow peas, chickpea, yellow soybean and black-eyed pea. Although the anti-
oxidant properties of polyphenols have been extensively investigated, more recently
their real impact as antioxidants has been reconsidered and questioned. In fact, there
is an emerging view that these molecules may act not only by scavenging reactive
316 F. Sparvoli et al.

Fig. 10.3   A simplified model representing how different pulse compounds can protect from can-
cer. SCFA short-chain fatty acids. (Adapted from Hayat et al. 2014)

oxygen and nitrogen species or suppressing their production but also by enhancing
the endogenous antioxidant capacity of cells/tissues (e.g. glutathione synthesis) or
by influencing signalling pathways through interaction with proteins, enzymes and
nuclear receptors, as recently reviewed (Martin et al. 2013). The anticancer activity
of polyphenols has been associated with lower leukocyte immobilization, apoptosis
induction, cell proliferation and angiogenesis inhibition (Garbisa et al. 2001; Ni-
jveldt et al. 2001).
Phytic acid is a broad-spectrum antineoplastic agent, playing an important role
in cancer prevention as well as in control of experimental tumour growth, progres-
sion and metastasis. Phytic acid seems to be responsible for the epidemiological
link between high-fibre diets (high phytic acid content) and low incidence of some
cancers. Phytic acid, after its rapid intake and dephosphorylation, enters the pool
of inositol phosphates and acts as a strong antioxidant, enhances immune function,
elicits anti-inflammatory activity, modifies phase I and II metabolizing enzymes,
modulates oncogene expression, normalizes abnormal cell proliferation, induces
cell differentiation, induces apoptosis and inhibits angiogenesis (Vucenik and
Shamsuddin 2006).
Saponins are another class of non-nutrient bioactive compounds for which epi-
demiological studies suggest anticancer activity (Shi et al. 2004). Soyasaponin I
properties have been mainly investigated, and its molecular activity was identified.
This compound is able to inhibit the transfer of sialic acids to the nonreducing
terminal positions on sugar chains of glycoconjugates, a process correlated with on-
cogenic transformation and metastatic potential (Chang et al. 2006). Moreover, sa-
ponins are able to regulate the apoptosis pathway enzymes, leading to programmed
cell death of cancer cells (Zhu et al. 2005).
10  Nutritional Value 317

Also, lectins present in legumes may play a key role in preventing certain can-
cers (Campos-Vega et al. 2010). In vitro studies demonstrated, for example, that Vi-
cia faba agglutinin (VFA) stimulated the morphological differentiation and reduced
the malignant phenotype of colon cancer cells (Jordinson et al. 1999). The inclusion
of PHA from raw kidney bean in the diet of a murine model for non-Hodgkin lym-
phoma tumour greatly reduced, in a dose-dependent manner, the growth rate of the
tumour, either as an intraperitoneal ascites tumour or as a solid subcutaneous one
(Pryme and Bardocz 2001). The number of Krebs II lymphosarcoma tumour cells
in the ascitic fluid of mice fed a PHA diet for 8 days was three times lower than in
mice fed a control diet (Bardocz et al. 1997). There is scientific evidence for differ-
ent anticarcinogenic mechanisms of action of lectins, including binding to tumoural
cell membranes, cytotoxic effects of lectins on tumour cells (decrease in protein
synthesis and induction of apoptosis), reduction of cell proliferation and stimulation
of the immune system (De Mejia and Prisecaru 2005).
Different in vitro and in vivo experiments have shown that protease inhibitors
have anticarcinogenic properties. Although the majority of these studies were per-
formed with soybean, as reviewed by Roy et al. (2010), more recently, the antipro-
liferative effects on human colon cancer cells of two recombinant wild-type Bow-
man–Birk inhibitors from pea seeds has been reported (Clemente et al. 2005).
All the molecules present in pulses having anticancer properties described so far
are soluble in aqueous–alcohol extracts, while RSs, present in high amount in puls-
es, together with nonstarch polysaccharides, are primarily insoluble residues from
aqueous–alcohol extracts. Colon carcinogenesis was induced by azoxymethane
treatment in obese ob/ob mice fed with diet containing cooked navy beans (whole
beans), the insoluble or soluble fraction of aqueous–alcohol extracts or a standard
diet (Bobe et al. 2008). In comparison to control-fed mice, the incidence rates of
various types of colon lesions were detected in fewer mice on either bean fraction
diet. Moreover, no significant differences in incidence rate of various types of colon
lesions between mice-fed diets containing bean residue or bean extract were ob-
served. These results suggest that the insoluble fraction, containing RS, contributes
in a similar way of the soluble fraction to the cancer-protective effect of cooked
navy beans (Bobe et al. 2008). The cancer-preventive effect of RSs and nonstarch
polysaccharides was previously shown (Bauer-Marinovic et al. 2006). As already
discussed, SCFA, mainly butyric acid, are the products of the bacterial fermentation
of resistant starches. They have protective effect against colon cancer as against the
majority of tumours developed in the distal colon. Butyrate was reported to induce
apoptosis, growth arrest and differentiation in colon cancer cell lines (Barnard and
Warwick 1993); its effect is due to its activity in histone hyperacetylation and down-
regulation of epidermal growth factor receptor (Archer et al. 1998). The low GI of
legumes attenuates the postprandial insulin response contributing to their cancer-
preventive effect as hyperinsulinaemia and hyperglycaemia are reported to increase
the risk of colon cancer (Jenkins et al. 2002).
318 F. Sparvoli et al.

Acknowledgment  This work was partially supported by the Programme FILAGRO “Strategie
innovative e sostenibili per la filiera agroalimentare”, as part of the activities defined within the
Accordo Quadro Consiglio Nazionale delle Ricerche and Regione Lombardia.

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Chapter 11
Seed Physiology and Germination of Grain
Legumes

Jaime Kigel, Leah Rosental and Aaron Fait

1 Introduction

Grain legumes belong to the Fabaceae (= Leguminosae) family in which the struc-
ture and anatomy of the seeds, as well as their main mechanism of dormancy and
germination control based on seed-coat impermeability to water, have been remark-
ably conserved, despite the prolonged time span since the origin of the family dur-
ing the early Tertiary, ca. 60 million years (Myr) ago (Cronk et al. 2006). The major
crop legumes are included in the Hologalegina and the Millettoid/Phaseoloid sister
clades of the Papilionoideae (= Faboideae) subfamily (Bruneau et al. 2013). Tem-
perate herbaceous legumes, such as the pulses chickpea (Cicer arietinum), lentil
(Lens culinaris), garden pea (Pisum sativum), faba bean (Vicia faba), sweet pea
( Lathyrus spp.), and pasture and fodder species ( Trifolium spp., Medicago spp.)
belong to the Hologalegina clade. In contrast, tropical and subtropical legumes
such as common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), runner
bean (Phaseolus coccineus), tepary bean (Phaseolus acutifolius), cowpea (Vigna
unguiculata), mungbean (Vigna radiata), pigeon pea (Cajanus cajan), and soybean
(Glycine max) belong to the Millettoid/Phaseoloid clade. Lupins ( Lupinus spp.) are
included in the minor Genistoid clade (Wojciechowski et al. 2004). Germination
responses of the different legume crops to environmental conditions are strongly
associated with taxonomic affiliation and climatic regions of origin.

J. Kigel ()
Hebrew University of Jerusalem, POB 10, 76100 Rehovot, Israel
e-mail: [email protected]
L. Rosental
French Associates Institute for Agriculture & Biotechnology of Drylands, Jacob Blaustein
Institutes for Desert Research, Ben-Gurion University of the Negev, 48990 Beer Sheva, Israel
e-mail: [email protected]
A. Fait
Blaustein Institutes for Desert Research, Ben Gurion University,
84990 Midreshet Ben-Gurion, Israel
e-mail: [email protected]
© Springer Science+Business Media New York 2015 327
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_11
328 J. Kigel et al.

2  Seed Changes During Domestication of Grain Legumes

The legume family has 41 species with domesticated crops, the greatest number
compared to any other plant family (Harlan 1992). Grain legumes are grown for
their highly nutritious seeds, already an important component of the human diet in
early civilizations (Zohary et al. 2012). As in other seed crops, the “domestication
syndrome” in legumes involved a set of traits that differentiate between domesti-
cated crops and their wild ancestors (Fuller 2007; Weedin 2007; Zohary et al. 2012;
Abbo et al. 2014). Regarding seed traits, the syndrome included loss of seed dis-
persal, an increase in seed size, and loss of seed dormancy. Loss of seed dispersal
through selection for non-dehiscent pods was a primary step in legume domesti-
cation. Together with synchronous seed maturation, it allows higher yields, since
losses due to pod dehiscence are prevented and harvesting can be carried out when
most seeds on a plant have matured. As to seed size, many wild legume species
produce relatively large seeds rich in proteins, presumably as a consequence of
symbiotic nitrogen fixation, making them an attractive food source. Large seeds
rich in nitrogen facilitate seedling establishment, particularly in nutrient-poor tropi-
cal soils. Furthermore, larger seeds generally produce bigger and more competitive
seedlings (Nakamura 1988; Al-Karaki 1998; Bond et al. 1999; Gan et al. 2006),
with higher stress tolerance (Westoby et al. 1992; Gholami et al. 2009) and able
to emerge from deeper burial due to greater seed reserves (Lush and Wien 1980;
Kluyver et al. 2013). Therefore, large seeds are also more suitable for sowing and
cultivation practices. Indeed, it has been proposed that increased seed size was se-
lected for during domestication by tillage and cultivation (Harlan et al. 1973). Seed
mass of cultivated grain legumes is often up to tenfold larger compared to wild rela-
tives, as in common bean (Gepts and Debouck 1991; Santalla et al. 2004), mung-
bean and azuki bean (Isemura et al. 2007), rice bean (Vigna umbellata; Isemura
et al. 2010), lablab bean (Lablab purpureus; Maass and Usongo 2007), and soybean
(Kluyver et al. 2013). The increase in seed mass of grain legumes with domestica-
tion is usually linked to a reduction in seed-coat thickness and increased seed-coat
permeability (Lush and Evans 1980).
Regarding seed germination, domestication resulted in the reduction and loss
of seed dormancy. Plant species usually possess dormancy mechanisms that dis-
perse germination in time, thus reducing the risk of catastrophic germination and
population extinction due to soil seed-bank depletion, as well as preventing sibling
competition, particularly in annual species (Kigel 1995; Baskin and Baskin 2014).
In domesticated crops, in contrast, fast and uniform germination is necessary to en-
sure early plant establishment before severe weed competition occurs and to allow
synchronous seed set and maturation before harvest. Loss of dormancy leads to si-
multaneous germination after sowing once water availability and temperature allow
it. In domesticated crops, lack of dormancy was most probably selected for by sow-
ing from harvested seeds, because seeds that do not germinate will not contribute to
the harvested seed population (Fuller and Allaby 2009). Moreover, in several grain
legumes, early domestication occurred independently at different locations, imply-
11  Seed Physiology and Germination of Grain Legumes 329

ing that in these species loss of dormancy took place several times in historical
times. The fact that seed-coat permeability in legumes is controlled by a few genes
(see Sect. 3.10) probably facilitated loss of dormancy in the different domestication
events (Zohary et al. 2012). This is the case for common bean that was domesti-
cated in South America and Meso America in different times and locations (Brucher
1988; Gepts and Debouck 1991; Kaplan and Lynch 1999; Gepts et al. 2005). Seeds
of Phaseolus vulgaris ssp. aborigineous, a wild relative of common bean, are dor-
mant due to seed-coat impermeability (Kaplan 1965).

3 Seed Structure and Germination Control

in Legume Seeds

3.1  Seed Dormancy and Hardseededness

A comprehensive classification consisting of five classes of seed dormancy has

been proposed by Baskin and Baskin (2014, 2004): physiological (PD), morpho-
logical (MD), morpho-physiological (MPD), physical (PY), and combinational
(PY + PD). MD refers to seeds that have an underdeveloped embryo that requires
time to grow and germinate. Physiological embryo dormancy, the most prevalent
type of dormancy, is regulated by hormonal action involving abscisic acid (ABA)
and gibberellins (GA). PY is due to the presence of water impermeable seed covers
that prevent water uptake by the seed, and can be broken by mechanical or chemi-
cal scarification. PY occurs in 18 evolutionarily advanced angiosperm families
(Gamma-Arachchige et al. 2013), sometimes combined with physiological embryo
dormancy, namely PY + PD (Baskin et al. 2000). PY is widespread in the legume
family and has been extensively studied. It usually occurs in the wild ancestors
of cultivated legumes and is one of the key traits modified through domestication
(Gepts and Debouck 1991; Abbo et al. 2011; Zohary et al. 2012). In recent years,
PY + PD has been found in an increasing number of papilionoid legumes—species
of Vicia, Trifolium, Medicago, Lathyrus (van Assche and Vandelook 2010; Hu et al.
2013). The presence of physiological dormancy is probably more common than
presently known in papilionoid legumes and in the wild ancestors of legume crops.
Seeds with PY that are unable to imbibe and germinate when in contact with
free water are defined as “hard (impermeable) seeds,” in contrast to “soft seeds”
which swell during imbibition. Thus, “hardseededness” is the inability of seeds to
absorb water and germinate. Legumes’ hard seeds may remain impervious to wa-
ter even after prolonged imbibition (Rolston 1978; Tran and Cavanagh 1984). The
biological and ecological advantages of the hard-seeded character are a longer seed
life span due to protection against seed decay under humid conditions and post-
ponement of germination, thus building up seed reserves in the soil (Baskin and
Baskin 2014; Taylor 2005). For instance, wild azuki bean is a summer annual with
impermeable seeds that during winter in wet soil at low temperatures inhibiting
330 J. Kigel et al.

germination. Permeable seeds that imbibe under these conditions are lost since they
fail to germinate in the cold soil and decay due to the action of soil pathogens (Kaga
et al. 2008). In wild legumes, degree of hardseededness varies within and between
cohorts of seeds produced by different plants in the natural population. Variation in
the fraction of impermeable seeds and in the duration of hardseededness arises from
genetic differences, environmental effects during plant and seed development and
from intra-plant somatic effects related to nodal position of the pods and to position
of the seeds within the pods (Nakamura 1988; Kigel 1995). Thus, wild legumes pro-
duce seed cohorts that vary in their propensity to imbibe water, spreading germina-
tion of their offspring over time. However, hardseededness is an undesirable trait for
cultivation of grain legumes and for the food processing industry (Ross et al. 2010).
Seed soaking is the initial step in the thermal processing of raw legume seeds in spe-
cies with toxic compounds and antinutritional factors in their seeds. Furthermore,
impermeable seed coats delay seed imbibition, increasing cooking time and fuel use
and may lower the quality of the food product. On the other hand, the association
between increased seed-coat permeability and thinner seed coats make seeds more
susceptible to mechanical damage during harvest and seed processing. Hence, past
selection and current breeding programs aim at creating lines with seed coats that
are fairly permeable to water and yet reasonably strong to reduce mechanical dam-
age to the seeds.

3.2  Legume Seed Structure and Seed-Coat Anatomy

Seed development has been thoroughly studied in grain legumes, particularly in pea
(van Dongen et al. 2003; Nadeau et al. 2011). Legume seeds develop from campy-
lotropous ovules covered by two integuments, have well-developed embryos with
storage cotyledons and a seed coat with a distinctive anatomy (Boesewinkel and
Bouman 1995; Werker 1997). The more massive, outer integument differentiates
into the seed testa that is composed by several cell layers with different functions
(Watson 1948; Chamberlin et al. 1994; Ma et al. 2004). The outermost layer, the
epidermis, develops into a uniseriate palisade layer of elongated and narrow mac-
rosclereids, the Malpighian cells oriented perpendicularly to the seed surface. The
palisade layer is covered by a thin cuticle and lacks intercellular spaces. The mac-
rosclereids are tightly packed, with thick and often lignified cell walls that partially
occlude the cell lumen. Their outermost cell wall is usually thicker and often suber-
ized, forming the macrosclereid cap. Cells of the subepidermal layer differentiate
into a continuous uniseriate sheath of osteosclereids (bone shaped, hourglass cells),
with uneven cell wall thickenings and an extensive network of intercellular spaces.
The macrosclereid and osteosclereid layers contribute to the mechanical strength of
the seed coat. Deeper layers, called the nutritional tissue, consist of branched par-
enchymatic cells with intercellular spaces and collapses during seed maturation due
to the pressure exerted by the expanding cotyledons. The thinner, two-layered inner
integument is obliterated during seed ontogeny, but may produce a cuticle on the
11  Seed Physiology and Germination of Grain Legumes 331

surface facing the endosperm (Werker 1997). In species that retain an endospermic
layer in the mature seed, this internal cuticle adheres to the aleurone layer when the
inner integument degenerates.
A main function of the seed coat during seed development is to supply nutrients
arriving via the funiculus and distribute them to the growing embryo through a vas-
cular net embedded in its parenchyma tissue (Patrick and Offler 2001). The vascula-
ture of the seed coat differs among papilionoid legumes—in soybean and common
bean (Phaseoleae tribe) the seed coats have an extensive vascular network, whereas
in pea and faba bean (Vicieae tribe), as well as in Medicago truncatula, it consists
of a single chalazal vein with two lateral branches (van Dongen et al. 2003; Wang
and Grusak 2005). The parenchyma tissue is responsible for the post-phloem sym-
plastic transport of nutrients from the seed coat to the embryo cotyledons. Solutes
imported by the phloem, such as sucrose, amino acids, and minerals, are unloaded
first into the parenchyma cells, then released into the apoplast and conveyed there-
after through the endosperm to the growing embryo and storage cotyledons (Patrick
and Offler 2001; van Dongen et al. 2003).
In contrast to the relatively uniform structure of the testa, the presence, amount,
and function of the endosperm in the mature seed vary among papilionoid legumes
(Kirkbridge et al. 2003). About 66 % of the 452 genera in the subfamily have
endosperm in the mature seeds. In the other 154 genera, the endosperm is fully
consumed and obliterated during seed ontogeny and is characteristically absent
in mature seeds of the Phaseolae tribe (e.g., Phaseolus, Vigna, Cicer; Yeung and
Cavey 1990), but may remain as a uniseriate aleurone layer of unknown function,
as in soybean (Ma et al. 2004). In genera with endospermic seeds, the endosperm
typically encloses the embryo. Of these, in 97 genera, the endosperm is thick and
functions as a storage tissue with cell walls rich in galactomannans, thus comple-
menting storage in the cotyledons, as in fenugreek (Trigonella foenum-graecum),
crimson clover (Trifolium incarnatum) and lucerne ( Medicago sativa; Reid and
Meier 1972). In these seeds, the testa is separated from the embryo by a well-de-
veloped endosperm with an outermost aleurone layer, responsible for synthesis and
secretion of the enzymes involved in degradation of the galactomannans stored in
the cell walls of the inner, larger endosperm cells. This type of endosperm is well
developed in seeds of the legume Cyamopsis tetragonoloba, an important industrial
source of guar gum (McClendon et al. 1976). In addition to its storage role, swelling
of the strongly hydrophilic galactomannans in the endosperm during seed imbibi-
tion causes seed-coat rupture, thus facilitating radicle protrusion (Reid and Bewley
1979). Most interestingly, in 177 genera, the endosperm remains as a continuous
thin layer of living cells of unknown function. In some species, the endosperm is
reduced to an aleurone layer lacking intercellular spaces, while in others it includes
a few additional layers of endosperm cells (Watson 1948; Ma et al. 2004). In the
mature seeds of these species, the endosperm surrounds the entire embryo, enclos-
ing the radicle within a sheath. In Medicago truncatula (Bolingue et al. 2010) and
Trifolium repens (Jakobsen et al. 1994), this endospermic envelope is not fused to
the testa and is composed of several layers of living cells, similarly to the endo-
sperm cover involved in the physiological dormancy of Solanaceae and Asteraceae
332 J. Kigel et al.

(Finch-Savage and Leubner-Metzger 2006). However, its function in papilionoid

legumes has not been elucidated till now.
Altogether, development and differentiation of the seed coat in legumes is a
complex and highly regulated process. Cell layers differentiate rapidly according to
the changing role of the seed coat—from nutrient transport and metabolism, during
the embryo growth phase, to the structural and chemical characteristics required for
protection and control of germination of mature seeds after dispersal (Miller et al.
1999). This complexity is reflected in the transcriptome analysis of the seed coat in
Medicago truncatula, which contains more than 30,000 genes (Verdier et al. 2013).

3.3 Anatomical and Chemical Bases of Seed-Coat

Impermeability

Seed-coat impermeability in papilionoid legumes is due to structural and chemical

changes that take place during the last stages of seed maturation. Seed dehydration
and subsequent contraction of cells in the seed coat result in mechanical compres-
sion and a closer cell packing that drastically reduce rate of water uptake by the
seeds. Complete impermeability requires, however, the presence of hydrophobic
compounds in the cells and closure of all water gaps in the seed coat (Werker 1997;
Gamma-Arachchige et al. 2013). Yet, there is no consensus regarding the nature of
these hydrophobic compounds. The palisade layer is generally considered as the
main permeability barrier in legume seeds. The waxy layer and thin cuticle cover-
ing the palisade layer are not accountable for seed impermeability in many legumes,
since abrasion or organic solvents did not render the seed coat permeable to water
(Rolston 1978; Werker 1997). Nevertheless, cracks in the cuticle lead to permeable
seeds in some legumes, such as soybean (Ma et al. 2004). A subcuticular muci-
laginous layer is present in the outer cell wall of the palisade cells. It is composed
of pectic insoluble materials that contract upon desiccation and undergo chemical
changes that make them hard and hydrophobic, with a very low swelling capacity
(Werker 1997).
In Pisum, the mucilaginous layer is conspicuous in impermeable seeds of the
wild species Pisum elatius, Pisum fulvum, and Pisum humile, but barely distin-
guished in cultivated Pisum sativum with a permeable seed coat (Werker et al.
1979). In contrast, in Lupinus palaestinus, the mucilaginous layer remains soft, but
seeds are impermeable (Werker 1997). Impermeability of the palisade layer has
been attributed to the deposition of callose and suberin in the macrosclereids ( Meli-
lotus alba—Riggio-Bevilaqua et al. 1989), to suberized caps on the outer walls of
the macrosclereids ( Trifolium subterraneum—Aitken 1939), and to the presence of
phenolic compounds in the cell walls and lumen of macrosclereids and osteoscler-
eids ( Pisum spp., Werker et al. (1979)). In Pisum elatius and Pisum fulvum with im-
permeable seeds, catechol oxidase activity increases sharply during seed maturation
and is associated with a higher content of phenols in the seed coat. These changes
did not occur in cultivated Pisum sativum with permeable seed coat (Marbach and
11  Seed Physiology and Germination of Grain Legumes 333

Mayer 1974). When seeds of Pisum elatius were dried in the absence of O2 the seed
coat became totally permeable to water, thus suggesting that oxidation of phenolic
compounds renders the seed-coat impermeability (Marbach and Mayer 1975). In
black common bean, phenolic compounds such as tannins are associated to reduced
water uptake (Sievwright and Shipe 1986). Similarly, in permeable lines of com-
mon bean, seeds have less phenolic compounds in the osteosclereids at the site of
initial water entry in the raphe and chalazal region, compared to semi-hard lines
(Holubowicz et al. 1988). In navy lines of common bean, faster water uptake was
associated with seed coats containing lower levels of phenolics, tannins, and un-
saturated fatty acids, compared to pinto bean lines with slower water uptake (Ross
et al. 2010). Other studies, however, indicate that phenolic compounds and callose
do not play a role in seed-coat impermeability of other papilionoid seeds (Serrato-
Valenti et al. 1989).
The role of the internal cuticle of the inner integument in the control of seed
imbibition by legume seeds has been questioned and apparently does not impair
water uptake (Ma et al. 2004). Localization of the impermeability barrier has been
attempted by piercing the seed coat to different depth layers and correlating with
their anatomy and composition. Seeds became permeable after piercing the whole
palisade layer in Lupinus palaestinum (Werker 1997) and up to the endosperm in
Coronilla varia (McKee et al. 1977), while in Medicago rotata an intact osteo-
sclereid layer prevents water uptake (Russi et al. 1992). Thus, different tissues can
contribute to seed-coat impermeability in papilionoid legumes, even though the
palisade layer is generally viewed as the main barrier to water uptake. Moreover,
the fact that seed-coat impermeability can be achieved by different combinations of
structures and hydrophobic compounds in the testa shows the potential plasticity of
testa development and mechanisms of germination control in grain legumes, despite
their uniform morphology.

3.4 The Roles of the Micropyle, Hilum, and Lens

in Legume Seeds

The seed coat of papilionoid legumes has several specialized structures—the hi-
lum, micropyle, and lens (or strophiole)(Fig. 11.1; Kirkbridge et al. 2003), which
have different roles in the regulation of both seed dehydration and water uptake by
the dry seed. The micropyle is a narrow gap in the integuments through which the
pollen tube enters the ovule during the fertilization process. In some species and
cultivars, it remains open in the mature seed, while in others is closed by shrink-
age and compression of surrounding cells upon seed desiccation, or occluded by
hydrophobic compounds thus preventing water entry (Werker 1997). The radicle tip
is oriented towards the micropyle area and protrudes through it during germination.
The hilum is a specialized structure that differentiates in the seed coat at the place
where the seed detaches from the funiculus, the structure connecting the developing
seed to the fruit. An abscission layer differentiates that facilitates seed detachment.
334 J. Kigel et al.

Fig. 11.1   Structure of papilionoid legume seed and position of micropyle, hilum, and lens. (Kirk-
bridge et al. 2003)

In legume seeds, the funiculus is fused to the outer integument forming the raphe, a
ridge on the seed coat of the mature seed. The hilum of papilionoid legumes has a
complex structure in which the palisade layer is interrupted by a hilar groove with
a longitudinal slit—the hilum aperture (Fig. 11.2). In addition, a counter-palisade
layer of macrosclereid cells derived from the funiculus epidermis is fused to the
palisade layer of the hilum. A cuticle develops between the fused palisade layers.
Importantly, chemical composition of the cell wall differs in the palisade compared
to the counter-palisade macrosclereids, with a higher content of hydrophilic com-
pounds in the counter-palisade layer (Lush and Evans 1980; Werker 1997). This
leads to differential swelling and contraction of the two layers, causing opening
and closure of the hilum aperture in response to changes in humidity outside the
seed. The osteosclereids layer is absent under the hilum. Instead, a tracheid bar is
present and runs underneath the hilum groove. This is a unique structure found only
in papilionoid seeds (Lersten 1982). It has tracheid-like cells, larger than tracheids
in the vascular bundles of the seed. They are oriented perpendicularly to the hilar
groove and have cell walls densely pitted, with the pit membrane absent in many
pits. It probably plays a role as an avenue for water vapor loss during seed dehydra-
tion and later for water diffusion through the hilum during seed imbibition. The lens

Fig. 11.2   Median section of the hilum (a) and enlarged view of hilum structure (b) in the lupin
seed. (Adapted from Hyde (1954))
11  Seed Physiology and Germination of Grain Legumes 335

(or strophiole) is an area of modified seed coat, which often appears externally as
a lens-shaped structure on the raphe near the hilum, opposite to the micropyle. In
the lens, the macrosclereids of the palisade layer are longer and the osteosclereid
layer is absent (Werker 1997). The long macrosclereids can split apart more easily,
producing a small fissure—the strophiolar cleft, through which water can enter into
the seed (Hagon and Ballard 1970).
The function of the hilum in papilionoid seeds was described and experimen-
tally confirmed in a seminal paper by Hyde (1954). In legume seeds with imperme-
able seed coats, the hilum operates as a hygroscopically activated valve controlling
seed dehydration after seed dispersal: the hilum slit opens when the seed is in dry
air and closes when the outside air is moist ( Trifolium—Hyde (1954), common
bean—Hanma and Denna (1962), wild cowpea—Lush and Evans (1980)). Under
dry conditions, the slit opens by the contraction of the counter-palisade layer, allow-
ing water vapor loss to the dryer atmosphere. Under humid conditions, the coun-
ter-palisade expands, closing the hilum aperture and preventing entry of moisture.
Empty cells of the tracheid bar and the large intercellular spaces in the osteoscle-
reid layer most likely facilitate water vapor diffusion through the open hilum to
the atmosphere. Thus, papilionoid seeds can reach very low water content despite
wide fluctuations in external relative humidity and also prevent water entry due to
hilum closure. The lowest water content in legume seeds with a functional hilum is
established during equilibrium with the driest conditions to which the seeds were
exposed, thus prolonging seed longevity (Hyde 1954; Ellis and Roberts 1982). In
domesticated grain legumes in which the hilum is not functional, seed water content
fluctuates according to external humidity and seed longevity is shorter. Full hilum
closure can be prevented by incomplete development of the palisade layer or by
structures interfering with closure, such as a more outward position of the tracheid
bar as in cultivated pea (Werker 1997).

