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Morphology of Chickpea Seeds (Cicer arietinum L.): Comparison of desi and

kabuli Types

Article  in  International Journal of Plant Sciences · June 2011

DOI: 10.1086/659456

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Morphology of Chickpea Seeds (Cicer arietinum L.): Comparison of desi and kabuli Types
Author(s): J. A. Wood, E. J. Knights, M. Choct
Source: International Journal of Plant Sciences, Vol. 172, No. 5 (June 2011), pp. 632-643
Published by: The University of Chicago Press
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Int. J. Plant Sci. 172(5):632–643. 2011.
Ó 2011 by The University of Chicago. All rights reserved.
1058-5893/2011/17205-0002$15.00 DOI: 10.1086/659456

MORPHOLOGY OF CHICKPEA SEEDS (CICER ARIETINUM L.):

COMPARISON OF DESI AND KABULI TYPES
J. A. Wood,1 ,* E. J. Knights,* and M. Chocty
*Tamworth Agricultural Institute, Industry and Investment NSW, 4 Marsden Park Road, Calala, New South Wales 2340, Australia;
and yUniversity of New England, Armidale, New South Wales 2351, Australia

The morphology and composition of seeds of desi and kabuli chickpea (Cicer arietinum L.) genotypes were
studied using light microscopy with differential staining for protein, starch, b-glucans, and nonfluorescing
compounds. Kabuli seeds had a thinner seedcoat due to thinner palisade and parenchyma layers which
contained fewer pectic polysaccharides and less protein. The outer palisade layer varied in thickness from one
to two cells, leading to a textured and sometimes wrinkled appearance of the seed surface. In contrast, the desi
palisade layers were rigid and extensively thickened. Hourglass cells were homogeneous for both seed types,
but not in an interspecific desi line (containing Cicer echinospermum parentage), which had heterogeneous
cells. The inner surface of the seedcoat contained both pectic and proteinaceous materials. The cotyledon
comprised a single outer epidermal layer of protein-filled cells devoid of starch, with thickened outer cell walls;
cell size and shape differed on abaxial and adaxial faces. Subepidermal cells on the abaxial face were similar to
epidermal cells. These findings help explain differences in the processing behavior between the major chickpea
seed types.

Keywords: Cicer, seed morphology, seed coat, cotyledon, light microscopy, fluorescence.

Introduction ledons covered by a seed coat and are generally nonendosper-

mic (Anderson 1949). The seed coat has an outer integument
Chickpea (Cicer arietinum L.) is an important food legume (cuticle, palisade layer, hourglass cells, parenchymatous layer,
in many regions of the world, particularly in the Indian sub- inner epidermis) with or without the remnants of an inner in-
continent, west Asia, and northern Africa, where it is an in- tegument (aleurone layer and endosperm) at the abaxial in-
tegral part of human diets. The species is divided into two terface (between the seed coat and cotyledons), as observed
populations, on the basis of seed type. Desi chickpeas, which in some legumes (Bailey 1971; Wolf and Baker 1972; Yeung
dominate Indian (and world) production, typically have a col- and Cavey 1988; Koch et al. 1999). Yeung (1990) also sug-
ored (mostly brown), somewhat angular-shaped seed with gested that the cotyledons of some pulses may be covered by
a prominent, characteristic ‘‘beak’’ that houses the embryonic a thin cuticle. Singh et al. (1984) and Hassan (2000) exam-
axis. Depending on genotype, seeds normally fall within the ined the seed coat of chickpea using scanning electron mi-
range of 0.1–0.3 g. Some desi seeds are consumed whole, but croscopy (SEM); however, there are no published reports on
the majority are decorticated and the cotyledons cleaved to cotyledon structure or internal tissue junctions. Moreover,
produce split seed (dhal). Further processing produces flour light microscopy with differential staining has not been used
(besan). Kabuli chickpeas have contrasting characteristics; to examine the internal structure of chickpea seeds, although
they are white or cream colored, have a rounder shape with it has been used for cereal grains (Andersson et al. 1999;
a less pronounced beak, and, despite some overlap in size Autio and Salmenkallio-Marttila 2001; Choct et al. 2001).
with the desi range, are generally larger and heavier (0.2–0.6 g). Desi chickpeas vary considerably in their ease of decortica-
The seed coat is also much thinner in kabuli types, compris- tion and splitting (Wood et al. 2008), reflecting differences in
ing 5% of total seed weight compared with 14% in desi seeds. the adherence of the seed coat to the cotyledons and also be-
Kabuli seeds are normally cooked and consumed whole, and tween the cotyledons themselves. A major difference in seed
in the Middle East, the cooked seeds are often ground into a shape, determined by a single gene (Knights et al. 2010), has
paste (hommus). been shown to contribute to variation in the ease of decorti-
Microstructural information about cells and tissues is im- cation and splitting in desi chickpea, although other, as yet
portant in identifying physiological and structural inter- unrecognized factors are also assumed to contribute signifi-
actions that influence grain quality and processing behavior cantly. An additional source of variation in ease of decorti-
in industrial applications and food manufacture (Saio and cation and splitting appears to have been introduced from
Monma 1993). Pulse (grain legume) seeds contain two coty- outside the cultigen (group of bred or selected plants). Cicer
echinospermum is a close, but wild, relative of chickpea. In-
1
terspecific progeny derived from crosses of chickpea with C.
Author for correspondence; e-mail: [email protected].
echinospermum have been used in the Australian chickpea
Manuscript received June 2010; revised manuscript received December 2010. breeding program to increase disease resistance. Wood et al.