3.5  Seed Water Content and Hardseededness

Seed water content is a major factor affecting water-absorption characteristics of

legume seeds. Impermeability of legume seeds develops as they lose moisture dur-
ing maturation and after dispersal. Seed-coat permeability already decreases at 20–
25 % water content, reaching full impermeability once water content falls below a
threshold, for example, 7 % in Trifolium subterraneum (Fairbrother 1991), 9 % in
Lupinus varius (Quinlivan 1968), 10 % in wild cowpea (Lush and Evans 1980), and
12 % in lentil (Tang et al. 1994). Thereafter, water diffusion between seed and at-
mosphere takes place through the hilum functioning as a hygroscopically activated
valve in the impermeable seed coat. In general, impermeability is reversible in grain
legumes if water content is above 10–12 %, but becomes irreversible below 6–7 %
(Ellis et al. 1985).
Time course of water absortion depends on initial seed water content. In lentil,
seeds had high rates of absortion at 16–24 % water content, with water entering
through the whole seed surface. At 10–12 %, permeability is reduced by four orders
336 J. Kigel et al.

of magnitude as a result of cell shrinkage and decreased capillarity of the seed coat,
and the closure of the lens and micropyle. Below 10 % the hilum is closed and
seeds became impermeable to water (Tang et al. 1994). In wild Lima bean, seeds
are dispersed with 12–14 % water content near the end of the dry season; they lack
dormancy and germinate readily if water is available. Yet, since in their natural
environment no rainfall occurs at the time of seed dispersal and the seeds remain
on the soil surface exposed to high temperatures (60–65 °C) and low humidity, seed
water content is further reduced to 7–8 %, seeds became impermeable and dormant
and are incorporated into the soil seed bank thus persisting for several years (De-
greef et al. 2002). In semi-hard lines of common bean, seed-coat permeability is
reversible and varies according to seed water content. Dickson and Boettger (1982)
defined semi-hard lines as lines in which seeds do not imbibe during the first 24 h
when their water content is lower than 8 %, but imbibe readily if higher than 10 %.
In these lines, water entry is localized at the raphe and chalazal region of the seeds.
Below 6 % water content palisade cells at the raphe become wider and shorter pro-
viding better sealing and seeds become impermeable. Increasing the water content
to 12 % reversed the changes in shape and length of the palisade cells, rendering the
seed coat permeable to water (Holubowicz et al. 1988).

3.6 Physical Dormancy and Disruption

of Seed-Coat Impermeability

Under natural conditions seed-coat impermeability can be disrupted by exposure to

extreme diurnal changes in temperature and humidity, cycles of wetting and drying
such as after rainfall events or dew condensation, and freezing and thawing (Tran
and Cavanagh 1984). These changes cause expansion and contraction of cells and
eventually produce cracks in the seed coat or disrupt the testa at specific weak
points, such as the lens. Direct damage to the seed coat as by fire, abrasion by soil
particles, the action of soil microorganism and passage through the digestive tract
also lead to increased seed permeability and germination in legume seeds. Such pro-
cesses may take years to complete and are of ecological significance, since they af-
fect seed-bank density and the spread of germination over time (Baskin and Baskin
2014; Taylor 2005).
PY can be artificially removed by mechanical and chemical scarification (Rolston
1978; Tran and Cavanagh 1984; Argel and Parton 1999). Priming treatments are ap-
plied to commercial legume seeds in order to improve germination rate and unifor-
mity (soybean—Marwat et al. (2008), Medicago sativa—Zhang et al. (2007)). The
enhanced permeability due to environmental factors or priming treatments leads
to oxygen uptake and activation of metabolic processes (Bai et al. 2012) which
enhance energy production, redox balance, and the biosynthesis of compounds for
the full resumption of metabolism in preparation for germination (Benamara et al.
2003; Fait et al. 2006).
11  Seed Physiology and Germination of Grain Legumes 337

In regions with Mediterranean climate, breaking of PY has been attributed to the

wide amplitude of daily temperature fluctuation at or near the soil surface during
the warm dry summer, in the range of 30–60 °C (Taylor 2005). Rate of softening de-
creases with increasing depth of burial in the soil, concomitantly with the decreas-
ing amplitude of the diurnal temperature cycle. Less known are the factors involved
in the disruption of PY of papilionoid legumes in regions with temperate climate,
with much smaller daily fluctuations in soil temperature. In an intriguing study, van
Assche et al. (2003) showed that exposure of legume seeds to chilling renders the
seed coats sensitive to daily temperature alternation, with their combined action
breaking seed-coat impermeability. The effect of chilling is reversible and disap-
pears by exposing the seeds to higher temperatures during the warmer season. This
reversal results in seasonal cycling in the ability of legume seeds to respond to the
alternating temperatures necessary to break seed-coat permeability and, therefore,
in seasonal germination (Baskin 2003).
Softening of hard seeds is a very slow process at low temperatures, and prob-
ably contributes to persistent legume seed banks in temperate regions. In Medicago
arabica, rate of seed softening increases exponentially with increasing tempera-
tures, with a Q10 higher than two (van Assche and Vandelook 2010). This suggests
possible involvement of a chemical reaction in the breakdown of impermeability in
the seed coat, in the lens or in the hilum. In Mediterranean annual pasture legumes,
thermal degradation and loss of lipids in the seed coats of seeds exposed to high
soil temperatures during the summer caused seed-coat fractures, thus increasing
permeability (Zeng et al. 2005). Thus, changes in the quality and quantity of the
lipid components may modulate seed-coat permeability.

3.7 Physiological and Combinational Dormancy in Legumes

In legumes with combinational dormancy (PY + PD), PD prevents accidental ger-

mination in case of failure of seed-coat impermeability (Baskin and Baskin 2014).
In hard-seed species, PD can be detected in fresh seeds by seed-coat scarification,
since a period of after-ripening in dry conditions is required for germination of
the scarified seeds. Accumulating evidence shows that PD is quite common in
papilionoid legumes, particularly in winter annuals and in species from semiarid
and Mediterranean climates (Thomson 1965; van Assche and Vandelook 2010). In
Medicago truncatula, imbibition at 4 °C of scarified seeds releases PD, allowing
earlier germination (Faria et al. 2005). In Trifolium subterraneum, PD is a heritable
characteristic and its expression is temperature dependent (Morley 1958; Ballard
1961). Scarified imbibed seeds do not germinate at 30 °C, but germinate readily
at 15 °C, or after transfer from 30 to 15 °C (Katznelson and Carpenter 1972). High
temperatures (25–30 °C) also inhibit germination of several Mediterranean annual
legumes of semiarid rangelands ( Onobrychis spp., Astragalus spp., Medicago spp.)
(Young et al. 1970; Katznelson and Carpenter 1972) and temperate regions (van As-
sche and Vandelook 2010) that, otherwise, are able to germinate at low temperatures
338 J. Kigel et al.

(5–10 °C). van Assche and Vandelook (2010) proposed that PY + PD of papilionoid
legumes evolved in Mediterranean climates, where seedling survival is highest dur-
ing the wet and cool winters, and was maintained in species that migrated to temper-
ate regions. However, PY + PD has been found also in annual and perennial species
of Vicia in the Tibetan Plateau, in which PD is lost after 1 year of dry storage (Hu
et al. 2013). The presence of PY + PD can differ in closely related taxa—in Vicia
sativa it is present in subspecies macrocarpa but absent in subspecies nigra (Uzun
et al. 2013).
Synthesis of ABA during seed imbibition appears to play a key role in main-
taining PD in Medicago truncatula (Bolingue et al. 2010) and in Vicia angusti-
folia (Hu et al. 2013). ABA does not inhibit testa rupture, but inhibits subsequent
radicle growth by hindering cell-wall loosening and cell expansion by water uptake
(Finch-Savage and Leubner-Metzger 2006; Gimeno-Gilles et al. 2009). Application
of fluridone, an inhibitor of carotenoid and ABA synthesis, to fresh scarified seeds
increased the rate of germination, suggesting that ABA synthesis maintains PD.
Fluridone also reduced the inhibitory effect of continuous light on germination of
Medicago truncatula (Bolingue et al. 2010). In contrast, application of paclobutra-
zol, an inhibitor of ent-kaurene oxidase and GA synthesis, reduced the rate of ger-
mination of fresh seeds, indicating that endogenous GA’s promote germination of
Medicago truncatula seeds. However external application of GA3 did not increase
germination, implying that endogenous GA’s are present in imbibed seeds of Medi-
cago truncatula at levels enabling germination. Yet, in Vicia angustifolia GA3 ap-
plication increased rate of seed germination (Hu et al. 2013).
Hardseededness and physiological dormancy are affected by high temperature in
opposite directions—it accelerates impermeability breakdown, but keeps embryos
in a dormant state, that is, thermodormancy. Both effects are required to prevent
summer germination in species inhabiting regions with frequent summer rains. Un-
der these conditions, strong selection for hardseededness may occur if PD is lack-
ing (Katznelson and Carpenter 1972). Thus, presence of physiological dormancy
may act as a buffer against selection for hardseededness. Thermodormancy can be
expressed as inhibition of germination during exposure to supraoptimal tempera-
ture or by induction of secondary dormancy preventing subsequent germination at
lower temperatures (Baskin and Baskin 2014). Thermodormancy is associated with
increased levels of ABA and increased embryo sensitivity to ABA (Toh et al. 2008;
Leymarie et al. 2009). High-temperature inhibition of germination can be alleviated
by suppression of ABA synthesis with fluridone and by exogenous GA (Toh et al.
2008).
In chickpea, inhibition of seed germination by high temperature (30 °C) and
ABA was associated to lack of transcription of specific expansin genes needed for
cell-wall loosening and subsequent water uptake by the embryo radicle (Hernan-
dez-Nistal et al. 2010). Furthermore, high temperature (30–35 °C) also inhibited
ethylene production and germination in chickpea, and its inhibitory action was al-
leviated by ethylene (Gallardo et al. 1991). In this case, thermoinhibition was due to
increased conjugation of the ethylene precursor 1-aminocyclopropane-1-carboxylic
acid (ACC) to 1-(malonylamino) cyclopropane-1-carboxylic acid (malonyl-CoA)
11  Seed Physiology and Germination of Grain Legumes 339

and inhibition of the ethylene-forming enzyme ACC synthase. Similarly, ethylene

increases germination of thermoinhibited seeds of Medicago truncatula and Trifoli-
um subterraneum (Globerson 1978). Inhibition of ethylene biosynthesis by 2,5-nor-
bonadiene strongly hinders seed germination in pea, and this effect was counter-
acted by ethylene (Petruzelli et al. 1995).
Combinatorial dormancy in hard-seeded species acts as a double safety mecha-
nism preventing germination of seeds dispersed with incomplete impermeable seed
coats or of early softening seeds (Hu et al. 2013). Furthermore, hydrothermal model
analysis showed that seeds of Vicia species possessing PD had higher median water
potential for germination, implying that PD can prevent germination of permeable
seeds under low water availability (Hu et al. 2013). But, since PD is transitory and is
lowered by winter temperatures, maintenance of a permanent seed bank in legumes
is achieved mainly by hardseededness. In regions lacking summer rains, hardseed-
edness is probably enough to control germination. However, germination control
based on seed-coat permeability is usually an irreversible process. Once the imper-
meability barrier is lost, seeds may readily germinate under unfavorable conditions,
thus increasing the risk of seedling death (Kigel 1995). Alternative mechanisms to
prevent germination of buried seeds during unsuitable seasons are the induction
of secondary dormancy by unfavorable conditions such as high temperature, or by
cyclic changes in PD coupled with seasonal variation in climatic conditions (Baskin
and Baskin 2014). Yet, the induction of secondary dormancy and cyclic dormancy
has not been reported for legumes.

3.8 Ports of Water Uptake and Pathways of Hydration

in Legume Seeds

Germination begins with the uptake of water by the dry seed, followed by meta-
bolic activation and embryo expansion. Water uptake is a triphasic process, with
a fast initial uptake (phase I, i.e., imbibition), followed by a plateau phase (phase
II) and a further increase in water uptake (phase III) taking place concomitantly
to embryo expansion, splitting of the seed coat and root protrusion (i.e., germina-
tion) (Finch-Savage and Leubner-Metzger 2006; Bewley et al. 2013). Papilionoid
legumes differ in the location of main ports of initial water uptake through the seed
coat after seed softening. Detection of ports of water entry and evaluation of the rel-
ative importance of the hilum, micropyle, and lens compared to the whole seed-coat
surface have been studied using magnetic resonance imaging (MRI), fluorescent
tracer dyes, or by occluding different ports of water entry. The hilum functions as
the main port in lentil (Tang et al. 1994), Vicia spp. (Aswathaiah 1988), and in wild
Lima bean (Degreef et al. 2002). In cultivars of Lima bean with permeable seeds,
the micropyle is open and hilum closure is prevented under humid conditions due to
an incomplete counter-palisade layer. In contrast, in lines with impermeable seeds
the micropyle is occluded and a well-developed counter-palisade layer allows tight
closure of the hilum (Stienswat et al. 1971).
340 J. Kigel et al.

In other grain legumes, the primary region of water entry is the lens, as in Lupi-
nus albus (Perisse and Planchuelo 2004) and azuki bean ( Vigna angularis; Isemura
et al. 2007), or the hilum and micropyle as in Lupinus luteus (Garnczarska et al.
2007). In soybean, the lens is the main port of water entry (Koizumi et al. 2008),
but in cultivars with nonfunctional lens water uptake takes place through cuticular
cracks in the seed coat (Ma et al. 2004). Thus, cultivars within the same species
might differ in the seed-coat ports involved in initial water uptake. In Lupinus an-
gustifolius either the micropyle (Serrato-Valenti et al. 1989) or the lens (Perisse and
Planchuelo 2004) were reported as ports of water entry. In common bean, 80 % of
the water uptake in Great Northern lines was through the micropyle, compared to 18
and 2 % through the hilum and the testa, respectively. In contrast, in Red Mexican
lines, the major venues of water uptake were the raphe and hilum, with the micro-
pyle playing a minor role (Kyle and Randall 1963). In other cultivars of common
bean, the main ports of water entry were the hilum (Varriano-Marston and Jackson
1981; Heil et al. 1992), the hilum and the lens (Kikuchi et al. 2006), or the hilum
and micropyle (Deshpande and Cheryan 1986). This intraspecific variation in the
location of initial water entry reported in different studies can be partly due to the
diverse methods and time scales used in these studies.
Pathways of seed hydration are structurally organized in legume seeds and are
based on changes in the seed coat during early imbibition that directs subsequent
water distribution within the legume seed. In permeable seeds, an important role of
the seed coat is to slow down the rate of water entry and, at the same time, facili-
tate water movement within the imbibing seed. In common bean, autoradiographic
studies using 3H2O showed that water enters the seed through the hilum and dif-
fuses to the periphery of the cotyledons via the parenchyma cells of the testa, thus
allowing uniform hydration of the whole embryo and preventing imbibition damage
(Varriano-Marston and Jackson 1981). Micro-MRI showed that in common bean
and adzuki bean, water entered through the lens as the sole water port was first dis-
tributed throughout the testa and then delivered to the radicle followed by hydration
and swelling of the cotyledons (Kikuchi et al. 2006). In common bean water uptake
was immediate, with rapid hydration of the whole testa and cotyledon swelling. In
adzuki bean, in contrast, water entry was delayed by c. 7 h, and the slow hydration
was required to activate the lens. In soybean, water entered near the raphe, diffused
first towards the dorsal side of the seed and then to the hilar region, followed by
expansion of the radicle and hypocotyl and later by swelling of the cotyledons (Koi-
zumi et al. 2008). In soybean cultivars in which seed-coat permeability is due to
cracks in the seed cuticle, permeability of isolated seed coats was five times lower
in hard-seeded compared to soft-seeded cultivars, as measured by pressure probe
(Meyer et al. 2007). The critical difference between these cultivars was the conti-
nuity of a 0.2-µm-thick cuticle covering the palisade layer—small cracks 1–5 µm
wide and 20–200 µm long occur in permeable cultivars, but not in impermeable
ones (Arechavaleta-Medina and Snyder 1981; Ma et al. 2004). Once water entry
starts in permeable seeds of soybean, the dry seed coat increased in volume causing
local wrinkles that detach the coat from the cotyledons. These wrinkles are initially
small and perpendicular to the long axis of the seed. Further imbibition enlarges
11  Seed Physiology and Germination of Grain Legumes 341

the wrinkles that reach the ventral side of the seed, thus channeling water around
the seed. Lateral water movement also occurs through the intercellular spaces of
the osteosclereid layer and is more extensive at the hilar side due to larger spaces
between its cells (McDonald et al. 1988). As the seed coat becomes wet it absorbs
water by c. four times its weight, partly due to the large water-holding capacity of
the osteosclereid layer. This structural arrangement of the seed coat allows lateral
water distribution in the seed and uniform wetting of the embryo, thus preventing
imbibition damage due to fast uneven hydration that might cause embryo fracture
and solute leakage.
Soybean lines with permeable and hard seeds differ in the strength of the outer
cuticle secreted by the palisade cells. In hard-seed lines, the cuticle is more resistant
to cracking and richer in hydroxylated fatty acids, while in cultivars with permeable
seeds the cuticle lacks mid-chain hydroxylated fatty acids and is prone to cracking
(Ma et al. 2004; Shao et al. 2007). These compounds probably modify either the
degree of cross-linking between components of the cuticle or its integration with the
underlying cell wall. The critical stage at which hardseededness is established dur-
ing seed development in these soybean lines apparently occurs when cuticle deposi-
tion was completed, but the embryo is still expanding (Ranathunge et al. 2010). In
cultivars with soft permeable seeds, continued embryo expansion after cessation of
cutin secretion causes microscopic cracks in the seed cuticle, while in cultivars with
hard non-permeable seeds, these cracks do not appear despite the same pattern of
cuticle deposition and seed expansion. Thus, seed permeability depends on whether
the external cuticle can withstand the internal forces generated by rapid embryo
expansion in the absence of cuticle deposition.

3.9  Imbibition Damage in Grain Legumes

Slow and controlled hydration of the dry seed is essential as the first step in the
reconstitution of cell membranes and organelles and reactivation of metabolic pro-
cesses in the seed (Bewley et al. 2013). Seed imbibition damage is generally attrib-
uted to rapid water uptake by the embryo, leading to disruption of cell membranes,
solute leakage, and even cell death, thus negatively affecting germination and em-
bryo growth (Powell and Matthews 1978; Duke and Kakefuda 1981). Imbibition
damage takes place in the early stages of water uptake (Parrish and Leopold 1977)
and affects both the cotyledons and the embryonic axis (Ashworth and Obendorf
1980; McDonald et al. 1988). In the relatively large legume seeds, as water enters
and hydrates the outer embryo tissues they begin to swell while internal tissues stay
dry, resulting in physical strains that can rupture the embryo.
One of the functions of the seed coat is to restrict water flow during seed imbibi-
tion, so that hydration does not proceed too rapidly and imbibition damage is pre-
vented (McDonald et al. 1988; Bewley et al. 2013). Damage due to seed imbibition
at low temperature is compounded by chilling damage causing impaired membrane
reorganization and solute leakage (Bewley et al. 2013). A particular outcome of dam-
342 J. Kigel et al.

age upon water uptake can be seen in the reorganization of organellar functions. Dur-
ing imbibitions mitochondria gain proteins, lipids, and important active components
such as cytochrome oxidase and malate dehydrogenase (Nawa and Asahi 1971). Pea
mitochondria were already able to oxidize reduced nicotinamide adenine dinucleo-
tide (NADH) and tricarboxylic acid (TCA) cycle intermediates 12 h after imbibition.
At this stage, however, phosphorylation was still not efficient because of membrane
damage (Benamara et al. 2003). After 22 h, the outer mitochondria membranes were
reconstituted and phosphorylation was improved. Studies of mitochondrial changes
during seed germination in diverse plant species—among them pea and mungbean—
revealed similar phenomena (Botha et al. 1992; Howell et al. 2006).
A relationship between seed-coat permeability and imbibition damage is often
reported in grain legumes. Cultivars with high seed-coat permeability and high in-
cidence of testa injury imbibe more rapidly and may show extensive imbibition
damage, as in chickpea (Legesse and Powell 1996), common bean (Powell et al.
1986; Legesse and Powell 1996; Chachalis and Smith 2000), faba bean (Powell and
Matthews 1978; Chachalis and Smith 2000), cowpea (Legesse and Powell 1992,
1996; Asiedu and Powell 1998), longbean ( Vigna sesquipedalis; Abdullah et al.
1991), pea (Chachalis and Smith 2000), and soybean (Oliveira et al. 1984; Toledo
et al. 2010). However, imbibition damage is seldom observed in seeds with initial
water content above 15–20 % (Toledo et al. 2010), in which respiration and meta-
bolic activity rapidly increase with the increase of water content upon imbibition
(Bewley et al. 2013). Light-colored seeds of grain legumes generally have faster
water uptake and higher propensity to imbibition damage, as found in common
bean (Powell et al. 1986), cowpea (Legesse and Powell 1996), pea (Powell 1989),
and soybean (Chachalis and Smith 2001). The relationship between seed-coat color
and seed water uptake has been studied in isogenic lines of common bean (Wyatt
1977), soybean (Tully et al. 1981), Lima bean (Kannenberg and Allard 1964), and
pea (Powell 1989). Absence of pigment in the seed coat is often associated to a syn-
drome of traits, as shown by comparing pigmented and white seeds: (1) thinner seed
coats; (2) shorter and broader macrosclereids; (3) greater seed-coat permeability;
(4) the testa is more easily damaged; and (5) seeds germinate more rapidly.
The way by which pigmentation in the testa reduces the rate of seed imbibi-
tion is not clear. Pigmented testae have higher levels of lignins and tannins (Mor-
rison et al. 1995), waterproofing substances that may reduce permeability. On the
other hand, lack of pigmentation is frequently associated with less adherence of the
testa to the cotyledons, thus creating a void between them during initial imbibition
(Wyatt 1977; Powell 1989; Nakayama and Komatsu 2008). This void is filled with
water that is in direct contact with the embryo, leading to faster water uptake and
increasing the risk of imbibition damage. In pea lines with white seeds, the seed
coat loosened rapidly in the imbibing seeds facilitating diffusion of water between
the testa and cotyledons, while in lines with pigmented seeds the seed coat remained
closely attached to the cotyledons (Powell 1989). Similarly, faster imbibition in
lines of yellow-seeded soybean was not due to differences in the permeability of
the seed coat, but rather to the increased movement of water between the seed coat
and the cotyledons (Nakayama and Komatsu 2008). Adherence of the testa to the
11  Seed Physiology and Germination of Grain Legumes 343

cotyledons is also the major factor contributing to the slow imbibition of Desi-type
chickpea (Legesse and Powell 1996). However, since several lines of legumes with
white and light-colored seeds also have impermeable seed coats, seed-coat pigmen-
tation is not a condition necessary for hardseededness. These contrasting observa-
tions are not necessarily contradictory, since genetic linkage between loci control-
ling pigmentation and permeability might be present in different genotypes. Genes
affecting pigmentation may also have pleiotropic effects on permeability, due to
possible involvement in biosynthesis of polyphenols that impregnate the cell wall.

3.10  Genetics of Seed-Coat Impermeability in Grain Legumes

The seed coat normally develops from maternal tissues, namely from the integu-
ments of the fertilized ovule and from the endosperm (Esau 1977). In legume seeds,
the inner integument is obliterated by the expanding embryo and the endosperm is
entirely consumed by the embryo in many species (Werker 1997). Thus, perme-
ability of the seed coat is generally controlled by maternal genes expressed during
the differentiation of the outer integument into the testa of the mature seed. Evi-
dence supporting embryo effects on seed-coat permeability in legumes is scarce,
even though coordinated growth of the embryo and seed-coat tissues is necessary
to prevent seed-coat rupture and gaps enabling water entry. Pressure exerted by fast
embryo expansion can cause cracks in the outer cuticle of the seed or even in the
testa, if their rate of expansion is slower compared to embryo expansion (Qutob
et al. 2008).
Few genes are involved in the control of seed-coat permeability in grain legumes,
a fact that allowed fast selection for dormancy loss during domestication (Zohary
et al. 2012). The site of action of these genes varies among species and cultivars.
It can be localized at potential ports of water entry, namely the hilum, micropyle,
or lens, or might affect the whole testa. However, the nature of the structural and
chemical changes controlled by these genes is less clear. In several papilionoid
legumes the hard-seed trait is controlled just by one or two genes. For instance,
crosses between cultivated Lens culinaris and two hardseed wild species showed
that seed-coat impermeability is controlled by a single recessive gene in the homo-
zygous condition in Lens orientalis and by a single dominant gene in Lens ervoides
(Ladizinski 1985). In Vicia faba seed testa-imposed dormancy is a monogenic trait
(Ramsay 1997), while in Vicia sativa it is controlled by a two gene system (Donnel-
ly et al. 1972). One gene (A) is dominant for hardseededness, a second gene (B) is
dominant for softseededness when the A locus is homozygous recessive (aa), while
the double recessive genotype (aabb) is hardseeded. In Lupinus hispanicus (Arrieta
et al. 1994), Lupinus angustifolius (Forbes and Wells 1968), and Lupinus luteus
(Mikolajczyk 1966), seed-coat permeability is controlled by one recessive gene.
However, Serrato-Valenti et al. (1989) found that in Lupinus angustifolius, where
the micropyle is the port of water entry in soft seeds, permeability is controlled by
two genes. In common bean, seed-coat permeability is controlled by several genes
344 J. Kigel et al.

and the soft-seed trait is incompletely dominant (Dickson and Boettger 1982). Lo-
cation and genetic control of permeability differs among cultivars of common bean
(Kyle and Randall 1963). In Great Northern lines, the micropyle was the port of wa-
ter entry, while in Red Mexican lines the raphe and hilum were the main ports and
their permeability is controlled by a single recessive gene pair. In mungbean, the
number of genes controlling hardseededness varied from one to a few, depending
on the genotypes used in the crosses (James et al. 1999; Humphrey et al. 2005). In
soybean, crosses with Glycine ussuriensis showed that permeability is controlled by
one gene (Marjüshkin et al. 1987), while in crosses with Glycine formosana two or
four genes controlled permeability (Shahi and Pandey 1982; Verma and Ram 1987),
and in both cases genes for permeability are recessive. Mapping of quantitative
trait locus (QTL) for hardseededness in soybean using a cross between a cultivar of
Glycine max ssp. max with permeable seeds and wild Glycine max ssp. soja with
impermeable seeds, showed that few major QTL account for most of the variation
in this trait (Sakamoto et al. 2004; Liu et al. 2007). Similarly, QTL analyses of a
recombinant inbred population of mungbean (Vigna radiata) derived from a cross
between a soft-seeded and a hard-seeded line revealed four loci for hardseededness
(Humphrey et al. 2005). In azuki bean ( Vigna angularis; Kaga et al. 2008) and yard-
long bean ( Vigna unguiculata ssp. unguiculata; Kongjaimun et al. 2012), five and
six QTLs, respectively, were identified for seed dormancy-related traits and occur
in the same linkage group. In common bean, four unlinked QTLs were identified
for seed dormancy in a cross between a cultivar and a wild accession (Koinange
et al. 1996).
Altogether, hardseededness of grain legumes should be considered a quantitative
trait, with a relatively few genes affecting seed-coat permeability, acting at different
locations in the seed coat according to species and cultivars. Analysis of the avail-
able information suggests that several processes under genetic control, acting at the
biochemical and structural level during seed-coat development, modulate seed-coat
permeability, and henceforth seed dormancy, as well as rate of seed hydration. The
challenge now is the identification of the genes and the specific processes under
their control that take place during seed-coat development, modulating seed perme-
ability by acting at specific locations in the seed coat.

4 Effects of the Parental Environmental on Seed Size

and Germination

Seed dormancy and seed size are determined by the genotypes of the maternal
plant and the embryo interacting with parental environmental conditions during
seed development and maturation (Roach and Wulff 1987; Baskin and Baskin
2014; Donohue 2009). Temperature, day length, light intensity, and quality as well
as availability of water and nutrients can change the proportion of dormant seeds
(Fenner 1991; Baskin and Baskin 2014) and composition and weight distribution
of offspring seeds (Fenner 1992). Yet, some effects can be indirect, such as through
11  Seed Physiology and Germination of Grain Legumes 345

pod and seed abortion that may increase seed mass of the remaining seeds by com-
pensatory mechanisms activated under stress conditions (Stephenson 1981; Gross
and Kigel 1994; Gusmao et al. 2012).
Despite numerous agronomic studies related to the effects of climatic conditions
on yield components, including individual seed mass, few studies focused on the
germination behavior of seeds produced under different environmental conditions.
Seed size of grain legumes is often modified by temperature and water availabil-
ity during the seed filling stage. Seed mass is the combined result of rate of seed
growth and duration of seed growth. Notably, increasing temperature has opposite
effects on these processes: It enhances seed growth rate but shortens the seed-filling
period, thus buffering temperature effect on seed mass (pea—Poggio et al. (2005),
soybean—Egli et al. (2005)). Chilling (< 10–15 °C) during seed development re-
duces accumulation of seed reserves such as starch, proteins, and minerals, resulting
in smaller seed mass (chickpea—Kaur et al. (2008), soybean—Egli et al. (2005)).
Processes underlying these chilling effects may involve loss of chlorophyll and de-
creased photosynthesis, restricted availability and/or mobilization of assimilates as
well as inhibition of enzymes related to the biosynthesis of storage compounds.
Low water availability during the seed-filling stage reduce pod and seed number in
chickpea (Behboudian et al. 2001) and in common bean (Boutra and Sanders 2001)
due to pod abortion, but did not affect seed mass and even increased protein content
in chickpea. In pea, in contrast, seed mass was reduced by water scarcity during
seed filling, but germination remained high (Fougereux et al. 1997).

5  Environmental Control of Seed Germination

Area of cultivation of different grain legumes is continuously expanding beyond

their original climatic regions of adaptation, thus exposing these crops to subop-
timal climatic conditions for germination, growth, and yield. This trend, together
with climate change effects, is leading to earlier sowings after winter in temperate
regions or after summer in regions with Mediterranean climate, in order to avoid
drought and heath stress during reproductive and seed-filling stages. Warm as well
as cold weather can have negative impacts on seed germination and emergence,
particularly in grain legumes grown continuously throughout the year, which may
be sown during periods of high or low temperature. In grain legumes in which
seed dormancy and seed-coat impermeability have been lost through domestication
and breeding, germination is mainly controlled by water availability and soil tem-
perature. Light has generally no effect on their germination, even though osmotic
stress may induce a light requirement, as in common bean (Lopes and Takai 1987).
The interactive effects of temperature and water availability in the seedbed are of
cardinal importance for seed germination. It is well known that each plant spe-
cies has its own specific temperature requirements: minimum (i.e., base-Tb) and
maximum temperature (Tmax) below and above which no germination occurs, and
an optimum temperature (Topt) at which germination rate is fastest. Germination
346 J. Kigel et al.

rate is often linearly related to temperature, allowing temperature and time to be

combined into thermal time. Regarding water availability, both the rate of germina-
tion and the proportion of germinating seeds decrease with decreasing soil water
potential, but each species has its own threshold—base water potential (Ψb)—for
germination. However, since water and temperature interact the threshold water po-
tential depends largely on the temperature optimal for germination (Bradford 2002).
Variation in Ψb, To, Topt, Tmax, and thermal time to 50 % germination ( TT50%)
among grain legumes of temperate, tropical, and subtropical origin are presented
in Table 11.1.
Temperate and tropical clades of grain legumes did not differ in threshold wa-
ter potential for germination ( Ψb -1–2 MPa). In contrast, clades differ in germina-
tion responses to temperature. The Phaseoloid clade with tropical and subtropical
species has a much higher Tb for germination compared to the Hologalegina and
Genistoid clades with temperate species. Seeds of pea are able to germinate even
on ice (Macherel et al. 2007). Furthermore, at lower temperatures rates of germina-
tion where faster in the Halogalegina clade compared to the Phaseloid clade (Topt).
Thus, below 25 °C pea germinates more rapidly than common bean, while above
25 °C common bean germinates faster than pea. Accordingly, thermal time to 50 %
germination (TT50%) was shorter in the Phaseoloid clade. On the other hand, Tmax
did not differ among the clades. Thus, differences in cardinal temperature param-
eters To, Topt, and TT50% reflect the different climatic and ecogeographic regions
of origin as well as the phylogenetic relationships among papilionoid legumes.
Domestication and breeding broadened the range of temperatures that allow ger-
mination of grain legumes. For instance, cultivars of cowpea (Lush et al. 1980),
common bean, and tepary bean (Scully and Waines 1987) germinate at lower tem-
peratures than their wild relatives. In common bean, a warm-season legume origi-
nating in Central and South America, some commercial lines show some level of
germination down to 7–10 °C (Kooistra 1971; Dickson and Boettger 1982; Scully
and Waines 1987; White and Montes 1993; Zaiter et al. 1994), while no germination
below 10 °C occurred in Phaseolus aborigineus (Kooistra 1971). Lower Tb predicts
germination at low soil temperatures in the field. However, cold tolerance at germi-
nation is not necessarily associated with cold tolerance at later stages of plant devel-
opment. Germination base temperatures frequently differ from base temperatures
for vegetative and reproductive development (White and Montes 1993). In common
bean and pea seedling, elongation requires 2–3 °C higher minimal temperature than
germination (Raveneau et al. 2011).
Importance of salinity effects on germination of grain legumes is increasing due
to expansion of their cultivation into more marginal land. Salinity can affect seed
germination through osmotic effects (Welbaum et al. 1990) or ion toxicity (Munns
2002). Even though it is difficult to separate between the two effects, low water po-
tential is the main limiting factor at low and moderate salinity levels. Grain legume
crops are adversely affected by relatively low salt levels (Maas and Hoffman 1977)
and possess limited genetic variability for salinity tolerance (Johansen et al. 1988).
Common bean is salt sensitive (Maas and Hoffman 1977), but has wild relatives
from arid regions possessing higher salinity tolerance—Phaseolus angustissimus,
Table 11.1   Relationships between taxonomic affiliation, temperature, and water potential requirements for seed germination of grain legumes. ( Tb base tem-
perature, Topt temperature of highest rate of germination, Tmax temperature inhibitory for germination ( <  empe, TT50 % thermal time (°C day), Ψb base water
potential)
Clades Common Species Temperature Water potential References
Name
Tb germ T opt T max TT50% Ψb
(oC) (oC) (oC) (oC day) MPa
Hologalegina Field pea Pisum sativum − 1.8–0.6 25–30 40 42 – Olivier and Annandale (1998)
− 1.9–0 21–27 29–40 22–40 − 1.7 – ­– 2.5 Raveneau et al. (2011)
Chickpea Cicer arietinum 0 32–33 48 42 – Covell et al. (1986)
0 31 – 32–43 – Ellis et al. (1986)
5.5 – – – − 2 Finch-Savage et al. (2005)
11  Seed Physiology and Germination of Grain Legumes

Lentil Lens culinaris 2.5 24 32–34 21 – Covell et al. (1986)

1.5 – – 25 − 1.7 – ­–  2.5 Ellis and Barrett (1994)
Barrel Medicago 2–3 20–25 30 13–16 − 0.7 – ­– 1.3 Brunel et al. (2009)
medic truncatula
Faba bean Vicia faba − 4 – ­– 7.5 20–25 42 13 – Ellis et al. (1987)
0.4 25 37 – – Dumur et al. (1990)
347
Table 11.1 (continued)
348

Clades Common Species Temperature Water potential References

Name
Tb germ T opt T max TT50% Ψb
(oC) (oC) (oC) (oC day) MPa
Phaseoloid Common Phaseolus 5.1–9.6 31–36 46–50 11–15 − 1.9 – ­–  2.4 Raveneau et al. (2011)
bean vulgaris
7–14 27–35 31–39 16–26 – Machado-Neto et al. (2006)
7.3 27 – – – White and Montes (1993)
15 30 40 – – Scully and Waines (1987)
12 – – – − 0.8 Hucl (1993)
10–12 – – 20–30 – Nleya et al. (2005)
12 – – – – Otubo et al. (1996)
– – 45 – – Pena-Valdivia et al. (2002)
Tepary bean Phaseolus 8.3 35 40 – – White and Montes (1993)
acutifolius
10 35 40 – – Scully and Waines (1987)
Cowpea Vigna 8.5 40  > 45 29 – Covell et al. (1986)
unguiculata 6.1–10.5 30–36 40–44 – – Craufurd et al. (1996)
Mungbean Vigna radiata 10 40  > 45 15 − 2.2 Fyfield and Gregory (1989)
Soybean Glycine max 4 34 47–55 32 – Covell et al. (1986)
Genistoid Narrow- Lupinus − 0.8–0.7 20 30 – − 1 – ­– 1.5 Dracup et al. (1993)
leafed lupin angustifolius – – – – − 0.8 Perisse et al. (2002)
Lupin Lupinus albus – – – – − 1 Perisse et al. (2002)
J. Kigel et al.
11  Seed Physiology and Germination of Grain Legumes 349

Phaseolus filiformis, Phaseolus leptostachyus, and Phaseolus microcarpus. Acces-

sions of these species had relatively fast germination under high salinity (120 mM
NaCl), and were able to germinate even at 180 mM NaCl. These wild Phaseolus
species may represent a genetic resource for improvement of salinity tolerance in
common bean (Bayuelo-Jimenez et al. 2002).

6  Metabolic Aspects of Seed Germination in Legumes

6.1 Metabolic Reorganization upon Imbibition

Is Challenged by Slow Oxygen Diffusion

Metabolism dominates cellular activity throughout the process of germination (Be-

wley et al. 2013), with some pathways activated from the beginning of imbibition
while other processes are initiated later on. The major function of cellular metabo-
lism at the onset of imbibition is to generate a redox state that promotes the activity
of essential enzymes and produces energy to support germination (Weitbrecht et al.
2011). In addition, change in the cellular redox level also enables the reduction
of protein disulfide bonds, through the activity of thioredoxin (Alkhalfioui et al.
2007). This protein modification contributes to starch and protein degradation in
Medicago truncatula by promoting the susceptibility of protease and amylase in-
hibitors to proteolysis. The metabolic events upon imbibition are tightly linked to
previous events at the end of seed development, that is, seed desiccation, when
molecular and physiological processes enter a quiescent state. Storage polymers
synthesized during seed development are stored in the dry seed and constitute most
of the seed dry weight. The most abundant storage polymers in legume seeds are
starch and storage proteins vicillin, legumin, and convicillin (Gallardo et al. 2003).
Their accumulation is influenced by the availability of nitrogen during seed devel-
opment (Weber et al. 2005). Also enzymes, gene transcripts and free metabolites to
be used during early stages of germination are present in the dry seeds (Angelovici
et al. 2010) and have likely the function to initiate metabolic processes minimizing
energy input. For example, mitochondria isolated from dry pea seeds were partially
functional and were able to oxidize succinate (Botha et al. 1992). Moreover, ribo-
somal proteins, RNA-binding proteins, and translation initiation factors were also
found in dry pea seeds, in preparation for essential protein synthesis during germi-
nation (Wang et al. 2012).
The first hours of imbibition are characterized by increasing oxygen uptake and
parallel increase of ATP levels (Botha et al. 1992). Full aerobic respiration includ-
ing the TCA cycle and the electron transport chain potentially provides 38 mol of
ATP per mol of glucose. As mitochondria activity improves during imbibition, the
TCA cycle enzyme malate dehydrogenase (MDH) highly increases in activity dur-
ing early germination (Morohashi et al. 1981; Soeda et al. 2005). In several spe-
cies, including chickpea, soybean, and mungbean, the role of cytochrome oxidase
350 J. Kigel et al.

in mitochondrial respiration during germination was demonstrated by inhibition of

total respiration by cyanide (Botha et al. 1992). Nevertheless, in slowly imbibing
dry seeds where oxygen is supplied through water uptake, an environment far from
being fully aerobic, ATP can be produced by alternative pathways. For instance,
cytosolic glycolysis followed by fermentation produces readily available but low
levels of ATP—only two mol ATP per mol of glucose. Fermentation is activated in
imbibed seeds when utilization of pyruvate by other pathways is limited (Al-Ani
et al. 1985).
In soybean as well as in other high-oil seeds, germination was correlated to res-
piration and energy charge at relatively higher oxygen pressures, with less consis-
tent relations at lower oxygen levels, suggesting the presence of additional limiting
factors acting at low oxygen (Al-Ani et al. 1985). In pea seeds, in contrast, lower
threshold of oxygen was needed to promote germination. Furthermore, its rate was
not correlated to respiration levels (measured as oxygen uptake) nor to adenylate
energy charge under low oxygen (based on ATP/ADP and ATP/AMP ratios), em-
phasizing the relevance of other pathways, such as fermentation, providing energy
for germination to proceed. During germination, ethanol fermentation increases in
many seeds (Botha et al. 1992), possibly due to an excess of glycolysis over mi-
tochondrial respiration during early germination (Morohashi and Shimokoriyama
1975). Soybean and pea have higher rates of fermentation and overall ATP produc-
tion compared to other nonlegume seeds under aerobic conditions, which could be
related to their larger seed size associated with lower oxygen diffusion (Raymond
et al. 1985). Also under anaerobic conditions, fermentation levels were relatively
higher in these two species, with ethanol production increasing in soybean but not
in pea and lactate dropping very low. Pea displayed the highest tolerance to anoxia
and was able to produce 47 % of ATP generated under aerobic conditions. Under
aerobic conditions alcohol fermentation accounted for 43 % of the ATP regeneration
in pea and 5 % in soybean (Raymond et al. 1985).

6.2 Amino Acids Are Substrates for Energy Buildup

During Imbibition

Several studies showed that amino acids can provide an alternative substrate for
energy production (Galili 2011). Manipulation of lysine metabolism had a major
effect on the TCA cycle metabolism, revealing a strong interaction between ly-
sine metabolism and cellular energy metabolism (Angelovici et al. 2011; Kirma
et al. 2012). Also the carbon skeleton from glutamate released from storage proteins
in yellow lupine is utilized for respiration, and the amine group is transferred to
form asparagine, which accumulates to high levels in germinating seeds (Lehmann
and Ratajczak 2008). Pools of free amino acids stored in the dry seeds are readily
used during the first hours of imbibition. In Arabidopsis, the nonprotein amino acid
γ-aminobutyrate (GABA) was shown to provide a substrate of energy metabolism
during seed imbibition via the GABA shunt (Fait et al. 2008). A significant role of
11  Seed Physiology and Germination of Grain Legumes 351

this amino acid in metabolic reactivation during germination was found also in faba
bean. The activity and transcript levels of glutamate decarboxylase (GAD)—the
entry enzyme of the GABA shunt—were shown to increase in germinating seeds
(Yang et al. 2013). Hypoxic conditions contribute to GABA accumulation in early
stages of germinating faba bean (Yang et al. 2013). At first, it was thought that
GABA per se could improve the tolerance of the seed to low oxygen conditions by
an unknown mechanism. However, it can be safely suggested that the activation of
the GABA shunt provides a metabolic continuity to an impaired TCA cycle. The
combined effect of high sensitivity to redox balance of 2-oxoglutarate dehydroge-
nase—the enzyme responsible for glucose incorporation into the TCA cycle—and
the long-known induction of GAD during early germination, likely by Ca2+ released
during the reorganization of the cellular components of the seed, promotes the GA-
BA-shunt activity, maintaining the TCA cycle functional integrity (Fait et al. 2008).
Under hypoxia, faba bean also increase polyamine and glutamate content and di-
amine oxidase (DAO) activity, all of which are lowered after relief of hypoxia.
Polyamine degradation supplies about 30 % of GABA in hypoxia conditions, as
shown by DAO inhibition (Yang et al. 2013). Taken together the GABA pool can
play an important role for the initiation and maintenance of TCA cycle functionality
in imbibed seeds, when energy and oxygen levels are limited and storage reserves
are not yet readily available.
Pathways in which amino acids provide alternative substrates to sugars for en-
ergy production in germinating seeds are regulated by a sugar-sensing mechanism
(Lehmann and Ratajczak 2008). For example, in germinating yellow lupine (Lu-
pinus luteus) seeds, arginine is catabolized by amino transferase into glutamate,
which can enter the TCA cycle (Ratajczak et al. 1996). Arginase and urinase activi-
ties, the enzymes of arginine catabolism, increase during germination of seeds of
several plant species, and the pathway is induced by sugar starvation (Borek et al.
2001). Under sugar starvation, higher levels of urea are also measured in the seeds,
a byproduct of elevated rate of nitrogen compounds degradation for use as energy
and carbon substrates.
De novo protein synthesis is required for germination. However, the level of free
amino acids in dry seeds is not sufficient for the needs of protein synthesis during
germination (Bewley et al. 2013) and storage protein degradation in the embryo
starts already during the first hours of imbibition. Approaching radicle protrusion,
degradation of storage proteins increasingly provides amino acids for de novo pro-
tein synthesis and cellular metabolism in the growing embryo (Wang et al. 2012). In
legumes, storage proteins are concentrated in protein storage vacuoles (PSVs) in the
embryonic axis and the cotyledon cells. A number of enzymes with different roles
are involved in the PSV breakdown at different stages during germination (Bewley
et al. 2013). The peptidases, which initiate this process during the first hours, are
stored in dry seeds while additional proteases accumulate during imbibition (Yomo
and Varner 1973). In black gram (Vigna mungo), cysteine endopeptidase SH-EP,
which is involved in the mobilization of storage globulin, reaches its peak 4 days
after imbibition and decreases thereafter (Okamoto and Minamikawa 1998). Protea-
some subunits identified in the pea embryonic axes increased during germination,
352 J. Kigel et al.

and probably have a role in storage proteins degradation (Wang et al. 2012). Since
amino acids from storage protein are also used as substrates for respiration, degra-
dation of storage proteins to amino acids is enhanced under sugar depletion condi-
tions (Borek et al. 2001, 2012).
On the other hand, amino acid mobilization and usage are inhibited by applica-
tion of exogenous sucrose (Okamoto and Minamikawa 1998; Lehmann and Rata-
jczak 2008). As their incorporation into energy metabolism or protein de novo bio-
synthesis decreases and free amino acids accumulate, the degradation of protein
reserves is inhibited (Yomo and Varner 1973).

6.3 Raffinose and Other Sugars Provide Early Carbon

Resources for Pre-Germination Energy

Major mobilization of starch reserves starts after radicle protrusion in most seeds.
In pea, amylase activity increases during imbibition (Yomo and Varner 1973). En-
ergy carbon resources at imbibition include the raffinose family oligosaccharides
(RFOs), which are present as soluble storage reserves in seeds of diverse species
(Peterbauer et al. 2001). Their degradation requires α-galactosidase and invertases.
During seed development, raffinoses accumulate in the cytoplasm of starchy (Be-
wley et al. 2013) and oily seeds alike (Baud et al. 2002; Fait et al. 2006), while the
degrading enzymes are partitioned in the protein storage vacuole (pea- Blöchl et al.
(2008)). They come together during germination. The small and soluble nature of
RFOs makes them a more accessible storage reserve than starch. Half of raffinoses
in pea seeds are degraded by the time of radicle emergence (Blöchl et al. 2008).
Impaired raffinose breakdown substantially lowers germination rate in pea (Blöchl
et al. 2007), while it is not essential for germination in soybean (Dierking and Bi-
lyeu 2009).
In legumes the production of mannose from manno-oligosaccharides during
imbibition by the activity of β-mannanases and β-mannosidases secreted by the
aleurone layer is long known (Reid and Meier 1972, 1973; McCleary and Mathe-
son 1975). Nonogaki et al. (1995, 1998) investigated their role at the tissue level
during imbibition in tomato seeds. A galactomannan-hydrolyzing enzyme was
localized specifically to the micropylar region of the endosperm of tomato seed
prior to germination. The enzyme was endo-β-mannanase and it hydrolyzed ga-
lactomannan into oligosaccharides with no release of galactose and mannose. This
pre-germination enzyme was identified 18 h after imbibition and increased up to the
time immediately before radicle protrusion, 6 h later. The profile of activity of this
enzyme corroborates the hypothesis that the physiological role of pre-germination
galactomannan-hydrolyzing enzyme(s) is to weaken the endosperm cell wall al-
lowing the radicle to protrude. Later studies (Nonogaki et al. 2000) showed that
distinct mannanases are involved in germination and post-germination processes,
with LeMAN2 being associated with endosperm cap weakening prior to radicle
emergence, whereas LeMAN1 mobilizes galactomannan reserves in the lateral en-
11  Seed Physiology and Germination of Grain Legumes 353

dosperm. Similar studies on the role of the endosperm in the control of germination
of endospermic legume seeds have not been performed yet.

6.4  Lipids and Early Fatty Acid Metabolism

Storage lipids of yellow lupine seeds in the form of triacylglycerol are first hydro-
lyzed to glycerol and free fatty acids by lipases, which are inhibited by application
of exogenous sucrose to the germination media (Borek and Nuc 2011). Fatty acids
are then fed to the β-oxidation and glyoxylate pathways to produce acetyl CoA, suc-
cinate, and malate (Pritchard et al. 2002). The β-oxidation and glyoxylate pathways
take place mostly in glyoxysomes, which are a specialized form of peroxisome
found in seeds. In dry seeds, they are present in a small underdeveloped form, and
then during germination, grow and accumulate essential enzymes, such as malate
synthase (MLS) and isocitrate lyase (ICL), which are unique to the glyoxylate cy-
cle. The glyoxylate cycle enzymes cytosolic aconitase and ICL are active in both
the embryo and the cotyledons of germinating yellow lupine seeds (Borek and Nuc
2011). Their activity is enhanced by exogenous sucrose by effecting gene expres-
sion. The products of fatty acid degradation via these pathways can be converted
to sugars, amino acids or can be utilized for energy via the TCA cycle (Eastmond
and Graham 2001; Borek et al. 2003), or for amino acid biosynthesis. Asparagine,
glutamine, and glutamate are also synthesized from lipid-derived carbon skeletons
during germination of yellow lupine (Borek et al. 2003).

7 Conclusions

Altogether, better information is required on the effects of stress conditions dur-

ing seed development of grain legumes on subsequent seed germination as well
as on the genetic and physiological control of germination responses to soil water
potential and temperature. This knowledge is needed to successfully confront the
challenge of climate change in regions of legume cultivation and for further expan-
sion of cultivation into regions with more extreme climatic conditions. The inte-
grated view of seed germination combining seed structural morphology, physiology
of dormancy and germination metabolism remains fragmented, mainly because of
barriers between disciplines. In the post-genomics era, with the development of
marker-assisted breeding strategies, a deeper understanding of the links between
seed-coat permeability, seed metabolism (e.g., energy-related or modulating perme-
ability), seed dormancy, and seedling vigor will greatly contribute to the production
of improved varieties. Concomitantly, these studies will lead to the identification
of genes functionally related to key processes in metabolic resumption upon water
imbibition and in the control of germination.
354 J. Kigel et al.

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Chapter 12
Reproductive Biology of Grain Legumes

María José Suso, Penelope J. Bebeli and Reid G. Palmer

1 Introduction

Regulations and consumer attitudes lead agricultural practices towards an ever

more environmentally friendly production. There is an increasing interest of legume
breeders in using bee pollinators for insect-aided outcrossing for heterosis-mediated
exploitation in hybrid seed production and in heterotic open-pollinated populations.
The global widespread decline of pollinator populations and diversity continues to
cause international concern in commercial agriculture (Burkle et al. 2013). Also,
this approach should preserve bee pollinator fauna by providing suitable floral re-
sources within the legume crops themselves. This requires in-depth knowledge of
the reproductive biology of legumes.
The reproductive biology of legumes has never been an easy topic. It has been an
old and traditional topic never solved. This chapter is not a comprehensive review
on legume reproductive biology; it was not possible nor was the aim. Valuable re-
view materials including estimations on pollen-mediated gene flow (PMGF) from
genetically modified (GM) crops to wild species are compiled by Andersson and Vi-
cente (2010). Their focus was on the following legume species: chickpea, common
bean, cowpea, pigeon pea, peanut and soybean. Also, there has been an excellent

Reid G. Palmer is deceased.

M. J. Suso ()
Plant Breeding. Instituto de Agricultura Sostenible (CSIC), Alameda del Obispo s/n,
14080 Córdoba, Spain
e-mail: [email protected]
P. J. Bebeli
Department of Crop Science, Laboratory of Plant Breeding and Biometry,
Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece
e-mail: [email protected]
R. G. Palmer
Department of Agronomy, Iowa State University, Ames, IA 50010, USA
e-mail: [email protected]
© Springer Science+Business Media New York 2015 365
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_12
366 M. J. Suso et al.

section on the reproductive biology of legumes in the chapter entitled “Biology of

Food Legumes” in the book Biology and Breeding of Food Legumes (Chaturvedi
et al. 2011). Readers, however, do not get core material on pollination and floral
biology for breeding strategies. Nowadays, most of the new concepts and research
on reproductive biology tends to be confined to ecologists and biologists. The ap-
plication of their knowledge is necessary to both make progress in breeding and
to improve the ecological services of legumes. We have tried to provide important
references on significant works published to date relevant to different aspects of
legume improvement. Bearing in mind the scope of the chapter, slight overlapping
is possible both because of the paucity of the data and because the same topic is
viewed from several perspectives.

2  Flower Attributes and Functions

2.1 Flower Morphology and Structure and Why It Matters.

Mechanical Fit to Pollinators

Taking into account that bee pollinators are able to distinguish between complex
floral traits, discriminate flower visits depending on floral design and display, and
reward and attraction traits, the flower attributes and functions should play an es-
sential role in legume breeding. In this section, we provide clues and analyse ques-
tions of relevance in relation to how the plant might best manage pollinators to
modify (increase or reduce) the level of outcrossing, how to design a crop to sustain
pollinators and the effectiveness of different types of flowers to support crop-pol-
linating wild bees and honeybees and at the same time discuss breeding strategies.
Grain legumes have a typical papilionate type of flower. Flowers have a pen-
tamerous ground plan. The calyx consists of five sepals and the corolla comprises a
standard two wings and two lower petals to form a keel. There are ten stamens sur-
rounding the pistil, which is superior in position and differentiates into a gynoecium
with stigma, style and ovary (Chaturvedi et al. 2011). According to Tucker (2003),
the successive and overlapping order of organ initiation in some legume flowers is
intriguing developmentally because of its conflict with the prevailing interpreta-
tion of hypotheses concerning timing of determination of organ identity. The ABC
model hypothesis of floral organ identity applies to flowers in which all organs of
a whorl initiate simultaneously; the order of initiation is sepals, petals, stamens and
carpels, and where the whorls do not overlap in time of initiation (Tucker 2003).
It does not satisfactorily explain a system in which more than one type of organ is
being initiated at the same time, such as that in papilionoid legumes that have over-
lapping whorls. The ABC model also does not explain the concurrent initiation of
the carpel at the same time as petals or stamens, which is typical in legumes. Thus,
Tucker (2003) concluded that legume flowers fail to conform to the ABC model.
The basic branching pattern in legume inflorescences is racemose, but systems with
pseudoracemes and cymose branching do occur (reviewed by Prenner (2013)).
12  Reproductive Biology of Grain Legumes 367

Fig. 12.1   Vicia faba flower

showing the nectar guides.
(Photo: JL Ubera)

The typical papilionated flower has a zygomorphic symmetry. Zygomorphic

flowers have one plane of symmetry where one half mirrors the other half, also
referred to as monosymmetric or bilaterally symmetric (Kalisz et al. 2006). Floral
zygomorphy (flowers with bilateral symmetry) typically manifests two kinds of
asymmetries, dorsoventral (DV) and organ internal (IN) asymmetries in floral and
organ planes. Zygomorphic development involves the establishment of different
body planes with distinct developmental axes, where various asymmetries are gen-
erated and superimposed under the control of different regulators. The pea ( Pisum
sativum L.) possesses a conspicuous zygomorphic flower, with an abundant mutant
collection, and provides an ideal experimental system to analyse the key regulators
in control of floral symmetry. Wang et al. (2008) investigated the loci affecting
these asymmetries during the development of floral zygomorphy in pea. Two genes,
LOBED STANDARD 1 (LST1) and KEELED WINGS (K) constitute the DV asym-
metry, and SYMMETRIC PETALS 1 (SYP1) control IN asymmetry. Genetic analysis
demonstrates that DV and IN asymmetries could be controlled independently by
two kinds of regulators in pea, and their interactions help to specify the type of
zygomorphy. Based on the genetic analysis in pea, they suggested that variation
in both the functions and interactions of these regulators could give rise to a wide
spectrum of floral symmetries among legume species.
Floral symmetry is a conspicuous cue. It has a particular signal value that is
evidently perceived by insect pollinators. Apart from the external symmetry (i.e.
symmetry of the outlines of the flower), flowers also display a symmetry of nectar
guides which are usually situated near the centre of the flower (Fig. 12.1). This
“internal” symmetry is perceived when the insect is very close to the flower. Thus,
the insect has two opportunities for detecting the symmetry of the flower: The first
opportunity arises when the insect is still at some distance, so that it can perceive the
image of the flower as a whole, and the second opportunity arises when the insect
comes close enough to resolve the pattern of the nectar guides (Giurfa et al. 1999).
The papilionate flowers also termed keel or flag flowers are very distinctive and
commonly involve specializations for triggered pollen release (Fig. 12.2; Willmer
2011). When a visitor lands on such a flower (Fig. 12.3), it will normally grip onto
368 M. J. Suso et al.