632
 WOOD ET AL.—MORPHOLOGY OF CHICKPEA SEEDS 633

(2002) noted that these progeny consistently had reduced allowed to dry. Acid fuchsin was used as a counterstain.
decortication and splitting output. Kabuli chickpeas, on the (Calcoflur/Fluorescent Brightener 28 binds to b-glucans within
other hand, are not decorticated or split commercially despite intact cell walls to fluoresce blue green and acid fuchsin com-
a lower seed coat content that affords a much higher theoret- bines with proteins to fluoresce red brown.) Sections for bright-
ical dhal yield. Practical experience has shown that the force field observation were stained with 1% light green (2 min) and
required to overcome the strong seed coat–cotyledon adher- blotted dry without rinsing. The slide was flooded with 1%
ence of kabuli seeds results in an unacceptable fracture of the Lugol’s iodine solution. A coverslip was added, pressed down
cotyledons (J. A. Wood, unpublished observations). to expel excess solution, and sealed around its edges using
This study was undertaken to provide a comprehensive de- nail polish. Light green stains protein green, and Lugol’s io-
scription of the morphology of the seeds of two desi geno- dine solution stains starch blue brown.
types (one of which contained C. echinospermum parentage) Image capture. The sections were examined by a Leica
and one kabuli genotype and to examine the chemical com- DLMB microscope (Leica Microsystems, Wetzlar, Germany)
position of different structures through selective staining tech- at 320 magnification and captured with a Spot RT digital
niques. In conjunction with other investigations, these results camera (Diagnostic Instruments, Sterling Heights, MI). The
will then be used to provide a better explanation of the differ- microscope configuration for fluorescent imaging used filter
ences in the ease of decortication and cotyledon cleavage ob- cubes with a Leica D UV and violet excitation range (mer-
served between and within the two main types of chickpea. cury light source 50W). The excitation filter allowed a wave-
length band of 355–425 nm to pass through to a dichromatic
mirror (455 nm, transparent for the longer-wavelength fluo-
Material and Methods
rescent light emitted). The light finally hit a suppression filter
(470 nm, to allow fluorescent light to pass while suppressing
Sample Preparation
any excitation wavelength still present). Similarly, the micro-
The test material comprised three genotypes: Amethyst, scope configuration for bright-field imaging used a Leica A
a commercial desi chickpea cultivar; 90101-57Q, a desi-type UV excitation range (tungsten lamp) with an excitation band
interspecific breeding line with a theoretical 25% of its ge- pass (340–380 nm), a dichromatic mirror (400 nm), and a
nome derived from Cicer echinospermum; and Bumper, a com- suppression filter (425 nm) with the digital camera.
mercial kabuli cultivar. Seeds were harvested in December
2001 from a genotype evaluation trial grown at Moree, New
Measurement of Tissues
South Wales, Australia, allowed to equilibrate to moisture con-
tent of 10%–11%, and then stored in sealed containers at 4°C. Thickness of seed coat and constituent tissues in desi and
kabuli genotypes was calculated from the average of five
measurements from microscopy images.
Light Microscopy
Microscopy techniques designed for animal nutrition stud-
Results
ies, as previously described by Dürrenberger et al. (2001) and
modified by Autio and Salmenkallio-Marttila (2001), were
Seed Structure
used to identify various tissues in the chickpea seeds.
Preparation of microscopy samples. Seeds were divided Figure 1 illustrates the structures of a chickpea seed. The
into two equal sections by a transverse cut at the midpoint on largest fraction is the embryo, which comprises two cotyle-
the longitudinal axis, perpendicular to the cotyledon-cotyledon dons joined at their adaxial surfaces, a small hypocotyl (em-
interface. The opposite end of each half seed was then cut at bryonic axis), and a radicle (embryonic root) located in the
an angle to allow for the best penetration during subsequent chickpea ‘‘beak.’’ The embryo is enclosed by a seed coat (testa)
processing. The grains were fixed in a 1% glutaraldehyde- that acts as a protective coating. The most prominent exter-
phosphate buffer for 3 d at 4°C, washed in distilled water nal structures on the ventral side are the hilum, a funicular
(2 3 30 min) and dehydrated through an ethanol series: 50% scar marking the point at which the seed was attached to the
(2 3 45 min), 70% (16 h), and 95% (24 h). Infiltration with pod wall during development, and the micropyle, a minute
resin (Leica Historesin) was effected by placing samples in pore controlling moisture entry to the seed. Both are sur-
50% ethanol and 50% resin (48 h) and then in 100% resin rounded by a corona (hilum rim). The raphe extends in a line
(6 d). The samples were then positioned in molds, embedded from the bottom of the corona to the spermotylium (arillate),
in resin, and mounted on blocks for sectioning. Sections were which contains the chalaza (ovule base). These structures are
cut to a thickness of 4 mm (Leitz 1516 microtome with mo- present in both desi and kabuli seeds but may differ slightly
tor drive), and each section was floated on a drop of distilled in their appearance (color, size, and prominence).
water on a slide and dried on a hot plate at 60°C. Two stain-
ing methods were used: fluorescence and bright field. Sections
Seed Coat
for fluorescent observation were flooded with 0.1% acid fuch-
sin (1 min), rinsed off with distilled water, and flooded with Table 1 shows significant differences between the seed coat
a distilled water rinse (1 min). The slide was allowed to drain thickness of desi and kabuli chickpeas. The kabuli chickpea,
and dry slightly (;5 min); any excess water was blotted Bumper, has a much thinner seed coat overall than the desi
off. It was then flooded with a 0.01% solution of Calcoflur/ genotypes, and all constituent tissues are thinner except for
Fluorescent Brightener 28 (1 min), rinsed as before, and the hypodermal region.
634 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 1 Chickpea seed (Cicer arietinum L.). A, Ventral view showing external features. B, Ventral view with seed coat removed, showing major
internal features. C, Lateral view with seed coat removed, showing major internal features.