Fig. 12.2   Floral morphology

of Vicia faba ( side view).
(Photo: JL Ubera)

Fig. 12.3   Floral visitors of

Vicia faba. Eucera numida
the most important pollina-
tor of Vicia faba in southern
Spain. (Photo: JL Ubera)

the wing petals and insert its tongue (proboscis) between the more or less erect
standard petal and the upper edges of the keel, sliding the tongue inward to reach
nectar in the staminal tube. There is a secondary pollen presentation system where
the style is sharply bent upward terminally, with a brush of fine hairs near the tip and
just below the stigmatic surfaces. Normally, the stamens dehisce inside the closed
bud, and therefore shed their pollen onto this brush or on the inside of the end of the
keel, where the brush picks it up. When the flower opens, the stamens are already
withered, but the stigma brush is exposed by the first visitor and carries the pollen
onto the visitor’s underside. In these flowers, only quite large visitors with long
tongues (bees, some hoverflies) can reach the nectar, and their weight is enough to
depress the keel and ensure that the brush is exposed. Secondary pollen presentation
occurs independently if the flowers are chasmogamous or cleistogamous, in differ-
ent legumes such as the genera Phaseolus, Vicia, Lathyrus, Lens and Pisum (Lavin
and Delgado 1990).
The morphology of legume flowers is complex, with each component serving
a specific function. According to Westerkamp and Weber (1999), the papilionate
flowers possess three elementary functional structures: the flag, the keel and the
12  Reproductive Biology of Grain Legumes 369

wings. (1) The flag is for visual attraction. Besides its visual role as a semaphore,
the flag has at least two other important roles: (a) formation of a tongue guide and
(b) function as an abutment. In certain species, nectar guides mark the pattern of
the flag (Fig. 12.1). (2) The keel also has two essential functions: (a) it hides pol-
len from the collecting bees and (b) it has to provide structures that help to release
the hidden pollen and deposit it onto a pollinating bee. (3) The wings facilitate the
landing of a pollinator as well as the required active handling (lowering and open-
ing) of the keel. The hiding of pollen within a keel necessitates subsequent pollen
presentation. In secondary pollen presentation, it is usually the style which acts as
the pollen presenter.

2.2  Functional Flower: Advertisement, Discovery and Rewards

2.2.1  General Approach, Advertisement and Discovery

Palmer et al. (2009) summarized the relative importance of floral functional mor-
phology and structure in relation to pollination aspects that are applicable to plant
breeding programmes. The significant question is how do floral traits influence bee
behaviour? The reason why an insect forages on a particular flower can be partly
attributed to differences in floral design and floral display. Flower shape and size
can be considered as a function of one flower or of an inflorescence. Thus, it is
helpful to deal with the following terminology. Floral design: describes the charac-
teristics of individual flowers including their size, structure, sex condition, colour,
scent, nectar production and degree of herkogamy and dichogamy. Floral display:
the number of open flowers on a plant and their arrangement within and among
inflorescences. The important functional unit for pollination is usually daily inflo-
rescence size (Barrett 1998).
A flower’s design and display will affect not only which animals can feed there
but also which animals visit and make return visits. Hence, the morphological de-
sign and display features are only part of the story (Willmer 2011). Other floral
traits are related to the distant and local recruitment by advertising the flower and to
management by payment of reward to ensure that the visitor gets sufficient benefit
to encourage further visitation. To receive the service of pollen transfer, plants often
offer rewards to flower-visiting animals, such as nectar, oil, resin, pollen, breeding
sites, etc. Flowers attract pollinators via various stimuli, whether they are olfactory
or visual cues acting from a distance or tactile cues at close vicinity to guide pol-
linators to rewarding resources. Floral traits, resource distribution and cognitive and
learning abilities of pollinators influence their behaviour, which in turn is strongly
linked to plant mating patterns and gene flow within and among plant populations
(Mayer et al. 2011). One aspect of flowers to be considered is the nectar guides.
These colour-based guides may help to highlight the architecture of the flower dur-
ing the approach, making foraging more efficient. Lines converging towards the
corolla entrance are common in Fabaceae, effectively directing pollinators towards
the rewards (Fig. 12.1; Delgado-Salinas et al. 2011).
370 M. J. Suso et al.

Texture in petals and other floral parts may offer both visual and tactile cues to
visitors (Kevan and Lane 1985; Whitney et al. 2011; Alcorn et al. 2012). Whitney
et al. (2011), reviewed the importance of conical petal epidermal cells to enhance
pollination success and concluded that conical epidermal cells significantly in-
creased tactile handling of the flower by pollinators and hence their preference. The
production of conical cells is controlled by an MYB-related transcription factor and
the mutant mixta that has a null allele of this factor was found in Antirrhinum majus.
In legumes, conical petal epidermal cells have been used as a marker for petal iden-
tity but there is no evidence of mixta homologues that play a role in differentiation
of petal conical cells (Ojeda et al. 2009; Çildir et al. 2012).
It is not appropriate to look only at a flower colour and shape as attractants, given
the ability of most flower visitors to detect and respond to scents or odours as well
as other cues (Farré-Armengol et al. 2013). Floral scents mostly result from the pro-
duction of small amounts of simple volatile organic compounds. Farré-Armengol
et al. (2013) reviewed the emission of diverse biogenic volatile organic compounds
(BVOCs; such as terpenoids, phenylpropanoids/benzenoids, fatty acid derivatives
and amino acid derivatives) that plants produce and emit from different floral parts
such as petals, sepals, pollen and nectar. The blends of the BVOCs are usually pro-
duced in petal conical cells (Whitney et al. 2011) and in scent glands or osmophores
during anthesis (Farré-Armengol et al. 2013). Marinho et al. (2014) localized the
sites of fragrance production and release in legume flowers in the perianth and
particularly the petals, in which scent plays an important role in pollination. An
analysis of cut flower scent in Lathyrus odoratus showed that the aroma was almost
exclusively produced by the standard and the wings while the keel petals and other
floral parts emitted very little (Sexton et al. 2005).

2.2.2  Reward: Pollen and Nectar

Regarding the reward traits, pollen, because of its inherent traits (containing small,
easily managed nutrient packages), makes it a useful resource to exploit as food.
Pollen is a crucial reward for pollen-eating and pollen-gathering visitors including
virtually all bees. But for many visitors, it is functionally secondary; an extra reward
for animals that primarily forage for nectar sources and pick up pollen incidentally
(Willmer 2011).
Nectar is the reward commonly presented by plants to attract potential pollina-
tors, thereby ensuring outcrossing and/or efficient pollination. Floral nectar is a
sugar-rich fluid dominated by the hexoses glucose and fructose and the disaccharide
sucrose (Brandenburg et al. 2009 for more details). Nectar allows flowers to “out-
source” the pollination business to animal vectors, which assure a directional, accu-
rate and efficient transfer of pollen compared to wind pollination. From the plant’s
perspective, in an ideal scenario, pollinators carry the maximum amount of pollen
from one plant to the stigma of a conspecific while consuming minimal nectar.
Limitation of nectar availability persuades pollinators to forage on a larger number
of flowers and enhance pollen distribution which in turn may increase outcrossing,
12  Reproductive Biology of Grain Legumes 371

as showed in faba bean (Suso et al. 2005). It is also worthy to point out that flow-
er depth also affects the pollinator foraging and that deeper flowers often contain
more nectar (Harder 1985). Floral tube length is of prime importance to faba bean
because longer tube length increases seed and fruit set (Davis 2001; Suso et al.
2005). Pierre et al. (1996) studied two spring-type faba beans, self-fertile and non-
self-fertile, in open field conditions for their nectar amount, sugar composition and
their attractiveness to bees. Nectar secretion of the self-fertile line was higher than
that of the nonself-fertile line. However, the nonself-fertile line was as attractive
as the self-fertile line to honeybees; in contrast, Bombus terrestris preferred self-
fertile line. In soybean, differences in nectar production and composition, and floral
morphology among soybean cultivars are affected by the environment. However, a
genetic component probably exists as shown by insect-mediated cross-pollination
with phenotypic recurrent selection (Ortiz-Perez et al. 2008). Recurrent selection
in soybean with male-sterile, female-fertile mutants was reviewed by Lewers and
Palmer (1997). These genetic differences in soybean should be amenable to plant
breeding manipulation similar to what has been with alfalfa (Teuber et al. 1983,
1990).
Floral nectar is produced by the nectary. The term nectary does not indicate a
well-defined anatomical structure, because there are various types of nectaries with
different anatomical origins and positions. The term has ecological significance, in
that nectaries are the place where liquid substances involved in interactions with
animals are produced and offered (Pacini et al. 2003).
A detailed description of the vascular and ultrastruture of the floral and stipular
nectaries of Vicia faba can be found in Davis et al. (1988) and Davis and Gun-
ning (1992). In addition, mechanisms of nectar secretion in the context of selection
for high nectar production, including Vicia faba, are summarized by Davis (2001).
Plants also offer a nectar reward for protection. Recent studies (Nepi et al. 2012)
demonstrated that nectar may have other functions in addition to attracting pollina-
tors: defence against microbial invasion. Nectar has been considered a major floral
reward for animals because it is predominantly composed of sugar, but also protein
and nonprotein amino acids and essential and nonessential amino acids have been
detected. It was discovered that floral nectar contains a large, heterogeneous as-
semblage of defence proteins. Thus, nectar is much more than the main floral food
reward for pollinators. It not only attracts insects but also must defend itself and the
gynoecium against invading microorganisms and eventually pathogens.
Nectar is produced in the flowers as a reward for pollinators, and thus plant pol-
lination is assured. Extrafloral nectaries (EFNs) are common (Narbona and Dirzo
2010) and are offered as a food source to predators that defend plants against herbi-
vores (Wäckers et al. 2013). In particular, EFNs are attractive to ants because of the
sugary reward, and many plants are visited and patrolled by ants that feed from their
extrafloral nectaries. For instance, lima bean bears EFNs on the stipules of its leaves,
three pairs of stipules per leaf (Heil 2004). Apart from the leaves, lima beans also
have EFN in the inflorescences (Hernández Cumplido et al. 2010). In the case of
faba bean, phenotypic plasticity enables many damaged plants to produce addition-
al EFNs or to increase nectar secretion rates to attract natural enemies of herbivores.
372 M. J. Suso et al.

Fig. 12.4   View of Vicia faba

extrafloral nectary. (Photo: JL
Ubera)

Ants often increase plant survival and fitness by deterring herbivore damage (Heil
et al. 2001). As EFNs of Vicia faba are visually conspicuous (Fig. 12.4), additional
nectaries may present a greater visual stimulus for ant attraction (Mondor and Ad-
dicott 2003). It is important to note that Vicia faba plants never produce more than
two EFNs per leaf, but rather produce multiple EFN-bearing stipules on the apical
meristem prior to the leaves’ unfolding (Mondor et al. 2006).
Most floral traits (attraction and mating system) that have been investigated have
significant heritability and also a tendency for heritability to vary with mating sys-
tem (Ashman and Majetic 2006). On the other hand, although floral and extraflo-
ral nectar traits are important for plant reproduction, protection and for breeding
strategies, we know little about their genetic basis (Mitchell 2004). Brandenburg
et al. (2009) reviewed progress on nectar research. It is typical for nectar to vary
substantially in the environment in concentration, composition and volume between
populations, plants, also genders and even interfloral and intrafloral variability from
day to day. Studies demonstrate that, in addition to strong environmental variation,
there is also abundant genetic variation and thus a substantial opportunity for a
response to selection on these traits. Nectar production could be increased by breed-
ing (Delaplane and Mayer 2000). Breeding pollinator-friendly varieties requires us
to unravel the genetic component of floral characteristics that are associated with
pollinators. Genetic control of flower–pollinator specificity has been reviewed by
Yuan et al. (2013).

3  Patterns of Mating System and Breeding System

3.1  General Approach

The terms “mating system” and “breeding system” have long been used without
clear definition. According to Neal and Anderson (2005), a strong consensus has
12  Reproductive Biology of Grain Legumes 373

been reached for the following uses: (1) “mating system” should be used when
treating the genetic relatedness and pairings between individuals (e.g. levels of self-
ing vs outcrossing); (2) “breeding system” should be used with the anatomical/
morphological (e.g. gynodioecy and heterostyly) and physiological (e.g. self-com-
patibility) aspects of individuals and populations; and (3) when addressing general
aspects of the reproductive biology of plants, a more general term such as “repro-
ductive system” should be used.
Reproductive characteristics are particularly important in affecting micro-evolu-
tionary processes and patterns because they influence genetic transmission, popula-
tion genetic structure, selection response and patterns of evolutionary variability
(Charlesworth 2006; Barrett 2008). Knowledge about the reproductive system of
legume species is essential for choosing appropriate procedures to be used for cul-
tivar development in intensive conventional breeding but also to develop strategies
for low-input farming systems, including participatory plant breeding and evolu-
tionary populations.
Agricultural practices affect natural biotic pollination. Agricultural management
practices in low-input or organic systems, such as prohibition or reduced use of
chemical pesticides and inorganic fertilizers, protection of noncropped habitats and
preservation of farming, are particularly beneficial for farmland wildlife. Low-input
and organic (LI/O) systems that harbour a high diversity and abundance of flower-
ing plants have a greater floral attractiveness and resource availability, and therefore
attract more pollinators compared to conventional fields and are beneficial for pop-
ulation build-up of native bees (Morandin and Winston 2005; Greenleaf and Kre-
men 2006; Power and Stout 2011). There is clear benefit delivered by pollinators
on yield quantity for field bean, but it is not maximized under current agricultural
intensification (Bartomeus et al. 2014).
Thus, according to Richards (2001), seed crops with flowers visited by animals
have been classified as follows: (1) those not requiring animal visitation. Autoga-
mous plants. Flowers automatically self-pollinate (sometimes known as autofertil-
ity), so that fruits and seed set in the absence of pollinators (e.g. lentil, groundnut,
pea). However, some autogamous crops do set more seed or better quality seed after
insect visits; (2) those requiring an animal visit to set seed or maximize fruit set. Au-
togamous–allogamous plants with self-fertile flowers which can set some seed and
fruit in the absence of animal visitation. However, animal visitation increases either:
(a) the proportion of fruit set or (b) the quality of fruit set. This group contains crops
such as soybean and broad bean; and (3) allogamous but self-fertile plants. Her-
maphrodite flowers which require animal visits for fruit and seed to set, but seed can
set following insect-mediated self-pollination, that is, an isolated individual plant
can set good seed after visitation (e.g. runner beans, Phaseolus coccineus). Crops
that are completely dependent on animal pollination are the minority.
Being aware of the significance of selfing in legumes, the question is to under-
stand the mechanics of the pollination process by determining when, and how much
self and outcross pollen are transported to stigmas. Lloyd and Schoen (1992) and
Barrett and Harder (1996) classified the modes of self-pollination. These differ in
whether or not they utilize specialized flowers, whether they involve the transfer
374 M. J. Suso et al.

of pollen within or between flowers, whether they are autonomous or mediated

by vectors, and their timing relative to opportunities for outcrossing. Cleistogamy:
specialized closed flowers and pollinator not involved. Geitonogamy involves the
transfer of pollen between flowers of the same plant. Facilitated self-pollination
occurs when visitors transfer self-pollen within a flower. It is important to point out
that low level of autofertility (seed set of isolated plant/artificial cross-fertilization)
did not necessarily imply lack of self-pollination under field natural conditions. Pri-
or, competing and delayed self-pollination are similar in being autonomous modes
of selfing that occur without the participation of a pollinator. The three modes differ
in their timing. Respectively, they occur before, during and after opportunities for
outcrossing a flower. Prior selfing occurs when anthers dehisce and stigmas are re-
ceptive before anthesis and there is contact between them in unopened buds. Com-
peting selfing resembles facilitated selfing in that it occurs during the same interval
as cross-pollination, but it differs in being achieved autonomously and, hence, it is
more easily selected. Delayed selfing occurs when the movements of flower parts at
the end of anthesis lead to pollen stigma contacts and the fertilization of ovules that
have not been previously cross-fertilized. Results in Vicia faba (Suso and Maalouf
2010) suggested that self-fertilization is the result of all three major modes: autono-
mous and facilitated autogamy as well as geitonogamy. Also delayed autogamy is
frequent when pollinators are absent at the beginning of flowering. Delayed autog-
amy may be selected because it provides reproductive assurance. Manipulating the
different modes of selfing (prior and delayed), Saxena et al. (2013) obtained good
hybrid yields in pigeon pea.
Recognition of the significance of the patterns of mating systems prompted the
development of specific tools for measuring the relative frequency of selfing and
outcrossing. Palmer et al. (2009) examined empirical methods to study mating sys-
tems. Mating events are usually classified according to whether seeds originate
from outcrossing or selfing. A common descriptor of the mating system is the esti-
mated outcrossing rate (the proportion of offspring fathered by genetic individuals
other than their seed parent). Historically and conventionally, knowledge of mating
systems was based on controlled pollination and statistical approaches based on
the classical tools of visual markers, such as heritable differences in floral pig-
mentation. However, procedures used to estimate the degree of cross-pollination in
classical marker studies refer to the frequency of hybrids that would have resulted
from crossing between pollen donor plants with the dominant marker and the pollen
recipients with the recessive marker; that is inter-genotype crossing. Estimations of
the cross-pollination take the form of pollen flow source and pollen “sinks” trials.
Small groups of plants were planted and pollen receipt was measured and moni-
tored for heterozygote plants. However, the source and sink trials cannot necessarily
detect the shortest pollen movement. Source and sink trials typically examine pol-
len flow between spatially clustered groups of plants, usually separated by at least
one meter (Kouam et al. 2012). It has been demonstrated, more than 25 years ago,
the utility of allozyme markers to estimate mating parameters, proportion of off-
spring produced by selfing, or its complement, the outcrossing rate (t = 1-s) (Barrett
and Harder 1996). Among outcrossed progeny, it is possible to estimate the degree
of biparental inbreeding, the correlation of paternity and the correlation of selfing
12  Reproductive Biology of Grain Legumes 375

among families (Ritland 2002). DNA markers with high allelic variation, such as
microsatellite loci and more elaborate biometrical models facilitated the develop-
ment of multilocus approaches (Ritland 1990; Ritland 2002, http://genetics.forestry.
ubc.ca/ritland/programs.html web page). Multilocus approaches can use informa-
tion from all genotypic categories and numerous loci, and thus, more accurately
reflect the total amount of outcrossing in open-pollinated populations.
However, mating-system studies in legumes to date have adopted at largely an
intercrossing perspective instead of a population-level perspective. Most reports
(see below) in the level of cross-pollination are simplistically based on the common
intercrossing methodology. This is likely to change because of the development of
evolutionary breeding populations for LI/O farming systems.

3.2  Case Studies

Next, relevant studies on mating systems for different legume crops are described
and detailed information is provided.

3.2.1 Lentil (Lens culinaris)

Erskine and Muehlbauer (1991) by using allozyme markers and the multilocus es-
timator of Shaw et al. (1981) reported a rate of outcrossing varying from 2.2 and
2.9 % in Turkish and Greek germplasm and up to 6.6 % in genetic resources origi-
nating from Chile. A total of 6.6 % of cross-pollination is an order of magnitude
above previous estimates. As the lentil flower is normally cleistogamous, a search
for the causal vector insect is required to effect cross-pollination.
Horneburg (2006), using the complete dominance of orange cotyledons over yel-
low cotyledons as genetic marker, investigated the degree of intercrossing among
three varieties. The degree of cross-pollination ranged from 0.06 to 5.12 %. Results
were strongly influenced by cultivar, year and location. The outcrossing rate of indi-
viduals also varied within cultivars, the extremes being 0 and 22.2 %. They consid-
ered that the differences in flower size and colour may have led to different behaviour
of pollinating agents. The highest degree of outcrossing was observed in the cultivars
with the largest flowers. They explained that only a small number of insects have
been observed on flowering lentils, most of them were honeybees ( Apis mellifera L.)
followed by bumblebees ( Bombus species) and a few hover flies (Syrphidae).

3.2.2 Cowpea (Vigna unguiculata)

Cowpea is a self-pollinated crop, however, many insects visit cowpea flowers,

which have EFNs and in the process facilitate both self- and cross-pollination.
Anthocyanin pigment, a dominant trait, was used to measure the frequency of
intercrossing. Results obtained showed that intercrossing occurs in cowpea albeit
376 M. J. Suso et al.

at a low frequency of less than 1.0 % and that cross-pollination occurred up to 31 m
distance. The insects most likely to be involved in pollen movement in cowpea are
bumblebees and honeybees (Fatokun and Ng 2007).
Asiwe (2009) intended to identify insect pollinators of cowpea and to determine
the level of cross-pollination in the crop. To estimate cross-pollination, morpho-
logical markers, purple pigments versus no pigmentation at the base of the petioles
and leaf shape were used. Results obtained showed that level of intercrossing was
higher (0.5–0.85 %) when cowpea was planted in alternate rows than in concentric
rows (0.01–0.13 %). Insects associated with pollen movement (pollinators) were
carpenter bees ( Xylocopa virginica L.), digger bees ( Anthophora occidentalis L.),
honeybees ( A. mellifera L.), bumblebees ( Bombus grieocollis and Bombus pennysl-
vanicus) and leaf-cutting bees (Megachile latimanus). Among the insects observed,
only honey and bumblebees were found with cowpea pollen dusts on their legs and
abdomens and were responsible for the observed level of outcrossing. This was
because only heavy insects such as honey and bumblebees with powerful vibra-
tions from their wings could depress the wings of cowpea flowers and expose their
stamens and stigmas for pollination.
Kouam et al. (2012) examined the multilocus outcrossing rates in 35 wild cow-
pea ( Vigna unguiculata ssp. unguiculata var. spontanea) populations from West
Africa. Multilocus outcrossing rates ranged from 1 to 9.5 % (mean 3.4 %). These
outcrossing rates are markedly higher than previous studies based on pollen flow
source and sink trials. Cowpea pollinators either belong to the genus Xylocopa or
the family Megachilidae.

3.2.3  Vetch (Vicia spp.)

Zhang and Mosjidis (1998) used the degree of polymorphism and the variability
distribution of seven enzyme systems in 31 accessions of 12 Vicia species to infer
their mating system. The results demonstrated that most of the Vicia species are
autogamous, but Vicia villosa ssp. varia is predominantly a cross-fertilizing species.

3.2.4  Faba Bean (Vicia faba)

Vicia faba has been considered a partially allogamous species. The level of allog-
amy is variable, ranging from 4 to 84 % with a mean around 30–60 % (Bond and
Poulsen 1983; Link 1990; Link et al. 1994; Suso and Moreno 1999; Suso et al.
2001; Gasim et al. 2004). Outcrossing may vary widely geographically (Link et al.
1994; Suso and Moreno 1999) depending on local environmental conditions, partic-
ularly the composition of the pollinator fauna (Bond and Kirby 1999, 2001; Pierre
et al. 1996, 1999). Growing faba bean enhances the diversity of flowering resources
and may help to maintain wild bee pollinators’ abundance and diversity. Thus, a key
environmental benefit of faba bean is its ability to facilitate diversification of the
agro-ecosystem by indirectly enhancing associated diversity of wild fauna that may
affect the sustainability of agricultural systems (Köpke and Nemecek 2010).
12  Reproductive Biology of Grain Legumes 377

Suso et al. (2001) found variation in plant daily available flowers, pollinator
abundance and pollinator foraging behaviour rates among the same populations
of Vicia faba growing in different regions (southern Spain and northern France).
They reported variation in outcrossing levels, showing that it was lowest in northern
France, the region with the lowest pollinator service. Pierre et al. (1999) reported
that honeybees (A. mellifera) and bumblebees (mainly B. terrestris) were the most
frequent pollinators in northern France while solitary bees (mainly Eucera numida)
were the most frequent in southern Spain. Moreover, the pollinator populations dif-
fered in their pollinating behaviour: in northern France only 40 % of the pollinators
were positive (i.e. entered the flower and induced tripping), because some B. terres-
tris behaved as “robbers” by collecting nectar through a hole they made at the base
of the corolla. In southern Spain, 99 ± 6 % of the pollinators were positive.
Benachour et al. (2007) and Aouar-Sadli et al. (2008) studied the pollinating
insects of Vicia faba in different regions of Algeria. Species of the Eucera genus
were the most abundant pollinators. Cunningham and Le Feuvre (2013) showed
that more stable crop yield of high quality through better pollinator management
could be achieved in Vicia faba. Nayak et al. (2015) found that the benefits of insect
pollination to the production of winter field bean variety ‘Clipper’, both through
increased pod set and seed set were clear. However, the dependence on pollinators
could vary between cultivars as has been shown (Suso and Río 2015). It is important
to understand varietal effects on the pollination ecology of faba beans and more
research into cultivar difference is needed.

3.2.5  Beans (Phaseolus spp.)

The five cultivated species in the genus Phaseolus are: Phaseolus vulgaris L., com-
mon bean, snap bean; Phaseolus lunatus L., lima bean; Phaseolus coccineus L.,
scarlet runner bean; Phaseolus polyanthus Greenmann, the year bean, and Phaseo-
lus acutifolius A. Gray, the tepary bean (Debouck and Smartt 1995).

3.2.5.1  Common Bean (Phaseolus vulgaris)

Among the cultivated species in the genus Phaseolus, the most economically impor-
tant is the common bean or snap bean (Phaseolus vulgaris), which is normally self-
pollinated. The outcrossing rates and the gene flow due to natural hybridization have
been the objectives of many studies with various results demonstrating low values in
a range of 0–1 % and also high values in a range of 6–10 %. These differences have
been attributed to the variability of the pollinator presence both in frequency and
pollinator species present among the experimental locations (Bliss 1980).
Efficient pollinators like carpenter bees ( Xylocopa brasilianorum L.) were shown
to raise the outcrossing level to 20 % in Puerto Rico (Bliss 1980). Other species of
Xylocopa ( X. valga, X. violacea, X. iris and X. olivieri) considered as valuable pol-
linators of some cultivated beans ( Phaseolus sp.) in Turkey (Özbek 2013). Thrips
have been reported as pollinators, but it has not been confirmed (Park et al. 1996).
378 M. J. Suso et al.

The benefit of foraging and tripping of bees and bumblebees on the yield of four
common bean cultivars in California was estimated under open visitation by insects,
in insect proof cages, and controlled visitation with bumblebees. Although the re-
sults varied in the different years and lines, two of them responded positively to
bee tripping and showed increased seed yield by up to one third (Ibarra-Perez et al.
1999). In a similar recent work, Kingha et al. (2012) studied foraging and pollinat-
ing activities in a region in Cameroon. Xylocopa olivacea was the most common
and nectar was the reward. The effect of the presence of X. olivacea on plant yield
components was evaluated and showed increasing pod and seed yields as well as
seed quality in open pollinated flowers.
Hypocotyl pigmentation, flower or seed colour, and protein markers have been
used as genetic markers for the detection of natural crosses in common bean. Mean
intercrossing rate of 6.9 % was estimated from the hypocotyls anthocyanin in their
progenies (Ibarra-Perez et al. 1997).
Ferreira et al. (2007) used bean cultivars with violet and white flowers as pollen
donor and receiver respectively. The heterozygote violet flowers derived from seeds
collected from different rows in the four cardinal directions were used to estimate
the intercrossing rate that showed the highest level (0.136 %) at a distance of 0.5 m.
In another experiment, also conducted in Brazil, the level of the outcrossing rate
was cultivar dependent and practically 0 % at a distance of less than 2.5 m. Two
pairs of transgenic cultivars resistant to the herbicide glufosinate ammonium were
sown surrounded by their non-transgenic counterparts as recipients of the transgene
(Faria et al. 2010).
The research carried so far in Phaseolus vulgaris shows that common bean can
present variable natural hybridization that could be used for increasing variability in
populations. The presence and the density of bees visiting beans, such as honeybee
A. mellifera and the two solitary bees, Xylocopa calens and Xylocopa incostans
varied in a farmland in Kenya depending on the management of surrounding area
(Kasina et al. 2009b).
Seed protein markers detected almost 0 % outcrossing in an experiment at Astur-
ias, Spain (Ferreira et al. 2000). Studies applying molecular markers in diverse bean
germplasm indicated that natural hybridization happened with variable frequencies
and led to introgression events between Andean and Mesoamerican bean genepools
(Angioi et al. 2011).

3.2.5.2  Lima Bean (Phaseolus lunatus)

The lima bean (Phaseolus lunatus) is a self-compatible annual with a mixed mat-
ing system (self-pollination and cross-pollination), mainly autogamous with a low
allogamy rate (mean outcrossing rate less than 10 %), qualified as facultative al-
logamy (Baudoin et al. 2004).
Cross-pollination mechanism, described by Webster et al. (1979) cited by Bau-
doin et al. (2004), is mediated by honeybees, bumblebees and possibly thrips with
major pollinator A. mellifera (Delaplane and Mayer 2000; Hardy et al. 1997).
12  Reproductive Biology of Grain Legumes 379

Depending on genotype, growth conditions, plant spacing, prevailing wind direc-

tion and native insect populations the outcrossing rate can vary significantly and
may reach 48 % (Baudoin et al. 1998 cited by Martínez-Castillo et al. 2007).
Using isozyme markers, Zoro Bi et al. (2005) estimated that the outcrossing rate
ranged from 0.027 to 0.268 % across nine wild populations of lima bean (Phaseolus
lunatus) grown in Costa Rica.

3.2.5.3  Runner Bean (Phaseolus coccineus)

The scarlet runner bean (Phaseolus coccineus) is considered an allogamous species.

In an experiment carried out in Cameroon for two seasons, Pando et al. (2011) stud-
ied insects’ visitation in inflorescences of plants growing in an open field. Among
the 16 species that visited the flowers in the 2-year study, X. calens was the most
frequent. Nectar was the reward for X. calens, whose foraging activities increased
the fructification rate, the number of seeds per pod and the percentage of normal
seeds.
The biology of flower, nectar secretion and foraging by insects were studied
in four runner bean varieties in Poland. The flowers developed exclusively in the
morning and the nectar started to be secreted as soon as the bud stage. Runner bean
was visited mainly by honeybees and bumblebees for nectar. If the bumblebees pop-
ulations are short tongued, then their workers open holes and steal the nectar and
the honeybees will also steal from the holes, rather than pollinate (Koltowski 2004).

3.2.6  Grass Pea (Lathyrus spp.)

There are many Lathyrus species cultivated around the world and used as forage/
fodder and/or as pulses for human consumption depending on the region and the
people’s eating habits. The following species are cultivated as pulses in the regions
or countries given in parentheses; Lathyrus annuus (Europe, N. Africa), Lathyrus
blepharicarpus (Near East), Lathyrus cicera (S. Europe, N. Africa), Lathyrus cly-
menum (Greece), Lathyrus ochrus (Greece, Middle East), Lathyrus sativus (India,
S. Europe, N. Africa) (Kearney and Smartt 1995). The most economically important
and widely cultivated Lathyrus species is Lathyrus sativus. Other important species
are Lathyrus cicera, Lathyrus tingitanus, Lathyrus hirsutus and Lathyrus sylvestris.
Lathyrus odoratus and Lathyrus sylvestris are ornamentals.
The reproductive mode of 14 annual and perennial Lathyrus species was
studied in upper semiarid zone in Tunisia. The studied species did not flower at
the same period. Selfing, natural pollination, and natural pollination following
emasculation were applied on 75 flowers per each species, and the frequency of
flowers giving pods and the mean number of seeds per pod was measured. The
results showed that Lathyrus latifolius, Lathyrus sylvestris and Lathyrus tuberos-
us, perennial species, are strictly outcrossing and the main pollinators are bees
and bumblebees. These three species are characterized by large bright-coloured
380 M. J. Suso et al.

flowers. Selfing by bagging flowers showed that Lathyrus cicera, Lathyrus hirsu-
tus, Lathyrus annuus, Lathyrus ochrus, Lathyrus nissolia, Lathyrus aphaca, Lath-
yrus tingitanus, Lathyrus setifolius, Lathyrus articulatus and Lathyrus sativus, are
preferentially autogamous. Lathyrus odoratus is preferentially outcrossing (Ben
Brahim et al. 2001).

3.2.6.1  Lathyrus sativus

The grass pea (Lathyrus sativus) is predominantly self-pollinating although out-

crossing has been found to vary significantly between Lathyrus sativus genotypes
ranging from 9.8 to 27 % depending on the flower colour (Rahman et al. 1995) and
occurs on a larger scale in late-opening flowers in field conditions due to higher
levels of insect activity (Kearney and Smartt 1995). Pollen takes part in pollinators,
particularly bees, attraction (Ben Brahim et al. 2001). An even higher outcrossing
rate of 36 % was found by Gutiérrez-Marcos et al. (2006) that applied isozyme
analysis in a worldwide collection of Lathyrus sativus and studied the genetic struc-
ture of the populations.
In the experiment carried out by Rahman et al. (1995) in Bangladesh for two
years, plots with different recessive genotypes (red, pink and white flowered)
each, were surrounded by blue-flowered dominant genotypes. The genotype with
the red flowers showed the highest intercrossing rate (27.8 %) and the one with the
white flowers the lowest (9.8 %), while in the pink flowered variety an intermedi-
ate outcrossing rate (19.4 %) was recorded. The considerable intercrossing rate of
Lathyrus sativus may have negative implications in maintaining the varietal purity
in seed multiplication (Chowdhury and Slinkard 1997), in genetic contamination
of improved cultivars with landraces in farmers’ fields and vice versa and also
in collecting and conserving grass pea genetic resources (Rahman et al. 2001;
Gutiérrez-Marcos et al. 2006). On the other hand, outcrossing provides natural
variability that can be exploited in grass pea-breeding programmes (Hillocks and
Maruthi 2012).
The high intrapopulation variation detected with inter sequence simple re-
peats (ISSR) markers within Tunisian, Portuguese and Ethiopian cultivated
Lathyrus sativus and Lathyrus cicera populations and within wild Tunisian
Lathyrus ochrus could be attributed to rates of outcrossing (Belaïd et al. 2006).
Analysis of the diversity of 20 Ethiopian Lathyrus sativus accessions (12 plants
per accession) with expressed sequence tag-derived simple sequence repeats
(EST-SSR) showed an increase of the distribution of the alleles among popula-
tions that also could indicate outcrossing (Shiferaw et al. 2012). The observed
heterozygosity between 2 and 10 % in Italian grass pea landraces, detected with
six polymorphic loci, revealed with the application of SSR markers, may be at-
tributed to reproductive biology of grass pea and confirmed natural outcrossing
(Lioi et al. 2011).
12  Reproductive Biology of Grain Legumes 381

3.2.7  Lupins (Lupinus spp.)

There are many lupin species that are used by humans as crop plants. These include
large-seeded lupins from the Mediterranean region; Lupinus albus (syn Lupinus
termis), Lupinus angustifolius and Lupinus luteus, and species from the Americas
Lupinus mutabilis. Lupinus angustifolius is cultivated as a grain crop in Australia
and Lupinus mutabilis (Andean or Pearl lupin) is consumed traditionally by indig-
enous people in Ecuador, Bolivia and Peru (Hill 1995; Dracup and Thomson 2000;
Clements et al. 2008).
The genus Lupinus attracts pollinators with a display of multicoloured flow-
ers and rewards of pollen and fragrance (Kazimierska and Kazimierski 2002). The
genus is composed of many species that, depending on genetic and environmental
factors, present the whole range of pollination modes from strictly self-pollination
and self-pollination with facultative cross-pollination to prevailing cross-pollina-
tion (Kazimierska and Kazimierski 2002). Annual species tend to be self-fertilized
while perennial lupins are cross-pollinated. Even within each species, the outcross-
ing rates vary depending on the genotype, the location linked to the pollinator fauna
species and population. Grain lupin varieties, particularly sweet albus lupin, even
though self-pollinated, cross freely with the aid of bees (http://www.ogtr.gov.au/
internet/ogtr/publishing.nsf/content/42D3AAD51452D5ECCA2574550015E69F/$
File/biologylupin2013-2.pdf).
Most cultivated lupin species are regarded as self-pollinated although there is
a degree of outcrossing. When breeding lupins, adequate pollination barriers are
needed (Hill 1995; Kazimierska and Kazimierski 2002). The outcrossing rate of Lu-
pinus albus that can reach as high as 9 % and has caused contamination of the sweet
(low-alkaloid) cultivars, used for feed and snacks in Australia, with bitter seeds that
have high alkaloids and better fitness (Richards et al. 2008).
Gene flow due to outcrossing from cultivated sweet lupins (Lupinus angusti-
folius) to wild relatives that coexist in the same environment was evaluated in the
agricultural zone of Western Australia (Hamblin et al. 2005). The estimation of the
outcrossing level showed one cross in 3600 plants within the first 1.5 m from neigh-
bouring crops, while no outcrossing was detected beyond 2.25 m. Gene flow from
wild populations to cultivated forms of lupins has never been observed in fields and
the likelihood is extremely low (Hamblin et al. 2005).

3.2.8  Pea (Pisum sativum)

The pea has been considered in practice a strictly self-fertilizing crop (Bouwan
1992). However, Xylocopa and Megachile do visit pea flowers and can be responsi-
ble for natural outcrossing (Cousin 1997). Polowic et al. (2002) studied the frequen-
cy of outcrossing from a transgenic line of peas into three cultivars by using two
dominant traits, normal leaf form, and a highly expressed β-glucuronidase ( gusA)
gene as markers of pollen transfer. However, normal leaf form was considered a
382 M. J. Suso et al.

no reliable marker. 0.07 % mean of intercrossing ranking from 0.00 to 0.11 % was
found. Additionally, Vershinin et al. (2003) suggested that despite the predomi-
nant inbreeding of the genus, significant outcrossing between all the Pisum species
should have occurred.

3.2.9  Soybean (Glycine spp.)

3.2.9.1  Glycine max (L.) Merr

Soybean has been considered naturally self-fertilized. In a 2-year study with two
cultivars using flower colour, cross-pollination rates varied from 0.03 % to as high
as 6.2 % (Ray et al. 2003).
Chiari et al. (2005b) evaluated the effect of honeybee pollination on seed pro-
duction and quality of soybean seed using caged and uncovered honeybee plots in
Brazil. They found that seed production was higher in caged plots with honeybees
(50.64 %) than caged plots without honeybees and even higher in uncovered areas
(57.3 %). This increase in seed production was explained in a separate study by
the same investigators, by examining pollination by honeybees. They found that
honeybees visited 2.24 flowers on average in uncovered plots, and 1.58 flowers in
caged plots (Chiari et al. 2005a). Additionally, the percentage of flower abortion
was 82.91 % in caged plots without honeybees, 53.95 % in caged plots with honey-
bees, and 52.66 % in uncovered plots. Higher soybean yield in Brazil were achieved
with wild bee pollinators (6.34 % increased). The addition of honeybee colonies
further increased yield (18.09 %; de O. Milfont et al. 2013).

3.2.9.2  Wild Annual Soybean (Glycine soja)

The wild annual soybean, G. soja Sieb. et Zucc., is believed to be predominately

self-pollinating; however, little effort has been made to evaluate its breeding sys-
tem. Kiang et al. (1992) estimated an outcrossing rate of 2.3 %. Fujita et al. (1997)
analysed the genetic structure of four G. soja populations in Japan by examining
allozyme variation. They obtained higher within-population genetic variation and
lower genetic divergence among populations than would be expected for a selfing
plant species. The mean outcrossing rate estimate was 13 % ranging from 9.3 to
19 % among the four populations. This higher outcrossing rate was supported by
observations of frequent visits during flowering by honeybees and carpenter bees
( Xylocopa spp.).

3.2.9.3  Wild Perennial Soybean Species

The perennial wild relative of soybean, G. argyrea (Tind.), has both self-fertilized
cleistogamous flowers and chasmogamous flowers on the same plant (Brown et al.
12  Reproductive Biology of Grain Legumes 383

1986). The chasmogamous flowers are visited by insect pollinators and ranged
from zero to complete outcrossing, with an average of about 40 %. G. clandestina
(Wendl.) is a closely related perennial species to G. argyrea and has both cleistoga-
mous and chasmogamous flowers (Schoen and Brown 1991). The floral biology of
G. clandestina and G. argyrea allows chasmogamous flowers to spontaneously self-
fertilize when left unpollinated; for example, in the glasshouse and in the field when
conditions for insect-mediated pollination are absent or suboptimal. Schoen and
Brown (1991) sampled two populations of G. clandestina (1500 and 750 m eleva-
tion) and one population of G. argyrea. In the 1500 m population of G. clandestina,
approximately 60 % of the overall rate of self-pollination in chasmogamous flowers
was attributable to whole-flower selfing. This contrasts to the zero whole-flower
selfing in chasmogamous flowers recorded with the 750 m population of G. clan-
destina. The difference in cross-pollination of the chasmogamous flowers between
the two G. clandestina populations was considered to be related to the contrasts in
the environmental conditions for insect-mediated cross-pollination. The chasmog-
amous flowers that did not receive pollinators would self-fertilize spontaneously
(Schoen and Brown 1991). In the G. argyrea population, only about 4 % of the
chasmogamous self-pollination was attributable to whole-flower selfing (Schoen
and Brown 1991).

4  General Approaches to Manage Pollination

4.1  Basic Questions

Pollination is a critical step in breeding strategies if cross-pollination is required for

breeding purposes or for hybrid seed production or if reduction of cross-pollination
is necessary in seed stock multiplication. Pollination services depend on both man-
aged and unmanaged pollinator populations. Any reflection on how to manage pol-
lination should be focused at two levels; open field including farmer’s-fields and at
enclosed production systems. This section targets these two systems.
The pollination phenomenon was considered important for the production of
local crops to promote sustainable development (IPBES 2013). There is growing
evidence that improved pollination practices can help support higher yield. Garib-
aldi et al. (2013) suggested that new practices for integrated management of both
honeybees and diverse wild insect assemblages will enhance global crop yields. The
creation of habitat diversity within a farm in the form of shelters, nest sites, water,
larval food plants, or nectar plants can promote populations of suitable pollinators
(Blaauw and Isaacs 2014). Christmann and Aw-Hassan (2012) suggested farming
with alternative pollinators (FAP) as an integrated agro-ecological-socio-economic
approach and a self-sustaining win-win-strategy for farmers, agro-ecosystems and
climate change adaptation. Palmer et al. (2009) proposed the Crop-Design Sys-
tem in which breeders and farmers develop, by participatory plant breeding (PPB),
384 M. J. Suso et al.

cultivars with enhanced heterosis-mediated yield and resilience as a result of the

provision of floral resources within the crop for supporting insect pollinator popula-
tions to be used as agents of crossings to increase heterozygosity. Moreover, these
techniques can be much less expensive in terms of time and labour than the alterna-
tive of hand pollination (Westerkamp and Gottsberger 2000; Potts et al. 2011).
LI/O systems attract more bee pollinators compared to conventional fields (Mo-
randin and Winston 2005; Greenleaf and Kremen 2006; Power and Stout 2011;
Andersson et al. 2012) and can benefit bee biodiversity, insect–flower interaction
networks and insect-mediated pollination. However, usually, in LI/O farming, bees
come freely from nearby natural habitats. According to Kasina et al. (2009c), in
western Kenya, pollination in small-scale farms is unmanaged and supported by
nearby ecosystems; although, large horticultural farms have initiated beekeeping
projects within their farms, where they keep honeybees for pollination of legumes
such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata). However,
they do so without considering whether honeybees are effective pollinators of these
crops. Interestingly, Kasina et al. (2009a) investigated the dependence of some
crops on bee pollinators as a cautionary measure to inform on the need to manage
pollinators. They found that crops that are assumed not to need bee pollination such
as some legumes had significantly higher yields when provided with bees. Wild
bees, carpenter bees ( Xylocopa spp.) and leafcutter bees ( Megachile spp.) were the
most effective pollinators of beans and cowpeas. These bees are large, and their
weight causes flower tripping. A. mellifera “stole” nectar through the sides of the
flower without causing flower tripping and therefore did not affect pollination. Both
beans and cowpeas increase seed formation after exposure to pollinators. Also, it
was found that flowers under the exposure to pollinators produce seeds that have
higher protein levels compared to seeds from flowers without pollinators (Kasina
et al. 2009a).

4.2  Methods for Pollination Control: Enclosed Spaces

4.2.1  General Approach

The management of pollinating insects under the controlled conditions of enclosed

spaces, including field cages, growth chambers and greenhouses is applied to breed-
ing strategies such as hybrid seed production and seedstock multiplication or ex situ
germplasm regeneration.
Currently, a number of bee species are managed for pollination for a range of
different crops. Honeybees, A. mellifera L., are the major contributors to legume
pollination. The use of other bees is less common. Additional examples of managed
bees are Megachile rotundata, Bombus spp. and Osmia spp. (Delaplane and Mayer
2000). The issue of supporting native pollinators by designing a crop with appro-
priate flower traits has been proposed (Suso and Maalouf 2010) as a good practice
for long-term sustainability and it is being incorporated in international schemes to
improve agri-environmental practices (Suso et al. 2013).
12  Reproductive Biology of Grain Legumes 385

However, Westerkamp and Gottsberger (2000) consider that in spite of the high
diversity of flowers, which requires an adequate diversity of pollinators, almost all
animal pollination is simplistically ascribed to the manageable but often less effi-
cient pollinator, the European honeybee, A. mellifera L. It is not only the inappropri-
ate match between honeybees and the great diversity of flowers which often makes
them inefficient pollinators. Moreover, it is also especially the missing “know how”
in flower handling, which results in the malfunction of the honeybee at certain flow-
ers such as zygomorphic flowers. They proposed as the very first step of solution to
try to understand floral function.
The effectiveness of honeybees and leafcutter bees in cross pollination between
two cultivars of faba beans within cages was assessed by Currie et al. (1990). Hon-
eybees were more effective than leafcutter bees as pollinators of faba beans in caged
plots.
The aptitude of leafcutter bees to pollinate male-sterile soybean plants ( ms2
gene) in caged plots was evaluated in four experiments from both quantitative and
qualitative points of view. The seed set on msms plants was satisfactory (Roumet
and Magnier 1993).

4.2.2  Exemplar Case Analysis: Multiplication/Regeneration

To maintain the genetic integrity of accessions and to take special care to prevent
outcrossing between accessions but facilitate crossing between individuals within
accessions to avoid inbreeding depression during legume species regeneration or
seedstock multiplication is needed. Ideally, plant accessions should be grown out-
doors, in isolation fields, and separate from each several meters depending on the
species level of outcrossing. This allows natural pollinators to pollinate within ac-
cessions but not between them (Street et al. 2008). Alternatively, it is possible to
use isolation cages or screenhouses to limit the movement of pollinators between
accessions.
The survey carried out by the European Cooperative Programme for Plant Ge-
netic Resources (ECPGR; Grain Legume Management Survey 2005, (http://www.
ecpgr.cgiar.org/worksgroup/grainlegumes.htm, Suso et al. 2011) demonstrated that
legume breeders are aware of gene flow problems and practiced some form of polli-
nation control or isolation procedure with seed multiplication. Results of the survey
showed that spatial isolation is the most common practice in the legume commu-
nity. Though, the use of isolation facilities along with suitable insects as pollen
vectors is the method most recommended. Many gene banks therefore use isolation
facilities along with suitable insects as pollen vectors.
Respondents of the survey agree that regeneration needs gradually move towards
the promotion of protocols aiming at encouraging and exploiting the link between
seed multiplication and use. Seed multiplication protocols in gene banks must be
closely integrated with pre-breeding and anticipate user demands to develop cus-
tom-tailored materials, for instance, materials for LI/O farming systems. With seed
multiplication, emphasis needs to be taken in the neglected aspect of maintaining
386 M. J. Suso et al.

the level of heterozygosity and heterogeneity in landraces and in understanding how

pollinators and crops interact to shape the specific environments in which curators
make their multiplications.

4.3  Spatial Isolation

In legume species, PMGF cannot be ignored in seed multiplication. Actually, due to

potential release of transgenic crops and to avoid contamination, the intercrossing
even in strictly self-pollinating legumes such as pea (Pisum sativum) has been stud-
ied (Polowic et al. 2002). Generally, physical distance is used to prevent gene flow
(details for specific references at point 3.2). However, when dealing with a large
number of undersized and diverse germplasm stocks, the distance needed for isola-
tion could be very large and therefore the method is not usually practical because
the land required would exceed the capacity. For insect-pollinated species, pollen
flow can be further interrupted and reduced by planting barriers of the same species
or of another species.
Suso et al. (2006) and (2008b) had the objective of testing the effectiveness of
different isolation zones on the control of PMGF. The study was centred on Vicia
faba, but we present these results because it is considered a model plant due to its
alternatively outbreeder–inbreeder behaviour. Therefore, the analysis of the PMGF
dynamic might serve as a base line for similar studies in other legumes with simi-
lar pollination mechanisms. Suso et al. (2006) and (2008b) tested three isolation
strategies: (a) a barren zone, an isolation zone devoid of all vegetation; (b) the same
size isolation zone sown with two different trap crops: (1) a faba bean male-sterile
variety and (2) a tetraploid genotype; and (c) the same size isolation zone sown with
non-pollinated crop, a Vicia narbonensis population. The male-sterile variety does
not release viable pollen, and the tetraploid genotype does not cross with diploid
faba bean genotypes. Consequently, the trap crops were used as pollen sink for bees
to deposit pollen as they moved away from the test plots. Vicia narbonensis was
used because of its similarity to Vicia faba, but because it is an autogamous crop, it
would discourage insect pollinators from leaving the faba bean plots. Pollen-medi-
atated gene flow is largely dependent on floral traits. Floral advertisement seemed
to be important in explaining gene flow between plots surrounded by a barren zone.
With regard to plots surrounded by a Vicia narbonensis population, the role of a
reward trait, pollen production, was established. In contrast, in plots surrounded by
faba bean trap crops, ovary length played the most important and consistent role in
accounting for variation in gene flow among plots.
Pollen gene flow is genotype, border and site specific, and novel information
on isolation distances and good “safe” guidelines in legumes is needed specifically
for commercial seed production under organic cultural practices—either by seed
companies or by farmers producing seed for their own needs. Because most of the
organic farmers have much more biological diversity on their farms than the con-
ventional seed farms, they have many more insect pollinators (wild bees, wasps and
12  Reproductive Biology of Grain Legumes 387

syrphid flies). The current recommendations for isolation distances in the legume
self-pollinated species might be inadequate for avoiding crossing between varieties.

5  Pollination in Crop Breeding

5.1  General Approach

In the first section, we emphasized the importance of understanding how flowers

function in order to use pollinators as agents of outcrossing. Availability of out-
crossing mechanisms could be important in the development of hybrids and popula-
tion improvement breeding schemes such as recurrent selection for accelerating the
rate of gene recombination. The search of these mechanisms may be more effective
in legume plants with zygomorphic flowers and pollinator-dispersed pollen than
in species dependent on wind for outcrossing. Plants can exploit bee behaviour
by flower morphology and by using advertising signals and rewards to increase
the number of seeds. Legume zygomorphic flowers promote precise pollination
by restricting which pollinators can visit a flower, the direction from which they
can approach, and their movement within the flower. The success of a breeding
programme for insect-aided outcrossing depends on breeders mimicking flower
behaviour and advantageously adopting strategies that take advantage of the com-
plex interplay that exists between floral features and pollinators. For instance, one
means of controlling pollinator bee visitation behaviour is through flower reward
traits. But when using nuclear or cytoplasmic male sterility in F1-seed production,
the recipient line lacks pollen as a reward. However, it is possible to overcome a
lack of pollen with the reward of nectar and other attractive traits. Breeding pro-
grammes leading to parental lines for the production of hybrid seeds must be carried
out in the presence of pollinator bees to ensure that overall pollinator-related traits
are adequate. Ensuring suitable floral morphology and structure for efficient cross-
pollination must be part of such breeding programmes (Kobayashi et al. 2009).
Unfortunately, many vegetable and horticultural crops that depend on pollination
have tended towards greater reliance on self-pollination as they have been subjected
to modern breeding programmes (FAO 2008). The shift from outcrossing or facul-
tative selfing to strict inbreeding has been described as the single most common
trend in legume domestication (Rick 1988). The transition from outcrossing to high
levels of self-fertilization may be accompanied by a change in the traits functionally
related to pollinators (Suso and Río 2014). As floral trait values associated with au-
tonomous selfing rates are generally the opposite of those associated with efficient
outcrossing, selection for pollinator attractiveness and selection for efficient selfing
are unlikely to act in parallel. Selfing favours the evolution of plants with lower pol-
linator attraction and altered morphology and the evolution of plants with lower and
often inadequate pollination visitation (Fishman and Willis 2008). Thus, we face a
situation in which the recuperation and improvement of functional floral traits that
388 M. J. Suso et al.

may have been diminished or lost through domestication is more than suitable in
order to facilitate the conversion of selfing species to allogamy.

5.2  Breeding Hybrids

5.2.1  General Approach

Palmer et al. (2011) reviewed the phenomenon of heterosis in food legumes and
noticed that food legumes, in general, have not benefitted from male sterility sys-
tems to produce hybrids. Hybrid pigeon pea is the only success story in pulses
(Saxena et al. 2013). They concluded that there are a number of factors that are
crucial for the successful development of hybrids but the lack of an efficient pollen
transfer mechanism from pollen parent to pod parent is the major limitation. If this
methodology or other technology becomes viable, food legumes would be a major
beneficiary of this science. Also, Fu et al. (2014) stated that in order to utilize het-
erotic potential, there remains a need to develop high-efficient pollination control
technologies on a species-specific basis.
Saxena et al. (2013) also recognized that the commercial exploitation of hy-
brids is directly linked to the ease with which their hybrid seeds could be produced
and delivered economically to farmers. Thus, the efficiency of mass pollen transfer
from male to female parent through air or insects to affect cross-fertilization plays
an important role in commercializing the hybrids in different crops. In most food
legumes, the absence or low natural cross-fertilization is the major bottleneck in
exploiting hybrid vigour at a commercial scale.
Kobayashi et al. (2010) argued that cultivars are generally bred by the artificial
selection of agronomic traits that are of commercial interest but with little regard
to pollinator-related traits and preferences. Consequently, insect-pollinated culti-
vars may not be attractive to pollinators, resulting in low seed production. This is
a problem for commercial seed production of autogamous or partially allogamous
legume crops. To enhance F1-seed productivity through the efficient application of
pollinators, including honeybees and native insects, we must fully understand the
pollination process and the plant–pollinator interplay.

5.2.2  Case Studies

Considerations and studies carried out on specific species are now summarized.

5.2.2.1 Soybean (G. max)

In soybean, despite the existence of genetic male sterility and heterosis expres-
sion, no soybean hybrids are used in commercial production. Unless better or more
12  Reproductive Biology of Grain Legumes 389

efficient pollinator systems can be found, the genetic male sterility used to develop
hybrids will not suffice for the commercial release of hybrid soybean (Cober et al.
2010). Palmer et al. (2012) considered that the exploitation of the heterosis de-
pends on basic information available on different aspects of the interplay between
plant and pollinator. The limiting step in the study of soybean heterosis is the few
hybrid seeds available for the agronomic performance tests. To determine and iden-
tify heterotic combinations or associations requires that a large number of parental
recombinations be evaluated in multiple years, many locations within years and
adequate replications per location. The seed requirement for each parental combi-
nation is very large. The first step in soybean heterosis studies is to understand the
plant–pollinator interplay. Ortiz-Perez et al. (2008) indicated that bee preference for
certain parental lines was the key factor in hybrid seed production. To address these
issues, Suso et al. (2010), using M. rotundata as main pollinator and two soybean
inbred lines, reported that floral size and shape are of primary importance in guid-
ing pollinators’ foraging decisions. High and low seed-set lines differed in flower
size and shape. Longer and more lobed flowers increased seed set. Increasing the
attractiveness and facilitating the manipulation of the flowers, by less flattened stan-
dards, provide a useful means of improving seed set. From a follow-up experiment
(Pappas et al. 2012), the proboscis extension response system (PERS) was used
to determine if honeybees detected differences between the volatiles emitted by
flowers from high and low seed-set lines and parental lines. Honeybees responded
more favourably to the high set lines, and parents, than to the low seed-set lines.
Differences between highly pollinator attractive genotypes and poorly attractive
genotypes were likely the result of organic volatiles intensity, rather than one or two
unique volatiles. This preference was utilized in phenotypic recurrent selection to
produce large quantities of hybrid soybean seed (Pappas et al. 2012).
An open flower mutant, apetally, in soybean was also male sterile, but female
fertile (Palmer et al. 2004). The apetalous mutant might have utility as a female
parent in hybrid seed production. The manual cross-pollination success rate with
apetalous plants as female parent was comparable to cross-pollinations made with
fertile female sibling plants. However, the unprotected stigma of the apetalous mu-
tant may be more vulnerable to desiccation under low humidity, and outcrossed
seed set would be reduced.

5.2.2.2  Pigeon Pea (Cajanus cajan)

Saxena et al. (2013) analysed the development of seed production technology in pi-
geon pea. They argued that the benefits of hybrid technology cannot be realized un-
less sufficient quantities of genetically pure hybrid seed are commercially produced
and sold at affordable prices. To harvest good hybrid seed yields, it is imperative to
select suitable seed production sites with good insect pollinator activity. Thus, the
hybrid seed set on the male-sterile plants is chiefly determined by the availability
of bee populations in the vicinity. In pigeon pea, the main pollinating vectors are
Megachile lanata, Apis florea and A. mellifera. Good hybrid yield is obtained, up to
390 M. J. Suso et al.

25–30 % outcrossing. This is primarily attributed to prolonged flowering in pigeon

pea as an evolutionary consequence. Pollinating insects may visit the male-sterile
plants several times, and at each visit, a certain proportion of the flowers are pol-
linated to set the pods while the un-pollinated flowers drop. This is followed by the
emergence of new flowers on the same plant, and again a proportion of them set
pods through open pollination. This cycle continues, and at the end of the season,
plenty of crossed pods are observed on each male-sterile plant.

5.2.2.3  Mung Bean (Vigna radiata)

Mung bean is a self-pollinating crop that displays significant hybrid vigour in seed
yield of F1 hybrids offering the possibility to use hybrid varieties as a breakthrough
to raise the yield plateau of mung bean. Sorajjapinun and Srinives (2011) consid-
ered that changing flower form in mung bean may affect pollination rate and in-
crease outcrossing. They proposed to encourage hybrid seed set and to develop
potential characters that promote higher outcrossing rate such as open flower (chas-
mogamy). Thus, chasmogamy controlled by a single recessive gene can be used to
promote natural outcrossing rate as a step towards large-scale hybrid seed produc-
tion in mung bean. However, the mechanism of outcrossing requires pollinators;
they observed that major pollinators of mung bean flowers were bees.

5.2.2.4 Chickpea (Cicer arietinum)

In chickpea, open flower mutants also have been proposed to be used to increase the
level of outcrossing (Pundir and Reddy 1998; Srinivasan and Gaur 2012). Chickpea
is a highly self-pollinated crop. The outcrossing is reported to be in the range of
0.0–1.9 % (Tayyar et al. 1995; Toker et al. 2006). Thus, cleistogamy poses chal-
lenges in the development of hybrids. Flowers with all petals open (open flowers)
that expose male and female reproductive organs would facilitate cross-pollina-
tion. A study conducted to estimate the multilocus outcrossing in an open flower
population using microsatellite markers revealed a 5.9 % outcrossing rate (Rubio
et al. 2010). The higher frequency of cross-pollination of the open flower pheno-
type could be used for the production of hybrid seeds, if lines producing heterotic
hybrids could be identified.

5.3  Breeding Populations: Faba Bean as a Model Plant

Heterosis determines higher productivity, resilience and fertility. It is fully realized

in hybrid cultivars and partly in open-pollinated varieties (OPVs) or synthetics that
are obtained via inter-mating selected parental genotypes for high general com-
bining ability (GCA)/specific combining ability (SCA). The development of these
12  Reproductive Biology of Grain Legumes 391

populations will allow: (1) to fully exploit the “panmitic-mid-parent heterosis” of

crosses between genetically distant populations (heterotic groups) and to capital-
ize not only GCA but also SCA effects. In some legume crops, such as faba beans,
due to instability of male-sterile systems and where it is not economically practical
to obtain hybrid seed by manual crossing, managing pollinator behaviour through
selection for floral traits that enhance pollinator adequate visitation rates has been
proposed (Palmer et al. 2009, 2012) to increase the level of outcrossing. This strat-
egy, selection of pre-mating floral traits, has been advocated by other researchers
(Ceccarelli 1978; Abdel-Ghani et al. 2003) to generate OPVs with high levels of
heterozygosity. This also avoids the dangers of genetic uniformity associated with
the development of hybrid varieties and allows the beneficial effects of heterosis
available to low-income farmers in a faster way (Nandety 2010). Preliminary ex-
perimentations have been carried out to understand the relationship between out-
crossing and floral traits in order to determine if floral variation can be effectively
utilized for the development of almost exclusively cross-pollinated varieties (with
high heterozygosity levels; Suso et al. 2005; Suso and Maalouf 2010).
Suso et al. (2005) proposed that outcrossing might be enhanced by artificial se-
lection for appropriate aspects of floral design and display. Floral traits relevant to
increase the level of outcrossing include enhanced inflorescence production during
the beginning of flowering but with few flowers open simultaneously per inflores-
cence. In addition, low nectar reward and short floral tubes should be taken into
account. However, as outcrossing varies geographically (Suso and Moreno 1999;
Suso et al. 2008a), the most suitable floral traits for promoting outcrossing depend
on local environmental conditions (Vogler et al. 1999), particularly the composition
of the pollinator fauna.
Suso et al. (2010) carried out artificial selection for outcrossing in open-pollinat-
ed faba bean. They found that whether the selection was for increased or decreased
outcrossing, the selected groups shifted in the opposite direction of the selected
type. The patterns of increase and decrease in outcrossing in the selected popula-
tions were due to a simultaneous multidimensional change in floral traits, thus limit-
ing or contra-balancing the effects of artificial selection. They concluded that for the
improvement of faba bean populations, direct selection on outcrossing cannot be a
selection criterion. However, floral traits, in combination with pollinator behaviour,
should be used as indirect selection criteria to increase the level of outcrossing. This
approach, in turn, will allow the development of both pollinator-friendly varieties
and enhance the environmental services of faba beans.

5.4  Concluding Remarks

Strategies for using heterosis more widely to increase yield and yield stability in
legumes centre on finding ways of reducing the cost and increasing the efficiency
of producing hybrid seeds. High-efficient pollination insect-aided technologies
on a species-specific basis are lacking. Unfortunately the information available is
392 M. J. Suso et al.

insufficient for an in-depth assessment of the mating patterns in most grain legume
species. However, there is experimental evidence that pollinator behaviour influ-
ences plant mating patterns and that plants can modify pollinator behaviour through
changes in floral traits. To advance in the application of the understanding of the
reproductive biology tool for heterosis breeding, more multipopulational studies,
combining ecological and genetic factors, are required in order to know the variabil-
ity and relationships among floral traits, mating systems and floral visitors under
different local conditions and ecological contexts. Plant–pollinator interplay may be
expected to vary among populations, generating a complex pattern of differential
adaptation. Thus, studies are particularly necessary to assess the year to year and
location to location dynamics of the plant–pollinator interplay.

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Chapter 13
Grain Legume Cropping Systems in Temperate
Climates

Thomas F. Döring

1 Introduction

Grain legumes (or pulses) are of paramount importance as sources of protein. At

the global scale, the role of grain legumes as protein providers is currently mostly
fulfilled in their function as feed for livestock, but there is also substantial usage of
grain legumes for direct human consumption. This means that this group of plants
realizes a critical complementary function for human and animal nutrition, espe-
cially in comparison to cereals. The protein digestibility for dairy cattle and mono-
gastrids is particularly high in grain legumes. The key role of grain legumes for
feeding humans and animals is expected to increase in the future as current breed-
ing activities are aiming at optimising protein quality (e.g. in terms of amino acid
profile) and minimising the content of anti-nutritive substances such as glycosides,
alkaloids and phenol derivates in grain legumes (Kolbe et al. 2002).
However, the importance of grain legumes is not restricted to their role as a sup-
plier of high-quality protein. In particular, grain legumes exhibit a critical function
from an agronomic point of view as well (Köpke and Nemecek 2010). Because
of their ability to fix aerial nitrogen (N2) in symbiosis with soil-living bacteria,
grain legumes provide crops following them in the rotation with an essential plant
nutrient. Indeed, N can be seen as the main driver of biomass production and a key
determinant of crop yield. As pre-crops, grain legumes therefore play a central role,
especially in terms of N provision for organic and low-input cropping systems.
Further, apart from providing N in the crop rotation, grain legumes are known to
have additional beneficial effects on the following crop, in particular by helping to
break the life cycle of soil-borne diseases of cereals. Finally, grain legumes are also
important from a broader agro-ecological point of view, as they provide nectar and
pollen resources for pollinators.

T. F. Döring ()
Department of Agronomy and Crop Science, Humboldt University Berlin,
Albrecht-Thaer-Weg 5, Berlin, Germany
e-mail: [email protected]
© Springer Science+Business Media New York 2015 401
A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5_13
402 T. F. Döring

Despite the multiple benefits of grain legumes, many countries have experienced
a relatively steady decline in the area grown with grain legumes over the last few
decades. A major direct driver of this development has been a dramatic change in
the subsidies paid to farmers for growing grain legumes in the European Union.
In addition, numerous other factors have contributed to this shift, including the
replacement of the role of grain legumes as N provider in the crop rotation by the
usage of mineral N fertiliser and the replacement of grain legumes in human con-
sumption by meat as a source of protein. However, at a global scale, not all grain
legume species have experienced the same trend. In fact, over the past two decades,
there has been a substantial expansion of the area planted with the globally most
important grain legume, soybean (Glycine max). Specifically, the global area of
soybean has increased by 64.4 % between 1993 and 2012, according to data by the
Food and Agriculture Organisation. The combination of these trends has led to an
increasing monopolisation of the grain legume spectrum by soybean. Used mainly
as animal feed, soybean is a crop dominated by highly intensive production sys-
tems, increasingly based on large-scale monocultures and intercontinental transport
of the harvested grain.
In order to regain protein self-sufficiency at a regional and national scale, there
have therefore been repeated calls and initiatives to expand the area cropped with
various grain legumes in Europe. A source of innovation and experience and a driv-
er of research in this area has been the organic farming sector. Much of the research
that this chapter draws on is therefore focussed on grain legumes in organic sys-
tems. Interestingly, the yield gap between organic and conventional management is
relatively low for legumes in comparison to cereals (Seufert et al. 2012).
Further, for the past decade, there have been increasing efforts to expand the
climatic range of soybean and introduce this crop in new, that is, more northern, re-
gions including Central Europe (Fig. 13.1). Globally, main cropping areas of grain
legumes are northern France, India, west Brazil, Argentina, South Australia and

Fig. 13.1   A soybean crop

on a sandy soil at the field
research station of Humboldt
University in Dahlem, Berlin,
where demonstration trials
of soybean varieties have
been conducted since the late
1980s. (Photo: T. Döring.)
13  Grain Legume Cropping Systems in Temperate Climates 403

Russia (Leff et al. 2004). Future climate change is likely to result in major shifts
in the geographical distribution of grain legume growing areas. For managing and
supporting sustainable change in this dynamic situation, it is of high importance to
understand the role of grain legumes in cropping systems and to know the require-
ments for optimal grain legume management in the field.
With a focus on temperate climates, and a geographical bias towards Europe,
this chapter reviews the agronomy of grain legumes and their role in cropping
­systems, building on previous reviews of the area (e.g. Jensen et al. 2010). The
highly integrative science of agronomy generally investigates the interactions be-
tween crop plants and the biotic and abiotic environment under different crop man-
agement regimes. The agronomist’s tools include the selection of crop species and
varieties, the design of crop rotations, the management of seed densities, the design
of intercropping systems, the integrative management of plant nutrition and plant
protection measures and the choice of appropriate tillage systems. In this context, a
cropping system can be understood as pattern and sequences of crops cultivated on
a given piece of land over a given period of time, together with the entirety of the
pertaining management measures such as crop fertilisation and tillage operations.
Usually, the period of time refers to a minimum of one rotation, that is, typically
between 3 and 8 years. In addition, the view of the cropping system includes the
interaction of the cropping management with the farm resources and other farm
enterprises such as livestock production units.
The main task that cropping system design typically focuses on is to obtain
high yields and high-quality levels of the harvested product; in the case of grain
legumes a major aim is to achieve high protein contents. In addition, cropping
system design also targets high yield stability, and the ability of crops to perform
under stress situations such as drought. Indeed, instability of grain legume yields
has been identified as a major problem, impeding progress and wider adoption of
grain legumes in practice. In particular, grain legumes are perceived to be associ-
ated with high risk, especially in terms of their response to stresses such as low
water availability. A widely accepted view is that grain legumes are character-
ised by a low ability to compensate influences of the environment. Therefore, this
chapter investigates some agronomic determinants of yield stability and potential
solutions to the problem of “markedly fluctuating yields” in grain legumes (Kolbe
et al. 2002).
The chapter concentrates on seven major species of grain legumes, namely faba
beans (Vicia faba), field peas (Pisum sativum), soybeans (G. max), lentils (Lens
culinaris) and lupins (Lupinus angustifolius, L. albus and L. luteus). Occasionally,
it will also draw on examples from common bean (Phaseolus vulgaris) and chick-
pea (Cicer arietinum). The predominantly tropical and subtropical grain legume
­species, such as cowpea (Vigna unguiculata) and pigeon pea (Cajanus cajan) are
not covered.
404 T. F. Döring

2  Grain Legumes in the Crop Rotation

Crop rotations encapsulate the essentials of a given cropping system. This sec-
tion therefore summarises some general rules of crop rotation design and reviews
the effects of grain legumes on the soil and the following main crop. It further
describes effects of the main crop preceding the grain legume, elucidates the in-
teractions between grain legumes and subsidiary crops, such as green manures in
the crop rotation, and finally discusses some examples of grain-legume-based crop
rotations.

2.1  General Rules of Crop Rotation Design

The term crop rotation refers to the practice of growing a series of different crop
species on the same field over a number of years. With the general aim to maintain
and increase long-term soil fertility, and thereby achieve high and stable crop yields
over several rotations, there are some general rules for designing successful crop
rotations. These rules refer to (i) the selection of crop species, (ii) the assignment
of proportions of these crop species within the rotation and (iii) the design of the
particular sequence of the selected crops.
As a first step, the selection of crop species for the rotation needs to follow the
known requirements of the crop species with regard to soil and climatic condi-
tions that are found on the farm. For instance, typical crops following yellow lupins
are rye and potatoes, because all of these crop species require relatively light soils
(Zimmermann 1958). There are large differences among the grain legume species
in relation to, for example, tolerated values of soil pH (Fig. 13.2a) or water require-
ments (Table 13.1) and such information forms the basis for crop species selection
in the rotation.
Second, economic as well as ecological considerations need to be taken into
account when determining the proportion of various crop species in the rotation.
For instance, grain legumes are (often) economically outcompeted by other cash
crops such as cereals in terms of economic performance per unit area land. In ad-
dition, direct support payments may influence the decision on the crop proportions
as well.
Further, the proportion of a crop species in the rotation is restricted by effects
of pests and diseases on the crop. In particular, soil-borne pests and pathogens can
build up high population levels if the same crop species is grown on the same field
over successive seasons. Similar considerations apply to infestation with weeds. In
particular, because several grain legume species are relatively weak competitors,
weed species adapted to the growing patterns associated with grain legumes, for
example, spring sowing, may increase to intolerable levels over time if the propor-
tion of grain legumes in the rotation is too high.
 13  Grain Legume Cropping Systems in Temperate Climates 405

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Fig. 13.2   Recommendations for grain legume growing with regard to (a) soil pH requirements,
(b) break years in the rotation, (c) sowing depth and (d) seed density. The y-axis shows the propor-
tion of sources recommending a given value. (Data sources include Schlipf 1898; Döring et al.
1956; Zimmermann 1958; Kay 1979; Mahler and McDole 1987; Sperber et al. 1988; Franzmann
1992; Kolbe et al. 2002; Kahnt 2008; Guddat and Karalus 2009; Rühl et al. 2009; Jensen et al.
2010; Alpmann and Schäfer 2014)

Because of weed, pest and disease problems, it is therefore usually recommend-

ed that not more than about a quarter of the rotation is assigned to grain legumes
(Kolbe et al. 2002). Thus, a year of growing a grain legume needs to be followed by
a number of break years that is required to minimise pest, disease and weed prob-
lems. The number of break years might be reduced when the grain legume is used as
(the minor) partner in intercropping systems with cereals or when used for forage.
Importantly, different grain legume species differ in the number of recommended
break years (Fig. 13.2b). However, the available information also highlights that
the recommendations on the number of required break years for grain legumes vary
strongly among different sources. Because systematic investigations on the effects
of the number of break years on grain legume yield and quality are rare at the mo-
ment, the information available to researchers and practitioners carries a relatively
large degree of uncertainty.
A major criterion for designing the sequence of crops within the rotation is that
the harvesting time of one crop and the sowing or planting time of the following
crop cannot overlap but need to leave a time window for preparing the seedbed. For
406 T. F. Döring

Table 13.1   Soil and water requirements and agronomic management of various grain legume
species. (Data sources: Water requirements: Sperber et al. 1988, Horneburg 2003, Kahnt 2008,
Guddat and Karalus 2009; soil conditions: Döring et al. 1956, Franzmann 1992, Kolbe et al. 2002,
Horneburg 2003, Guddat and Karalus 2009; sowing and harvest times: Schlipf 1898, Döring et al.
1956, Sperber et al. 1988, Franzmann 1992, Kolbe et al. 2002, Guddat and Karalus 2009, Hiltbrun-
ner and Kessler 2009, Rühl et al. 2009)
Species Optimal soil Water demand Sowing time Harvest time
conditions
Vicia faba Deep medium–heavy High–very high; Very early spring: 8-III.9
(spring) or light with good requires even III.2-II.3(-I.4)
water availability distribution
Vicia faba Similar to spring High–very high Autumn: I.8
(winter) form II.9–II.10
Pisum sativum Light–medium High Early: (I.3-) II.7–II.8
(spring) humus-rich soil; no II.3–II.4
waterlogging
Pisum sativum Autumn: II.9–I.11 7
(winter)
Lens culinaris Quick to warm up, Can tolerate rel. Spring: I4-III.4 7
can tolerate stony, dry conditions
shallow, nutrient
poor soil
Lupinus Medium soil Moderate Early spring: (I.3-) 8–9
angustifolius conditions, between II.3–I.4
Lupinus luteus and
Lupinus albus
Lupinus luteus Light soil, sand to Moderate Early spring: 8–9
sandy loam (II.3-)III.3–I.4
Lupinus albus Typically on loam Moderate Early spring: 8–9 (late)
II.3–I.4
Glycine max Quick to warm up, Low–moderate Late spring: 9
rich in humus, no II.4–I.5
water logging

example, early sowing of a main winter crop such as oil-seed rape (e.g. in late Au-
gust) requires early harvest of the preceding main crop with enough time to perform
the necessary tillage operations. As a pre-crop of oil-seed rape, faba bean with its
relatively long vegetation time (150–180 days in temperate climates), is therefore
not suitable under these conditions; in this case, an alternative crop would be pea
with its shorter vegetation time; however, plant pathogens for which both peas and
oil-seed rape are hosts mean that this may not be an optimal cropping sequence
either (see below).
Further, when determining the order of crops in the rotation, plant nutrition ef-
fects need to be taken into account, with particular reference to N. In particular,
grain legumes usually leave some N in the soil for the following crop (see below).
Therefore, a highly demanding crop species such as winter wheat should be grown
in the season after the grain legume, in order to make ideal use of the soil c­ onditions
left by the grain legume. Also, rotation designers should avoid stringing crop spe-
13  Grain Legume Cropping Systems in Temperate Climates 407

cies together that share the same pathogen or pest species. This means that in most
cases grain legumes need to be followed by crop species from a different plant
family.
Generally, rotation design often follows the rule to alternate species with con-
trasting properties. This can refer to various traits such as rooting depth, water re-
quirements, spring or autumn sowing and effects on soil organic matter. These is-
sues are discussed in the following two sections.

2.2  Effects of Grain Legumes on the Soil

The most important effect of growing grain legumes on the following main crop is
the enrichment of the soil with N. The symbiotic bacteria enabling grain legumes
to fix N2 from the air belong to various species of rhizobia and live within nodules
formed by the plant root. However, grain legumes do not exclusively rely on sym-
biosis for N acquisition; in particular, when the soil is high in mineral N, the propor-
tion of N derived from the atmosphere through symbiosis decreases.
The N taken up by the grain legumes, either with or without symbiosis, follows
three main routes. Part of the N is converted into grain protein and harvested with
the grain, that is, removed from the field. A second part is present in the form of
organically bound N in above-ground and below-ground plant residues. When these
residues decay after the death of the plant, the nitrogen is mineralised and can then
be taken up by the following crop. However, there is also the risk that the miner-
alised N is lost due to either leaching as nitrate (NO3−) or in gaseous form as N2O to
the air. Cropping systems therefore need to be designed in a way to minimise these
potential losses (Jensen et al. 2010). A third fraction of nitrogen is released by the
living roots of the grain legumes. Here, the nitrogen takes the form of low-molecu-
lar-weight chemicals (soluble root exudates, amino acids, hormones and enzymes),
and high-molecular-weight substances (from mucilage, dead cells, cell lysates and
decomposed root material). The N released from roots, termed N rhizodeposition,
can constitute a significant proportion of the N balance in crop rotations (Mayer
et al. 2003).
With regard to the N effects of grain legumes in the rotation, there are four key
findings of agronomic research. First, the N supply from grain legumes is, in most
cases, lower than from legume-based leys, for example, grass clover or lucerne-
based pre-crops (Hossain et al. 1996). This is mainly because of the large content of
N in the product of the harvested grain so that a large proportion of the N taken up
by the grain legume is eventually taken off the field and is therefore not available to
the next crop. Also, grain legumes are not as long in the field as the perennial forage
legumes in fertility building leys.
Second, as indicated above, there is a negative correlation between mineral N in
the soil and the proportion of N in the crop from biological N2 fixation (Schwenke
et al. 1998; Schmidtke and Rauber 2000). This means that N2 fixation is not only low
when mineral N is applied as fertiliser but also when large amounts of organically
bound N are microbially mineralised during the growth of the grain legume plant.
408 T. F. Döring

Third, the N balance after grain legumes may occasionally also be neutral or
even negative. The N balance depends on several factors, including the identity of
the crop species. Crops that are able to achieve high levels of N2 fixation, for ex-
ample, faba bean and field pea, have been found to be more likely to lead to positive
N balances, whereas grain legumes that achieve only modest levels of N2 fixation,
for example, chickpea and common bean, can be either N neutral or lead to decrease
of soil N (Walley et al. 2007).
Fourth, the N-fixing ability and other parameters determining the amount of N
available for the next crop are notoriously variable and difficult to estimate. This
poses a serious problem for the evaluation of the pre-crop value of grain legumes.
This variability, evident from individual studies (Evans et al. 1989; Walley et al.
2007; Urbatzka et al. 2011) and from compilations in handbooks for practitioners
(Sperber et al. 1988; Franzmann 1992; Kolbe et al. 2002; Horneburg 2003), makes
it extremely difficult to quantify expected effects of grain legumes on the following
crop and give recommendations on the choice of legume species. One reason for
the high variability is that the amount of N released from roots (rhizodeposition) is
extremely difficult to quantify (Mayer et al. 2003); a further problem is the dynamic
response of N2 fixation by grain legumes to the mineral N fraction in the soil.
Several approaches have been developed for estimating the N fixed by grain
legumes at the farm level, in order to support rotation planning. However, some
models widely used in practice have been shown to be extremely poor in terms
of their ability to reproduce experimentally measured values. One model based
on grain yield and a species-specific N2 fixation factor was demonstrated to be
particularly poor, as it systematically overestimated N balances at low levels and
underestimated true values at high levels (Kolbe 2009). More accurate estimates
were obtained by using nonlinear functions and a larger range of input variables
including the grain legume species, grain yield, soil Nmin in spring and the N harvest
index (Kolbe 2009).
Apart from effects on soil N dynamics, grain legumes have several other effects
on the soil, thereby influencing the following crop. In particular, grain legumes im-
prove soil tilth and soil structure by their root systems. For example, in experiments
on the effects of various grain legume pre-crops of cotton, it was found that penetra-
tion resistance of the soil was lower after most grain legume pre-crops (including
faba bean and field pea) than when cotton was used as a pre-crop. However, effects
of soybean–cotton rotation in comparison with the cotton–cotton rotation yielded
inconsistent results with regard to penetration resistance (Rochester et al. 2001).
Grain legumes may also affect soil structure indirectly through accumulation of
soil organic matter. Depending on the amount of above- and below-ground plant
residues left after harvest, grain legumes may contribute to the maintenance and
enrichment of soil organic matter (humus). Grain legume species are generally
thought to be moderately positive with respect to soil organic matter balance in
the rotation. When the grain is harvested, their positive effects on soil humus are
lower than of grass–clover leys. A further effect of grain legumes on the soil is the
potential to mobilise nutrients from deeper layers of the soil, particularly in species
with relatively deep tap roots, such as faba beans and lupins. In contrast, lentils, for
example, have a relatively weak root system (Horneburg 2003).
13  Grain Legume Cropping Systems in Temperate Climates 409

2.3  Effects of Grain Legumes on the Following Main Crop

In most cases, cumulative effects of grain legume pre-crops on the yield and
quality of following cereals have been reported to be positive (e.g. Kirkegaard
et al. 2008). Grain legumes are usually recommended as good pre-crops for winter
cereals, ­especially winter wheat and winter triticale, but also for maize. Early-sown
crops such as winter rape and winter barley can be grown after peas. In a study
from Germany, yields of winter wheat were on average 0.92 t ha-1 higher after
grain legume pre-crop than after a cereal pre-crop (Albrecht and Guddat 2004). In
relative terms, yield increases were 5.6–18.2 % in winter wheat and 8.7–28.7 % in
other cereals. These values are similar to those obtained in the northern Prairies
where wheat and barley grain yields increased by 12–21 % after grain legume pre-
crops in comparison to cereal pre-crops (Wright 1990). However, it is probably
more appropriate to compare grain legume pre-crops with other noncereals when
assessing the rotation effects on cereals. In a French study, reporting the results
of 17 field trials, it was found that yields of winter wheat following either peas
or oil-seed rape did not respond significantly to the pre-crop species in 14 out of
the 17 trials (Plancquaert and Desbureaux 1985); out of the remaining three cases,
significant differences in wheat grain yields favoured peas as pre-crops in one case
and oil-seed rape in two cases.
Pre-crop effects of grain legumes are highly context dependent. For example,
the pre-crop benefit of grain legumes has been found to be higher in organic than
­conventional systems (Albrecht 2002); in line with this finding, a series of field tri-
als in East Germany showed that the effect of the grain legume pre-crop, measured
as the relative increase of yield in the cereal, decreased with increasing yield level of
the cereal. That is, when the cereal yield was close to its optimum, additional gains
obtained from a grain legume pre-crop were small (Albrecht and Guddat 2004).
Similar results were obtained in a long-term trial in the UK, testing pre-crop effects
of faba beans at different N levels (Dyke and Prew 1983). Thus, when comparisons
are made between treatments with optimal fertiliser levels, pre-crop effects of grain
legumes may disappear. This was observed for cotton lint yields where grain le-
gume pre-crops had no significant effects at optimal fertiliser level (Rochester et al.
2001). The pre-crop effects of grain legumes are considered to be mainly based on
N dynamics. However, there are also additional effects on soil-borne pathogens of
cereals (e.g. take-all, Gaeumannomyces graminis var. tritici) and on soil structure
(see above).
Unfortunately, effects of grain legumes on the stability and resilience of the fol-
lowing crop (e.g. cereals) have not been subject to extensive research so far. Effects
of grain legume pre-crops on the yield of the following cereal could be stabilising or
destabilising. On the one hand, it could be expected that a cereal following a grain
legume would be more stable than when following a cereal, because, after a cereal,
the unpredictable build-up of soil-borne cereal diseases may destabilise yields in
comparison to a grain legume pre-crop. On the other hand, however, the variable
and weather-dependent N availability after grain legumes may mean that stability
of the following cereal grain yield may actually be lower than after other pre-crops.
410 T. F. Döring

In accordance with these contrasting predictions, a long-term trial at Broadbalk

(Rothamsted, UK) found effects of a faba bean pre-crop on yield variability of
wheat over 10 years to be small and inconsistent (Dyke and Prew 1983); for ex-
ample, a measure of yield variability (the ratio of largest to smallest wheat yields)
was higher in a wheat–wheat system than in a bean–wheat sequence when farmyard
manure was applied (1.8:1 and 1.7:1, respectively), but the order was reversed when
mineral fertiliser was applied (1.4:1 and 1.7:1 respectively). In light of these incon-
clusive results, it is clear that more research is needed to clarify the question of grain
legume effects on yield stability in the rotation (see Sect. 5).

2.4  Selecting Pre-Crops for Grain Legumes

Grain legumes are generally not considered to be particularly demanding with

­respect to pre-crops. They can be grown after crops with high N demand, for exam-
ple, recommended pre-crops before faba beans include winter wheat, winter barley
and silage maize (Freyer 2003). As mentioned before, however, grain legumes are
self-intolerant, that is, they should not directly be grown in successive years. Yield
losses in direct succession of grain legumes have been reported to be substantial,
for example, 22 % in faba beans and 29 % in field peas in comparison to nonlegume
pre-crops (Bachthaler cited in Freyer 2003). Other sources report yield reduction in
peas of 25–50 % (Alpmann and Schäfer 2014). In a comparison between growing
peas every 2 years and every 4 years in a total of seven environments, it was found
that peas in the 2-year pattern had on average a grain yield reduction of −24.5 %
compared with peas in the 4-year pattern (Plancquaert and Desbureaux 1985; calcu-
lated by the author from published data). This yield reduction was mainly attributed
to soil-borne pea diseases ( Peronospora and Ascochyta). However, yield effects of
the number of break years ranged considerably around this mean, namely between
− 89 and + 19 %. Also, the effects of the number of break years were significant in
only three out of the seven environments.
One of the main reasons for required break years is the build-up of soil-borne
fungal pathogens over time. These can dramatically impact on the establishment
of the crop and therefore can have severely yield-reducing effects (Stoddard et al.
2010). For example, in faba bean, the minimum number of break years required to
reduce the risk of fungal infections to tolerable levels is considered to be 4 years
for Ascochyta blight, Cercospora leaf spot, chocolate spot (Botrytis fabae), and
faba bean rust (Uromyces viciae-fabae). Substantially longer intervals suggest-
ed for the control of powdery mildew, that is, at least 10 years, are unlikely to
be ­practical though (Stoddard et al. 2010). Peas and faba beans share the fungal
pathogen Sclerotinia sclerotiorum with oil-seed rape (canola, Brassica napus)
and sunflowers (Helianthus annuus), and direct successions of these crops should
therefore be avoided. Also, in rotations that include oil-seed rape and pea, popula-
tions of the fungal pathogen Fusarium avenaceum are believed to build up over
time (Feng et al. 2010).
13  Grain Legume Cropping Systems in Temperate Climates 411

In addition, weed levels may increase as well if breaks between grain legumes
are too short. Most grain legume species are prone to weed infestation because of
the relatively wide row width and comparatively low plant densities in currently
practiced cropping systems. Especially peas, soybeans and lentils are mentioned as
species with low competitive ability against weeds. Vulnerable stages include both
early development and late growth stages when seeds mature, and the ­vegetative
parts of the crops are dying off. Therefore grain legumes should be grown after
crops that have a high competitive ability against weeds or are at least complemen-
tary in their typical weed species community. When spring-sown grain legumes fol-
low a winter cereal, it is recommended to insert a highly competitive green ­manure
between the harvest of the cereal and the following legume in order to ­suppress
weeds. Weed reduction is also the main motivation for selecting mechanically
weeded row crops such as potatoes and maize as pre-crops before grain legumes.
For example, harrowed row crops have been recommended as suitable pre-crops
before lentils (Horneburg 2003). However, if both grain legumes and preceding row
crops are sown in spring, this direct succession might pose a risk of accumulating
weed species adapted to spring sowing. Of particular relevance for weed control in
grain legumes are broomrapes ( Orobanche sp.). However, because of long viability
of the seeds of these parasitic weeds and due to the broad host range of Orobanche,
crop rotation is of limited effect in this case (Stoddard et al. 2010).
Unfortunately, the most weed-suppressing cereal species, oats or rye, are not
ideal as pre-crops of grain legumes because both are hosts for plant parasitic nema-
todes that also infect grain legumes (Sperber et al. 1988; Freyer 2003). This view is
not unanimously shared and some authors do recommend rye and oats as pre-crops
of grain legumes (Kolbe et al. 2002). In any case, however, attention should be paid
to the host ranges of plant parasitic nematodes when selecting the pre-crop for grain
legumes. Further, in terms of the spatial planning of the rotation, it is also necessary
to avoid growing grain legumes as direct neighbours because of the risk of mobile
pest insects and insect-transmitted plant viruses (Jones et al. 2008).

2.5  Grain Legumes, Cover Crops and Green Manures

Cover crops have several functions in cropping systems (Clark 2008). Primarily,
they fill gaps in otherwise vegetation-free periods of the rotation, covering the soil
in the intervals between main crops. They help to reduce soil erosion, improve soil
structure and increase soil biological activity. They can contribute to the reduction
of weeds, soil-borne plant diseases and pests. For example, some brassica crops are
used for biofumigation to reduce soil-borne fungal diseases and nematodes (Larkin
and Griffin 2007). However, in organically and conventionally managed field tri-
als, it was recently found that a biofumigation crop (Brassica juncea) did not have
any significant effect on foot diseases or establishment in faba beans and peas and
effects on yields remained inconsistent (Jacob et al. 2014). Apart from this inves-
tigation, however, there is currently little experience with biofumigation in grain
412 T. F. Döring

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Fig. 13.3   Examples of simultaneous integration of grain legumes (GL) and cover crops (CC) into
rotations with nonlegume main crops (MC), for example, winter cereal and stubble (S); a spring-
sown grain legume following a cover crop. b Autumn-sown grain legume following a cover crop.
c Cover crop undersown into a spring-sown grain legume. d Summer-sown cover crop following
an early-harvested grain legume

legumes. More research is therefore necessary to explore the potential of cover

cropping for disease reduction in these crops.
A further function of cover crops, in case of leguminous species, is to increase N
levels in the soil for the subsequent crop, thereby acting as a green manure. Finally,
nonleguminous cover crops may be used to reduce the risk of N losses from the soil.
As catch crops, their main role is to retain the N and prevent it from being leached
or volatilised. After the catch crop has died off, the retained N is then released again
to be taken up by the next main crop.
The combination of grain legumes and cover crops can take several forms in the
rotation (Fig. 13.3). The cover crop can precede the grain legume, be grown simul-
taneously, or follow the harvest of the grain legume. The first option of a cover crop
preceding a grain legume applies mainly to spring-sown grain legumes following
a main crop that is harvested in the summer of the previous year (Fig. 13.3a). It is
particularly relevant in regions where autumn-sown forms of grain legumes cannot
be used because of harsh winters. Here, a cover crop will be essential because of the
long period that the soil is not covered by main crops. But also with autumn-sown
grain legumes, cover crops can be used when the preceding main crop is harvested
relatively early, that is, as in barley (Fig. 13.3b).
Further, cover crops can be undersown into grain legumes (Fig. 13.3c). For ex-
ample, faba beans can be undersown with brassica crops such as mustard in order
to keep soil nitrate levels low in the autumn (Köpke 1998). Thereby, losses of grain
legume-derived N from the soil are reduced. An underlying mechanism of this ef-
fect is that the nonleguminous companion crop of the grain legume has a wider
C to N ratio than the grain legume, so that the decomposition of plant residues in
the soil is slowed down in comparison to a pure grain legume crop. Also, trials
have been conducted on undersowing grass such as cocksfoot (Dactylis glomerata)
into young faba beans; after harvesting the faba beans, the grass was then kept
in the field either until winter wheat was sown in the autumn or until maize was
established in the following spring. While the grass did not have any effects on
the yield of the beans, there were effects on yields of the following crops. In the
above-ground biomass of the grass 20–30 kg ha−1 N were bound, but the release
13  Grain Legume Cropping Systems in Temperate Climates 413

of the retained N was r­elatively slow. This explains why the directly following
crop showed reduced yields in the undersown treatment, whereas increased main
crop yields were observed in year after that (Gröblinghoff et al. cited in Zerhusen-
Blecher and Schäfer (2013)). For undersowing, it is recommended to use a slow-
growing-partner variety with a small height, high tolerance against low light levels
and high diseases resistance levels.
A different function of undersowing in grain legumes is the suppression of
weeds. Experiments on subterranean clover ( Trifolium subterraneum, at 2000
seeds m−2) undersown into peas (90 seeds m−2) concluded that weed suppression
was an ­important benefit of this system. However, there was no effect on the yield
of the following wheat, nor on N2 fixation of the pea. Also, effects of undersowing
on weeds already disappeared in the following wheat crop (Köpke et al. 2011).
The risk of postharvest N losses can be reduced by growing catch crops after
grain legumes and before the next main crop (Fig. 13.3d). For example, because of
the relatively early harvest date of peas, it is useful to plant a fast-growing summer
catch crop such as mustard after the peas and before the following autumn-sown
wheat. The practice of following a cover crop after grain legume harvest is particu-
larly important when the period between harvesting the grain legume and sowing
the next main crop is characterised by high precipitation as it is often the case when
the main crop after a grain legume is spring sown. In this case, it is a recommended
practice to plant a catch crop that is killed by frost in the winter. The dead mulch can
then be incorporated into the soil in spring.
A major problem when combining grain legumes and leguminous cover crops
is that there is currently insufficient knowledge about how the two components
­interact in the field. In particular, host ranges of fungal and viral pathogens as
well as nematodes infecting both grain legumes and leguminous cover crops are
currently not well enough characterised to evaluate in how far the integration of
cover crops into grain legume cropping systems poses phytopathological risks.
One example of a fungal pathogen infecting both grain legumes and forage le-
gumes is Fusarium solani which attacks common bean and pea but also white
clover (Trifolium ­repens).

3  Growing Schedule for Grain Legumes as a Sole Crop

3.1  Sowing and Inoculation

Sowing times affect grain legumes in cropping systems in a number of ways. In

temperate climates, sowing times primarily depend on the frost hardiness and
minimal temperature required for germination, but water availability also plays a
role for choosing the right sowing time; in particular, because of their high seed
weight, grain legumes have a relatively high water demand at germination. Gener-
ally, e­ arlier sowing results in higher grain yield of the grain legume, for example,
as observed for autumn-sown chickpeas (Horn et al. 1996). This effect is mainly
414 T. F. Döring

due to the l­ onger time period available to the plant at early sowing. For instance, in
comparison to other grain legume species, faba bean as a long-day plant species has
a relatively long vegetation period (150–180 days) and therefore needs to be sown
particularly early (Table 13.1). Insofar as sowing times affect the amount of grain
legume biomass and of the nitrogen fixed, sowing times can affect the yield and
quality of a subsequent cereal crop (Heenan 1995). Early sowing can also be ben-
eficial in terms of weed control, because of stronger competition of the crop against
weeds. On the other hand, however, sowing later, that is, waiting until the soil tem-
perature is higher often results in more uniform and stronger crop emergence. This
can be of particular importance in organic systems. Also, sowing too early entails
the risk of damaging the soil when soil moisture levels are too high. With vulner-
able soils, it is therefore better to wait until later in spring. As lighter soils are less
prone to waterlogging and warm up more quickly, it is recommended to sow earlier
in lighter soils and later in heavier soils.
Sowing time can also be a potential tool for aphid and virus control. For ­example,
it is recommended to sow early so the plants are well developed before aphids start
to colonise the crop. However, in a study on faba beans in Germany, aphid infesta-
tion was not consistently reduced through early sowing; nevertheless early sowing
­resulted in lower virus incidence, possibly because of stronger mature plant resis-
tance in the early- than in the late-sown beans (Saucke et al. 2009). While grain
legumes grown to maturity are typically sown in the spring or autumn, summer
sowing is possible if they are used as cover crops. For example, a lupin cover crop
may be sown directly after the cereal harvest and can then be kept until the follow-
ing main crop is sown in the autumn or the next spring.
Sowing depth mainly depends on seed size and therefore recommendations vary
considerably, both among and within grain legume species (Fig. 13.2c). Generally,
species with hypogaeic germination (faba bean, pea and vetch) should be sown
deeper, whereas species with epigaeic germination (soybean, common bean and
lupin) require shallower sowing. Deeper sowing is known to reduce bird damage,
leads to lower damage from mechanical weed control, and it also reduces lodging
risk. As for all crops, it is important to achieve even sowing depth.
Optimal seed density is, at least theoretically, mainly a function of plant size, and
plant spacing should be as even as possible to optimise resource use and minimise
competition (Weiner et al. 2010; Olsen et al. 2012). As for sowing depth, ranges
for recommended seed densities vary (Fig. 13.2d). Generally, there is scope to in-
crease plant density above currently recommended levels in order to achieve high
weed suppression. However, the success of these “high-density” cropping systems
depends on high spatial uniformity, and appropriate genotypes to avoid that plant
resources are allocated towards individual competitiveness (shade avoidance) and
away from grain yield (Weiner et al. 2010). In practice, however, seed densities are
also bound by row width, which in turn depends on the sowing and weeding technol-
ogy available on the farm. Row widths recommended for grain legumes are wider
for soybeans (45 cm) and faba bean (30–40 cm) and narrower for lentils (15–25 cm
Horneburg 2003), peas (10–15 cm Sperber et al. 1988) and lupins (­12–24 cm). If
the crop is too dense, it is more sensitive to drought and produces more vegetative
13  Grain Legume Cropping Systems in Temperate Climates 415

growth and a lower number of pods. Plants in high seed densities are taller, which
can increase the risk of lodging, and there is also a higher risk of plant diseases
(Sperber et al. 1988) because of increased humidity and smaller ­distance between
plants. However, if densities are too low, yield potential is lowered because the
number of pods produced during the growing season cannot compensate the low
plant density. In addition, there is a higher risk of early weeds competing with the
young plants; finally, low seed density can lead to a longer flowering time and un-
even maturity (Sperber et al. 1988).
Because of the specific combinations of grain legume species and rhizobial
­species, inoculation of grain legume seed with rhizobia is recommended when the
naturally occurring rhizobia populations are likely to be low or absent from the
­target site. In fact, it was already recommended in the nineteenth century to in-
oculate soil where legumes do not grow by sprinkling some soil onto it from a
site where legumes do grow (Schlipf 1898). However, experimental evidence from
Canada shows that inoculation with rhizobia increased seed yield of pea only in
the minority of field trials (9 out of 22). At the same time, though, the study also
showed that the inoculation benefit was more than three times greater on fields with
no history of legumes than on fields with previous legume cropping (McKenzie
et al. 2001). From a practical point of view, it is possible for farmers to test the pres-
ence of suitable bacteria by growing grain legumes in pots; investigating the pres-
ence of root nodules can then be used as an easy diagnostic tool. More recently, it
has been shown that co-inoculation of legumes with rhizobia and mycorrhiza can be
beneficial for crop growth under low P and/or low N conditions (Wang et al. 2011).
However, more research is needed to elucidate the complex interactions between
these symbionts and the crops, as well as the applicability in practice.

3.2  Nutrient Management and Irrigation

Nutrient management in grain-legume-based cropping systems comprises four key

aspects. These are (1) nutrient requirements of the different grain legume species,
(2) effects of grain legumes on the nutrition of other crops, of either intercropping
partners (see Sect. 4.1) or the following crops in the rotation (see Sect. 2.3), (3) ef-
fects of other crops on the nutrition of grain legumes and (4) the interplay between
grain legumes and organic manure. In discussing these issues, this section mainly
concentrates on the key nutrients N and P. In addition, the dependency of grain le-
gumes on soil pH is briefly conferred.
Nitrogen fertilisation of grain legumes is usually not economic (Freyer 2003)
partly because N2 fixation is impeded by high levels of mineral N in the soil,
though, in a study with faba bean, it has been shown that N2 fixation is not strongly
suppressed at high Nmin levels (Hardarson et al. 1991). When applying organic fer-
tilisers to grain legumes, it is recommended to use materials with low availability
of N, for example, well-rotted farmyard manure or compost, whereas the applica-
tion of slurry is not advisable. Conversely, grain legumes provide a source of N
416 T. F. Döring

which can be brought back to the field as organic manure in the nonlegume parts
of the rotation. Usually, the organic manure is derived from livestock production,
that is, the grain legumes are fed to the animals. However, grain legume seeds can
also be processed into a grist to be used directly as organic manure in horticultural
(Heuberger et al. 2005) or agricultural (Heinze et al. 2011) crops. Faba bean grist
usually contains about 3–5 % N (Heuberger et al. 2005; Raupp 2005; Heinze et al.
2011). Grain legume grist has been suggested as a replacement of animal-derived
manure on organic stockless farms. Although long-term effects of this vegetal fer-
tiliser are yet to be determined, available evidence suggests that crop yields are not
significantly different between farmyard manure application and grain legume grist
fertilisation when N levels of both are comparable (Oltmanns and Raupp 2006;
Heinze et al. 2011).
Unfortunately, the N autarky of grain legumes comes at a cost. In particular,
symbiotic N2 fixation requires relatively high levels of phosphorus for nodule
­development and function (Cassman et al. 1981). In fact, N2 fixation is sensitive to P
deficiency (Tang et al. 2001; Olivera et al. 2004). Although grain legumes are gen-
erally known for their relatively good nutrient scavenging ability, P can be ­limiting
in young grain legume plants, and the P mobilizing efficiency can be low until
relatively late in the season (Kolbe et al. 2002). P acquisition and use in grain le-
gumes is based on several different mechanisms. These include acidification of the
rhizosphere through the release of organic acids; exudation of phosphatase, changes
of root architecture at low P levels, for example, the formation of proteoid roots in
lupins, improved transport of P and use efficiency, and symbioses with mycorrhiza
(Graham and Vance 2003). The specificities of mycorrhizal symbiosis can also in-
form the design of the crop rotation. For instance, it needs to be taken into ­account
that brassica crops do not form symbiotic relationships with mycorrhiza, and some
brassica cover crops are known to reduce mycorrhiza populations in the soil (Larkin
et al. 2011). Furthermore, different legume species differ in which ­fractions of P
they can access (Rose et al. 2010).
Nutrient management of both N and P are closely linked to the soil pH. Grain le-
gume species are known to differ in their requirements regarding soil pH (Fig 13.2a).
Optimal ranges of soil pH in grain legumes are partly a result of the symbiotic rhi-
zobia, which are sensitive to low pH. At high pH, grain legumes can suffer from Ca
chlorosis, which can lead to plant death in extreme cases. Through the process of
N2 fixation, grain legumes normally reduce the pH in the rhizosphere (Nyatsanga
and Pierre 1973; Hinsinger et al. 2003), but alkalization of the rhizosphere has also
be observed in grain legumes (Betencourt et al. 2012b). Changes in the soil pH
strongly affect the availability of various macro- and micronutrients. Soil acidifica-
tion caused by legumes can be partly compensated for by returning crop residues to
the soil (Yan et al. 1996).
Grain legumes, and especially faba beans with their long vegetation time, are
known to be drought sensitive during and after flowering. Therefore, irrigation
­directly before and during flowering can have positive effects, in particular, when
the dry period is before flowering. However, in temperate climates, irrigation in
13  Grain Legume Cropping Systems in Temperate Climates 417

peas before flowering has been shown not to affect grain yield (Sperber et al. 1988),
even in dry years. On the other hand, irrigation can also have negative effects on
grain legumes. In particular, late irrigation can lead to lodging, and this may reduce
grain yields.

3.3  Tillage and Weed Control

Cropping systems are intricately linked with issues of tillage. Broadly, three
forms of tillage can be distinguished that are relevant for cropping systems. In
conventional tillage (CT) systems, ploughing is used prior to the preparation of
the ­seedbed. With reduced tillage (RT), mechanical disturbance of the soil is not
as strong as with CT, either by reducing the depth of tillage or by leaving strips
untilled. In no-till systems (NT), the seed is directly drilled into the untilled soil.
Three major aspects of tillage are of particular relevance in grain legume cropping,
namely the mineralisation of soil organic nitrogen, water supply for the crop and
weed control.
In principle, it can be expected that decreased tillage intensity leads to lower
amount of available N, because of lower rates of mineralisation and nitrification,
and because of increased N immobilisation. Reducing tillage intensity can also
­increase available soil water. It is therefore reasonable to assume that both effects
together will stimulate N2 fixation in grain legume under RT. However, in a long-
term experiment conducted in southern Spain, the effect of no tillage versus CT on
N2 fixation in faba bean was not significant. In particular, there were no significant
effects of the tillage system on the percentage of nitrogen derived from the atmo-
sphere or on the total amount of N2 fixed (López-Bellido et al. 2006).
Weed infestation in grain legumes causes several problems. Most importantly,
they compete with the crop for water, light and nutrients, thereby leading to re-
ductions of grain yield. In organic cultivation, especially late weed infestation is
­considered to be a major problem in grain legumes. In addition to direct effects
of competition, weeds also indirectly affect grain legumes through uneven and
late maturation of crops and lower harvestability. Further, cleaning of the har-
vested seeds is necessary at high weed infestation levels, and higher moisture of
grain from weedy fields requires longer drying after harvest. While the competi-
tive ­ability of grain legumes early in the season is thought to be relatively strong,
weeds such as Chenopodium album can thrive later in the season when the crop
is senescing. ­However, also early weed infestation can be severe, especially when
soybeans are grown in colder climates where the crop’s early development is slow.
In Central E ­ urope, lentils are considered to be particularly weak competitors (Hor-
neburg 2003). Problem weeds in lentils include wild oats (Avena fatua) and cleav-
ers ­ (Galium aparine, on more fertile soils). However, in some regions, lentils, as a
late closing spring crop on nutrient-poor soils, also act as habitat for weed species
with nature conservation value, such as Adonis aestivalis, Caucalis platycarpos and
Misopates orontium (Horneburg 2003).
418 T. F. Döring

Because of the general vulnerability of grain legumes to weed infestation,

p­rophylactic mechanical weed control is considered to be necessary in many grain-
legume-based cropping systems. For weed control reasons, ploughing before grain
legumes is usually preferred over RT. If grain legumes are sown in spring, plough-
ing in autumn is recommended, because spring ploughing delays the sowing date,
leading to higher water losses in the soil than autumn ploughing, and is also thought
to be inferior in terms of weed control. NT systems are widespread in soybean
growing but rely heavily on the application of nonselective herbicides that have
been criticised because of developing resistance in some weed species (Waltz 2010)
and toxicity (Gasnier et al. 2010; Romano et al. 2010).
In order to increase crop competitiveness of grain legumes, a uniform seed-
bed is needed, and some time should be left for the soil to settle after ploughing.
­Mechanical weed control during the growing season can be done with various tech-
niques, including tine harrowing and ridging (Kolbe et al. 2002). Specific tech-
niques ­mainly depend on the growth stage of the grain legume and the row width.
­However, it should be observed that mechanical weeding might contribute to the
spread of ­fungal diseases in the crop, for example, anthracnose in lupins (Kolbe
et al. 2002). In addition to mechanical weeding, rotational means to keep weed
levels at bay in grain legumes, for example, by growing a tall competitive cereal
such as oats before or after the grain legume. Further, variety selection can also
contribute to reduce weed problems. Generally, it is advised to select tall early ma-
turing grain legume varieties with low lodging risk (Kolbe et al. 2002). In peas,
semi-leafless types have been shown to be worse competitors than full-leaf types
(Urbatzka et al. 2013), though semi-leafless peas are better for mutual support and
reduced risk of lodging (Sperber et al. 1988).

3.4  Pest and Disease Control

As in virtually all crops, pests and diseases pose great challenges to the production
of grain legumes (Emden et al. 1988). Pests in grain legumes are manifold. Insect
pests with high economic importance include the pea moth ( Cydia nigricana F.) in
peas, bruchid beetles such as Bruchus rufimanus, and Acanthoscelides obtectus, and
the weevil Sitona lineatus. Several aphid species infest grain legumes, including the
black bean aphid (Aphis fabae) and the pea aphid (Acyrthosiphon pisum). Further,
the soybean aphid (Aphis glycines) has caused substantial plant protection and pest
monitoring costs after it was accidentally introduced to North America (Ragsdale
et al. 2011). Among soil-borne pests, nematodes play a key role in limiting grain
legume production. For instance, in peas, the nematodes Ditylenchus dipsaci and
Heterodera göttingiana can cause severe damage. Finally, grain legumes are also
vulnerable to bird damage. Specifically, birds such as pigeons ( Columba palumbus
L.), carrion crows ( Corvus corone L.) and jackdaws ( Corvus monedula L.) may
often damage the germinating seed, breaking the young plant. Periurban areas are
especially vulnerable to bird damage (Kolbe et al. 2002).
13  Grain Legume Cropping Systems in Temperate Climates 419

Grain legumes are also affected by a large number of plant diseases including
plant pathogenic viruses such as the Bean leaf roll virus and the Pea enation mosaic
virus. Fungal diseases of high importance include Fusarium species, Pythium, As-
cochyta, as well as the leaf diseases false mildew (Peronospora viciae) in peas and
faba beans, Botrytis cinerea in peas and chocolate spot (Botrytis fabae) in faba bean.
The seed-borne fungal disease anthracnose, caused by Colletotrichum acutatum has
had devastating effects on lupin farming. In Germany, it affected the previously
preferred lupin species (white and yellow lupin) more than the narrow-leafed lupin
(L. angustifolius). Accordingly, the disease led to a complete change of lupin spe-
cies grown in the country.
Major efforts are being made to develop grain legume varieties with resistance
or tolerance to pests and diseases. However, both pests and diseases of grain le-
gumes can also be reduced by adjusted management of the cropping system. Such
indirect control measures include (1) crop breaks in the rotation (see Sect. 2.1);
(2) keeping large distances between fields where grain legume crops are grown,
or had been grown in the previous year; (3) keeping distance to forage legumes
such as lucerne and clover both in time and space; (4) deep incorporation of plant
residues before sowing to reduce fungal infection risk; (5) aiming for uniform crop
development, uniform flowering and maturation by diligent seedbed preparation,
and moderate row width; (6) using certified seed against seed-borne diseases; (7)
sowing early against pest infestation and virus transmission but avoiding very early
sowing into cold soils to reduce the risk of fungal infections and (8) using moderate
plant densities.

4  Agronomy of Grain Legume Intercropping

4.1  Benefits of Intercropping Grain Legumes

From various systems, it is known that increased plant diversity in the field has
multiple benefits (Cardinale et al. 2011; Döring et al. 2012; Costanzo and Bàrberi
2014), including increased productivity (Tilman et al. 2001), reduction of pests and
diseases (Finckh and Wolfe 2006), better resource use, and higher yield stability
(Tilman et al. 2006). As described in this section, the scientific and applied litera-
ture has confirmed that these advantages can also be observed when increasing the
diversity in grain-legume-based cropping systems, either by mixing them with other
species (intercropping; Hauggaard-Nielsen et al. 2008) or by using intraspecific
diversity in the field (cultivar mixtures and populations; Pyndji and Trutmann 1992;
Atik et al. 2012).
Cultivar mixtures in grain legumes have received relatively little attention from
research so far. In contrast, numerous intercropping combinations involving grain
legumes have been tried in research and practice. Combinations of grain legumes
and cereals include spring-sown faba bean with spring oats (Helenius and Jokinen
1994; Kahnt 2008), spring barley (Schlipf 1898; Agegnehu et al. 2006; Kahnt 2008)
420 T. F. Döring

and spring wheat (Bulson et al. 1997; Wolfe et al. 2013); field pea with spring oats
(Schlipf 1898; Zimmermann 1958; Rauber et al. 2001; Kolbe et al. 2002; Kahnt
2008; Urbatzka et al. 2011) or spring barley (Schlipf 1898; Jensen 1996; Haug-
gaard-Nielsen et al. 2001; Kolbe et al. 2002; Kahnt 2008); lentil with rye, spelt, oats
or barley (Schlipf 1898; Horneburg 2003); lupins with oats or rye (Zimmermann
1958), winter-sown pea with winter rye (Urbatzka et al. 2011); summer vetch with
spring oats (Kahnt 2008; Böhm 2013), or chickpea with durum wheat (Betencourt
et al. 2012a). Grain legumes have also been intercropped with oil crops, for ex-
ample, in combinations of faba bean and oil-seed rape (Jamont et al. 2013) or field
pea with false flax (Saucke and Ackermann 2006), and with grasses (Franzmann
1992; Kolbe et al. 2002). Finally, combinations of two grain legume species, such as
faba bean and field pea (Zimmermann 1958; Kolbe et al. 2002; Kahnt 2008), winter
faba bean with winter vetch (Kahnt 2008), or summer vetch with white or blue lupin
(Kahnt 2008) have been trialled.
Already in the nineteenth century, it was suspected that intercropping grain
­legumes with nonlegumes leads to more efficient resource use through comple-
mentation (Schlipf 1898). This view has largely been confirmed by research, for
example, for nitrogen use in various intercropping systems. In a mixture of peas and
oats, it was found that a higher proportion of N was derived from the atmosphere
by the intercropped pea than by the sole cropped pea. When intercropped with pea,
the oat plants took up more soil N from deeper layers. Thus, the N leaching risk was
lower after the intercrop than after the monocropped pea (Neumann et al. 2007).
This is supported by other studies reporting that N use is more efficient in the grain
legume–cereal mixtures and the N balance in the soil is closer to zero, that is, there
is less over- or undersupply (Hauggaard-Nielsen et al. 2008).
Although the intercropping partners also compete for resources, there is less niche
overlap than in monocultures. In addition to niche separation among the intercrop-
ping partners, resource use of one partner can also be facilitated by the other. The
most significant mechanism of facilitation in intercropping grain legumes is that ni-
trogen fixed by the legume may be transferred to a nonlegume intercropping partner.
Evidence for such N transfer has been found in several intercropping including grain
legumes and cereals (Aufhammer 1999). For example, N transfer was observed from
soybean to sorghum, in particular, when the planting pattern was such that the dis-
tance between the partners was low (Fujita et al. 1990); However, there have also
been cases where N transfer was not significant, for example, in an experiment study-
ing N-transfer from pea to barley (Jensen 1996) or in an intercropping system with
soybean to maize (Hamel et al. 1991). Surprisingly, N transfer can also take place
in the opposite direction. In a study testing intercropping rapeseed and faba bean in
rhizotrons, N was transferred in both ways, from faba bean to rapeseed and vice versa
(Jamont et al. 2013). Generally, the percentage of N derived from fixing is greater in
intercropping than in monoculture grain legumes (Jensen 1996).
Facilitation in intercropping has also been observed for phosphorus. For exam-
ple, in a mixture of white lupins and spring wheat grown in pots with and without
root contact between the partners, it was found (Horst and Waschkies 1987) that the
lupin increased availability of soil phosphorus through exudation of organic acids.
13  Grain Legume Cropping Systems in Temperate Climates 421

In particular, the lupin made three times more P available than it needed; as a result,
there was an increased yield of the mixture on P-deficient soil (dry matter of wheat
doubled). Another more recent example is an experiment on a chickpea–durum
wheat intercrop which highlighted the nutrient mobilizing ability of grain legumes
and their ability to make P better available for other crops (Betencourt et al. 2012a);
in this experiment, the intercropping of chickpea and durum wheat in a P-deficient
soil resulted in higher durum wheat biomass per plant compared to the monocrop,
whereas chickpea was not affected significantly. In the P-deficient soil, intercrop-
ping also led to significantly higher levels of P (water extracts and Olsen extracts)
in the rhizosphere than in the sole crops.
A further important benefit of intercropping grain legumes is improved weed
control. For instance, better weed suppression was observed when autumn-sown
faba beans were intercropped than when wheat or beans were sown in monoculture
(Wolfe et al. 2013). Similar observations of weed suppression in intercrops com-
pared to monocrops were made for lentil intercropping (Horneburg 2003) and peas
and false flax ( Camelina sativa; Saucke and Ackermann 2006). In a field experi-
ment, weed cover was strongly reduced in the intercrop in comparison to the mono-
crop of peas, but only at one of two sites in comparison to the other partner, false
flax ( Camelina sativa; Paulsen et al. 2006). A further consequence of better weed
suppression in the intercrop is that soil inorganic N is used for grain production of
the nonlegume intercropping partner instead of weed biomass (Hauggaard-Nielsen
et al. 2001).
In many cases, grain legumes are grown in intercropping systems with cereals
to reduce the risk of lodging; for example, a cereal can physically support a pea or
lentil crop which can use its tendrils to climb. This also facilitates the harvesting
process as the lentils climb higher when grown in a mixed stand with cereals than
when grown in monoculture (Horneburg 2003). Reduction of the lodging risk can
also be achieved by intercropping two legume species, for example, when faba bean
acts as the supporting crop for pea (Schlipf 1898). Further, because intercropping
increases soil cover in comparison to monocrops, it reduces the risk of soil erosion.
This risk is particularly high where rows are spaced relatively widely in the (mono-
crop) grain legumes.
Intercropping has also been shown to reduce the incidence of pests and diseases
due to effects of diluting hosts, non-hosts acting as physical barriers, induced resis-
tance, modification of microclimate and manipulation of host-finding behaviour of
pests. For example, in comparison to monocropped barley, net blotch (Pyrenophora
teres) infestation was significantly reduced when barley was intercropped with
lupins, peas, faba beans, or any combination of the three legume species (Haug-
gaard-Nielsen et al. 2008). In an intercropping field trial in Nigeria, pest damage in
soybean was consistently lower over 2 years when soybean was intercropped with
millet than in the soybean monocrop (Sastawa et al. 2004).
However, intercropping effects are not always reliable (Trenbath 1993) or can
even be counterproductive (Helenius 1990). For instance, while several practical
guides (Schlipf 1898; Zimmermann 1958; Franzmann 1992; Aufhammer 1999) rec-
ommend intercropping for the control of the black bean aphid in faba beans, other
422 T. F. Döring

sources reported that no effect of intercropping oats with faba beans on the black
bean aphid could be observed (Kolbe et al. 2002). Similarly, mixed results were
found for effects of intercropping cowpea on insect pests (Jackai and Daoust 1986).
Also, intercropping wheat with faba beans can lead to higher disease infection with
powdery mildew (Blumeria graminis) in wheat (Chen et al. 2007), possibly because
of higher levels of foliar N in wheat leaves, and also because of a moister climate in
the intercrop than in the wheat monocrop.
In terms of yield, effects of intercropping are usually quantified by the land
equivalent ratio (LER), which is the area of monocultures required to achieve the
same yield as obtained in the mixture. For example, in a two-partner intercrop, an
LER of 1.2 means that 1.2 ha of land would be needed for growing the two partners
separately (in monocultures) to obtain the same yield as harvested from 1 ha of the
intercrop. In intercrops of grain legumes and cereals, the LER has usually been
found to be above 1, with a range between 0.91 and 1.51 (Hauggaard-Nielsen et al.
2008). However, the LER is also dependent on N input, with the LER being lower
and the proportion of the legume partner being smaller at high N levels (Jensen
1996). This has also been confirmed by a study on intercropping lentil and naked
barley where the yield advantage of the intercrop over both sole crops was only ap-
parent at low levels of mineral soil N (Schmidtke et al. 2004).
Finally, intercropping grain legumes has also been shown to result in higher
yield stability of combined yields in comparison to sole crops (Jensen 1996). In
lentil–barley mixtures, it has been observed that yield stability is achieved through
complementarity; in dry years, there are more lentils, whereas the balance in wet
years is more towards barley (Horneburg 2003). While the view that intercropping
grain legumes leads to high yield stability is widespread (Schlipf 1898; Zimmer-
mann 1958; Aufhammer 1999), also the opposite effect has been found (Hauggaard-
Nielsen et al. 2008). Further, details about effects of intercropping on yield stability
in grain legumes are presented in Sect. 5.3.

4.2  Managing Intercropped Grain Legumes

Agronomic management of intercrops is more complex than sole cropping of the

partners, partly because requirements of both (or all) partners need to be balanced.
One of the most important criteria when the intercrop is grown for threshing is that
the components of the mixture have similar times in the season at which they ma-
ture. For example, for intercropping peas with cereals, it is recommended to select
pea varieties with suitable maturity so that the maturation is matched in time with
the cereals (Franzmann 1992). An alternative solution to the problem of different
maturation times is to harvest the intercrops before maturity, and to use either the
green crop for silage or with slightly later harvesting to thresh before the grains are
completely dry and conserve the combined intercrop in a process called crimping.
Such early harvests are particular relevant for cropping system design as they affect
the entire planning of the rotation.
13  Grain Legume Cropping Systems in Temperate Climates 423

Also, if intercropping partners are sown at the same time, the required seed
depths of the partners cannot be too different, unless specialised sowing machinery
for separate sowing depths of the different partners is available. In terms of seed
densities, experience shows that yield benefits are largest when the added densi-
ties of each partner are above 100 % (better than substitutive mixtures), but not
completely additive (i.e. below 200 %). A further issue in managing intercrops is
weeding. This refers both to the design of mechanical weeding in row crops such
as maize and, in conventional agriculture, to the selection of herbicides that can be
used in both crops simultaneously (Pekrun et al. 2013).
Generally, the design of intercropping systems with grain legumes is currently
a tedious case-by-case work. Although many general principles have been estab-
lished, transferability of experience from one system to the other is still limited.
Despite these limitations, however, research on intercropping is well advanced, in
that it provides detailed accounts of optimal agronomic management for many crop
species combinations. Still, adoption rates of intercropping are well below the po-
tential. The main underlying problem for the lack of implementation in practice
appears to lie in the supply chain, which, in the case of feed for farm animals is
structurally not well set up to deal with mixed grain products. Future work should
therefore aim to remove socioeconomic constraints to intercropping.

5  Yield Stability in Grain Legumes

5.1  Concepts of Stability

For virtually all crops, achieving high levels of yield stability is an important goal.
This goal is shared by farmers, plant breeders, cropping systems designers and gov-
ernments concerned about food security. However, yield stability is not a simple
concept. Instead, it comprises numerous different statistical approaches (e.g. Becker
and Léon 1988). Making progress towards greater yield stability therefore requires
specification of the kind of stability that is to be promoted. For example, when De-
ghani et al. investigated yield stability in lentils in Iran (Dehghani et al. 2008), they
calculated a total of 19 univariate yield stability measures. Some of the stability in-
dices used in the study gave strongly differing rankings for yield stability among the
tested genotypes. This finding indicates that the results of stability analyses strongly
depend on which specific statistical approach is employed.
Parameters of stability can refer to temporal variation (across years), variation
across locations, or both. When looking at temporal yield stability, it is crucial
to ­define the spatial area over which yields are aggregated. For example, farm-
ers’ ­interests are likely to be more directed towards temporal yield stability at the
farm scale than at the field scale, because within-field fluctuations over time can be
compensated within the farm (Porter et al. 1998). In addition, however, temporal
yield stability at the regional and national scale is also of importance as the farm’s
424 T. F. Döring

economic performance will be affected for example, through price fluctuations on

the market.
Some relatively simple measures of yield stability that have been used in the
context of evaluating yield stability in grain legumes include the ratio between
highest and lowest yield (Dyke and Prew 1983), the coefficient of variation (CV),
that is, standard deviation expressed as percentage of the mean (Smith et al. 2007),
or the frequency of years in which yields are below a given minimum.
Many of the other stability parameters currently in use belong to one of two
broad groups, being based either on variance components or on (linear) regression
(Annicchiarico 2002). Yield stability parameters based on variance components cal-
culate how much the yield of a genotype fluctuates in individual year-by-location
combinations (environments) around the mean yield of that element, that is, the
yield of the element averaged across all tested environments. Large fluctuations
indicate low stability.
Regression-based approaches to stability first calculate for each environment E
the average yield xE achieved across all genotypes G, and then determine the linear
regression of yields yG of individual elements G in all environments against x. One
suggested definition of yield stability is that a genotype shows maximal stability if
its linear function y = a + bx has a slope of b = 1, that is, slopes of b < 1 and b > 1 indi-
cate lower stability. For b = 1, there are no significant interactions between genotype
and environment.
Finally, a novel measure of yield stability is based on Taylor’s power law (Cohen
2013; Döring et al. 2014). This stability index is able to operate over a large range of
yield levels as it takes the frequently observed dependence of means and variances
into account. Therefore, it appears to be particularly suitable for comparisons of dif-
ferent crop species. Here, means m and variances s² of yields over environments are
calculated for each genotype (or species). Then, deviations from the linear regres-
sion of log( s²) against log( m) can be interpreted as an index of yield variability, that
is, the smaller these power-law residuals (POLAR), the higher the yield stability.

5.2  Are Grain Legume Yields Unstable?

Already in 1840, the German agricultural writer Alexander von Lengerke stated that
unstable yields are one of reasons why grain legumes are not being grown more by
farmers (von Lengerke 1840). Similarly, at the end of the nineteenth century, Johann
Schlipf mentioned in a handbook of general agricultural practice that grain legumes
show unstable yields because of their low ability of compensation (Schlipf 1898).
More recently, this view of grain legumes as being characteristically unstable was
reiterated (Sperber et al. 1988; Duc 1997; Horneburg 2003), and reasons given in-
cluded vulnerability to pests and diseases (Schlipf 1898) or the general response to
environmental and weather factors (Franzmann 1992). The view that grain legumes
are yield unstable is also held by farmers. In a European survey, von Richthofen
and colleagues asked farmers in Switzerland, Spain, Belgium and Germany to rate
13  Grain Legume Cropping Systems in Temperate Climates 425

reasons for not growing grain legumes. Out of 21 reasons given in total, high vari-
ability of yield was among the four most important reasons in all four countries, the
most important being the low (economic) performance in comparison to row crops
and cereals (von Richthofen et al. 2006).
Unfortunately, however, quantitative evidence of low yield stability in grain
legumes is surprisingly scarce. One reason for the lack of robust data is that analy-
ses of yield stability have predominantly been the domain of plant geneticists
and breeders, whereas agronomists have not engaged in this area with the same
enthusiasm. Most available data of yield stability in grain legumes refer to intra-
specific differences in yield stability (Bond 1987; El-Moneim and Cocks 1992;
Link et al. 1994; Dehghani et al. 2008). In contrast, comparisons among grain
legume species, and between grain legumes and other crops, are relatively rare.
Also, comparisons between different cropping systems in terms of their effects
on yield stability of grain legumes (Smith et al. 2007) have so far not been well
covered by research.
At least at larger levels of spatial aggregation, and for Central Europe, yield
­stability in grain legumes may possibly not be as bad as its reputation. Data from
the German official record over the years 1993–2012 indicate that temporal yield
­stability in faba beans and field peas is comparable with that of cereals when yields
are aggregated at the national level (Fig 13.4). In the two grain legume species, the
national average yield was below 90 % of the average yield in only 3 out of 20 years.

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Fig. 13.4   A simple measure of temporal yield stability for grain legumes (circles), oil crops (trian-
gles) and cereals (squares), calculated for national average yield data from Germany, 1993–2012.
The x-axis shows the number of years out of 20 in which the yield of a given crop was below 90 %
of the 20-year yield average of that crop. The y-axis shows the mean yield over the entire 20-year
period. FB field beans, FP field peas, M maize, SB spring barley, SF sunflower, SO spring oats,
SOSR spring oil-seed rape, SW spring wheat, T triticale, WB winter barley, WOSR winter oil-seed
rape, WR winter rye, WW winter wheat
426 T. F. Döring

This was the same result as observed for winter barley and spring barley. Also, it
was considerably better than for the oil crops (sunflower and oil-seed rape), where
yields were < 90 % of the mean yield in 5 or more years. An analysis of regression-
type stability of the same data confirms this picture, showing that field peas and
faba beans have similar regression slopes as barley and winter rye (data not shown).
Unfortunately, for other legume species such as lupins, continuous yield data was
not available for the investigated period.
According to a further analysis using regional yield data from Germany, tem-
poral yield stability of grain legumes is not consistently lower in comparison to
other crops (Fig. 13.5). While the CV over years was significantly higher in grain
legumes than in cereals ( p < 0.01), the difference in CV between grain legumes and
oil crops was not significant (Fig. 13.5a). The CVs of field peas and faba beans did
not differ significantly. However, as a measure of stability, the CV has the disad-
vantage that CV values negatively correlate with mean yields, that is, greater means
tend to lead to lower CVs. This bias is not observed when stability is calculated as
the residuals from the power-law regression line (Fig. 13.5b, c). For this measure of
stability, differences between grain legumes and other crop groups were not signifi-
cant. Data from experimental field trials show a similar picture (Fig. 13.5d).
It is clear from these analyses that the view of particularly unstable yields in
grain legumes may not be universally valid at all spatial levels. However, it is pos-
sible that at smaller spatial scales, grain legume yields do fluctuate significantly
more than yields of other crops (Reckling et al. 2015). In fact, as far as small-scale
environmental fluctuations may cancel each other out at higher spatial scales, yield
stability is expected to be decreasing with the level of spatial aggregation. This
is especially plausible where yields respond strongly to variations in precipitation
across years. While this has been suggested for some grain legumes, in particular
faba bean with its long vegetation time, further research is necessary to clarify the
situation.

5.3  Factors Affecting Yield Stability in Grain Legumes

There is a multitude of potential factors that can have an effect on yield stability.
Again, however, while suggestions on the nature of destabilizing factors abound,
quantitative evidence is as yet relatively scarce. Generally, it is believed that grain
legume yields are strongly dependent on weather, especially drought and heat. Fur-
ther, their compensatory ability through plasticity of yield components is consid-
ered to be low. Genetic variation in yield stability among grain legumes does exist
but stability is not strongly heritable. For faba beans, it was concluded that “due
to low heritability, yield stability of faba bean inbred lines is a recalcitrant trait for
practical breeding purposes” (Link et al. 1994). However, it was also found that late
maturation in faba beans, though associated with high yield potential, implied lower
yield stability due to the risk of lodging.
 13  Grain Legume Cropping Systems in Temperate Climates 427

 

 
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Fig. 13.5   Yield stability of grain legumes at regional and field trial level in faba beans (grey cir-
cles), field peas (white circles), oil crops (black triangles) and cereals (grey squares). a Mean yields
(in t ha−1) versus coefficient of variation (across years, in %), data from seven official regional
yield surveys in Germany (Baden-Württemberg, Niedersachsen, Oberbayern, Rheinland-Pfalz,
Sachsen-Anhalt, Schleswig-Holstein, Thüringen) where at least 10 years of data were available;
median number of years = 14; differences between groups of crops were tested with Tukey’s HSD,
using the Programme R, v. 3.0. b logarithm of the mean yield log( m) versus logarithm of the vari-
ance of the yield log( s²) (calculated across years, on data in dt ha-1), same dataset as in panel (a);
the regression line follows log( s²) = a log( m) + b, with a = 1.349 ± 0.171 (s.e.), b = –0.847 ± 0.288,
Adj. R² = 0.450, p < 0.001. c residuals of individual data points from the regression line displayed
in panel (b) (power-law residuals, POLAR). d POLAR calculated for experimental data of various
crops. (Sperber et al. 1988; Link et al. 1994; Jensen 1996; Duc 1997; Kolbe et al. 2002; Smith et al.
2007; Fikere et al. 2008) HSD honest significant difference, TPL Taylor’s power law

In terms of the effects of cropping systems, rotation will have a positive effect
on yield stability in comparison to monocultures (see Sects. 2.1 and 3.5). Also, the
variability of yields pooled from intercropping partners is generally lower than in
the constituent monocrops, because of compensatory and complementary effects.
Similarly, cultivar mixtures are associated with higher stability than single varieties,
for example, as recently shown for cowpeas in Uganda (Okonya and Maass 2014).
However, the yield variability of the grain legume partner in an intercropping
­mixture can be greater than in the sole crop (Böhm 2013). This could be because
of the weather sensitivity of the competitive balance between the intercropping
428 T. F. Döring

partners as the asymmetric competition amplifies weather effects between years.

Similarly, weeds can constitute a positive feedback mechanism, magnifying other
­destabilizing factors. If grain legumes show weak growth, weeds will fill the gap
left by the crop and reduce grain legume yield even more.
With a view to compare different cropping systems, Smith et al. (2007) measured
temporal stability of soybean yields under different management conditions using
CV as the measure of yield stability. In a 3-year rotation, four management systems
were compared, namely CT, NT, Low Input (LI) and Organic (ORG). While mean
yields of soybean were similar across the cropping systems, temporal yield stabil-
ity differed between the systems. The interannual variation of soybean yields was
lowest in the NT system and highest in the ORG system, with the other systems in
between. Yield variability of soybean was comparable to that of corn, but higher
than in wheat. However, the latter comparison is confounded by the fact that there
was a greater variation of precipitation in the wheat phase than in the soybean phase
of the trial. Thus, the study adds further, albeit indirect, evidence that the view of
grain legumes being inherently less yield stable than other crops may not be uni-
versally true.

6  Conclusions and Outlook

Designing cropping systems with grain legumes is complex and requires integra-
tive consideration of multiple issues, including aspects of soil physical, chemical
and biological properties, competition among crop plants and between crops and
weeds, as well as pest and disease epidemiology. Crop diversity in time, as in crop
­rotational design, and in space, such as in intercropping, provides ample opportuni-
ties to balance the needs from all these areas. In addition, currently underexplored
genetic diversity at the species level offers new chances for further developing
grain-legume-based cropping systems (Bell et al. 2011). This idea of diversification
also extends to more complex systems such as agroforestry, both in tropical (Red-
head et al. 1983) and temperate (Isaac et al. 2013) climates.
Unfortunately, the advance of molecular biology and the huge progress in the
understanding of genetic and physiological mechanisms in grain legumes have not
been paralleled by comparable advances in the agronomy of grain legumes and as-
sociated cropping system design. One area with particularly large gaps is the issue
of how stability and resilience of grain legume yields are influenced by agronomic
management. Thus, more research is clearly needed to optimise and innovate crop-
ping systems for better performance and stability of grain legumes.
However, at the global level, there is a massive and increasing bias of current
grain legume cropping towards a single species—soybean, as well as the wide-
spread use of simplified and de-diversified cropping systems associated with this
species. Research alone will not be able to turn this situation around. More is need-
ed than just developing recommendations for optimised grain legume cropping. In
fact, most of the key issues for successful cropping of grain legumes are already
13  Grain Legume Cropping Systems in Temperate Climates 429

well established. Instead, barriers to greater implementation and adoption of a di-

verse range of grain legume cropping systems need to be identified. In addition, the
demand for grain-legume-based products needs to increase.
Further, experience over the past few decades shows that political support can
dramatically increase acreage grown with grain legumes, but that waning support
can equally lead to a downward spiral of decreasing interest and an erosion of the
socioeconomic framework that is needed for growing grain legumes. To make use of
the considerable benefits of grain legumes both in agro-ecosystems and for human
nutrition, increased efforts in integrative agronomic research need to be matched by
critical analyses of whole food chains and enthusiastic and sustained public support.

Acknowledgments  I would like to thank Frank Ellmer, Wolfgang Köhn, Peer Urbatzka and Mar-
tin Wolfe for helpful discussions on grain legume agronomy.

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Index

A field pea-breeding program (Horsham,

Alkaloids 197 Australia) 67
Antinutritional compounds  292 for agronomic interest  147
in seeds  294 for CBB resistance  14
Antinutritional compounds also Alkaloids  197 for disease resistance  14
in dry bean  14
B for resistance  16, 153, 172
Bioactive compounds  165, 292 for tolerance  124
classes 316 gene pools  45, 53
saponins 316 grass pea  380
minor 307 hybrids 388
l-DOPA 307 ideotype 14
synergistic effects of  292 improvement techniques  196
Biodiversity  lentil  122, 126, 130h
conservation of  254 specific goals in  126
flower 26 lines  16, 20, 59, 66, 73
LI/O systems  384 major breeding achievements and specific
mine 52 goals in  90
for crop improvement  52 marker-assisted breeding strategies  353
Biotechnology 199 methods  20, 66, 96
and molecular biology  180 and specific techniques  20, 66
in breeding programs  199 gamete selection  20
use in pea-breeding programs  69 NDSU breeding program  73
use in plant breeding  158 of bean  7, 11
Black-eyed pea  315 in USA  11
Breeding  17, 61, 131, 144 of low alkaloid cultivars  191
achievements 120 pea  38, 51, 58, 60, 62–65, 67, 68
activities 69 achievements of  58
and domestication  346 in China  65
chickpea  89, 94 in Europe  62, 63, 67
programs 94 in France  65
conventional 18 in India  64
crop 387 in USA  60
pollination in  387 programs 64
faba bean  151, 152 plant  21, 373
specific goals in  152 goals of  21
pools 70

© Springer Science+Business Media New York 2015 435

A. M. De Ron (ed.), Grain Legumes, Handbook of Plant Breeding 10,
DOI 10.1007/978-1-4939-2797-5
436 Index

population 390 variety development programs  238

primary objectives of  65 viral disease of  236
programs  12, 13, 17, 21, 23, 25, 54, 65, Cropping system  1, 86, 141, 237, 277, 401,
66, 69, 71, 72, 96, 98, 100, 152, 403, 411, 413, 417
153, 156 designing of  428
for beans  13 management of  419, 427
use of molecular markers in  100 of soyabean  428
research 17 Crowder bean  223
strategies  12, 62, 383, 384 Cultivar improvement  183, 196
pollination  383, 384
system 372 D
USDA-ARS breeding program  73 Disease resistance  11, 12, 14, 20, 63, 64, 95,
use in improving disease resistance  12 99, 124, 129, 148, 153, 166, 238,
use of molecular markers in  14 255, 258
white lupin  192, 195, 381 breeding 65
specific goals in  195 fungal  206, 208
Domestication  5, 41, 42, 111, 115, 328
C definition of  130
Cancer  291, 308, 315 effect of  6
breast 2 self-pollinated 116
cell lines  315
cells 316 E
programmed cell death of  316 Ecosystem services  144
colon 317 Enzyme inhibitors  292
Cicer spp.  Evolution 
Cicer arietinum 403 diffusion of  6
Cicer arietinum L.  85, 87 of cpDNA  160
Cicer echinospermum 87 of hybridization  89
Cicer reticulatum Ladz.  87
Cowpea (Vigna unguiculata)  164, 219–221, F
244, 375, 376, 384 Faba bean (Vicia faba L.)  141, 252, 327, 376
bacterial disease of  236 Feed  46, 70, 141, 221, 235
breeding lines  227 Field pea  45, 60, 403, 408
breeding methods  237–239 AAFC’s 73
intercropping 237–239 characteristics of  58
breeding programme  226, 232, 233 cooperative  58, 59
in California  232, 233 CVs of  426
breeding programmes  240, 241, 243 disease of  58, 61
consensus map  242 Floral traits  366, 369, 386, 392
development using Bt gene  242 pre-mating 391
disease resistance  235 Food  11, 45, 330
diversity 224–226 anti-cancer effects of  315
germplasm  223, 224 consumption of  309, 311, 313
grain size  237 definition of  309
heat stress in  234 high-GI 310
in Uganda  427 micronutrient-rich 291
major breeding achievements of  227 thermic effects of  312
molecular breeding for  242
nutrient content of  235
origin of  222, 223 G
seed system  244 Genetic resources  3, 7, 8, 45, 47, 90, 119,
storage proteins and enzyme 147, 148, 184, 185, 254, 255
inhibitors  292, 298, 342 catalogue of bean  12
Index 437

center of IITA  227 seed-bank 339

collection of  224 seed-coat  332, 333, 335, 343
collections of  191, 196 SNF of  278
distribution of  223 Lentil  39, 45, 88, 111, 115, 135, 252, 314,
in USA  12 375, 423
molecular characterization of  159 pre-crops 411
Genetics  12, 142, 163, 210, 344 seeds of  301
from INRA  51 self-pollinated 116
of seed-coat  343, 344 spread of  116
Genome  98, 160 Lupin (Lupinus spp.)  179, 184, 258, 327, 381
cowpea  233, 234, 236
cpDNA  160, 161 M
draft pea  57 Marker-assisted selection (MAS)  21, 69, 71,
faba bean  163, 166 97, 130, 166, 257
genomics of  162, 163 Mating system  372
MABC 243 patterns of  372, 373, 375
sequence of  21, 22, 43, 87 Metabolic syndrome  308
Germination control  329, 330 Molecular genetics  144
seed-coat  327, 339 Molecular markers  5, 7, 14, 21
Germplasm  3, 13, 47, 126, 129, 130, 132, large-scale 97
223, 254 PCR-based 204
faba bean  163 types of  22, 164
high-throughput 72 Mutation  18, 23, 24, 51, 64, 194, 198, 303
in UK  153
non-duplicate pea  54, 57 N
Grain quality  66, 67, 227, 232, 238 Niébé Cowpea (Vigna unguiculata) 219
Grass pea  144, 251, 254, 379 Nitrogen fixation  86, 93, 152, 251, 273
breeding 256 Nitrogenase  270, 272, 273
distribution of  252 inhibition of  275
SSRs 258
O
H Osmotic  128, 154, 345
Hardseededness  329, 330, 335, 336, 338, 339, Oxygen 
343 diffusion  272, 274, 275, 279, 350
of grain legumes  344
P
I Phaseolus vulgaris L.  1, 7, 71
Imbibition damage  340–342 Phosphorus  7, 156, 277, 278, 420
deficiency  279, 280
L Phytic acid  18, 303, 311, 316
Lathyrus sativus  144, 251, 380 Pisum sativum L.  37, 252, 367
Lectins  17, 19, 292, 294–296 Pollination  25, 26, 366, 383, 384, 387, 390
bean-derived 313 Polyphenols  315, 316, 343
effects of  317 Protein  2, 17, 18, 304, 308, 328, 351
Legumes  7, 17, 87, 200, 220, 280, 308, 327, content of  235, 251
328, 337, 342, 407, 409 seed-storage 5
and non-legumes  269 source of  87, 112, 115, 402
biology of  365, 366 sulphur-rich 19
consumption of  291 vegetable-based 152
mention of  115 Protein crop  62
non-soy 314 Pulses  60, 71, 252, 312
of grain  330 consumption of  308, 311, 315
438 Index

R response of  275

Reproductive system  373 wild annual  382
Rotation  27, 38, 111, 152, 153, 172, 251, 401, yellow-seeded 342
403, 416 β-conglycinin  293
legume-based 404 Stability  14, 24, 65, 426
soybean cotton  408 concepts of  423, 424
of yield  403, 410, 419, 422, 428
S Stresses  39, 125, 154, 157, 259
Seed  3, 10, 25, 51, 71, 88, 101, 243, 337 abiotic  64, 86, 91, 93, 94, 96, 124
dormancy  42, 43, 45, 116, 127, 328–330, biotic  227, 251
353 situations of  155
genetics of  343, 344 Symbiosis  45, 112, 141, 251, 267
production  11, 27, 72, 73, 101, 102, 169, nitrate inhibition of  273, 274
208, 210, 243–246, 260 phosphorus requirement of  277–279
proteins  5, 71, 292, 294, 298 rhizobia legume  268, 276
Seed-coat permeability  328–330, 335, 336, Systematics  144, 145
339, 340, 342–344
Southern pea  5 T
Soybean  1, 112, 306, 382, 388 Tropical legumes II (TL II)  243–245
conglycinin 294
male-sterile 385 V
N2-fixing 279 Vigna unguiculata  279, 327