Figures 2–8 present a series of images showing the differ- chickpeas: it is thinner, is less stained, and has fewer tissue
ent regions of seeds from desi (Amethyst and 90101-57Q) layers.
and kabuli (Bumper) chickpeas. Figure 2 shows cross sections Cells in the outer layer of the palisade region are character-
of the seed coats. Figure 3 shows magnification of the hour- ized by a thickened outermost cell wall, most easily discern-
glass cells of desi genotype, 90101-57Q. Figure 4 shows the ible in the fluorescence image of kabuli seeds (fig. 2B) but
corner of a cotyledon, both abaxial and adaxial regions. Fig- also faintly visible in the bright-field images of all genotypes
ure 5 shows cross sections of the intercotyledon (adaxial) re- (fig. 2). Amethyst has either a proteinaceous material adhered
gion. Figure 6 shows cross sections of the inner cotyledon to this surface or a proteinaceous cuticle (fig. 2C, 2D). Very
cells, and figure 7 shows magnification of this. Figure 8 shows little staining is evident in the palisade region of the kabuli
cross sections of the hilar region of the seed coat. chickpea (fig. 2A, 2B). In the fluorescence images (fig. 2A),
Figure 2 clearly shows the seed coat morphology and that the outer palisade layer of kabuli seed coats is mostly a single
of the adjoining abaxial cotyledon region in bright-field and cell layer, although a double cell layer occurs in some places.
fluorescence-stained cross sections. The chickpea seed coat This intermittent double layer appears to be mostly responsi-
contains two distinct regions, an external palisade and an in- ble for the textured and sometimes wrinkled surface of the
ner parenchymatous region, both of which are multiseriate chickpea seed. The inner palisade region of kabuli seed coats
(comprising several cell layers). The palisade region is clearly is composed of a single layer of thin-walled elongated pali-
differentiated into outer and inner layers; the parenchyma- sade cells with a cell structure similar to that of the outer pal-
tous region is divided into an outer hypodermis and inner isade layer, although less intensive staining suggests weaker
parenchyma. The color pigments of desi seed coats are cell walls.
contained in the external palisade region and the inner paren- The entire palisade region of the desi genotypes is exten-
chymatous region is clear or white (fig. 1B). Bright-field im- sively thickened and heavily stained, indicating structural ri-
ages of the seed coat (fig. 2A, 2C) reveal a clear or pinkish gidity (fig. 2C, 2D). The outer palisade layer has thickened and
cell wall structure, and the traces of green staining reveal lignified palisade cells (sclerenchyma cells or macrosclerids)
some proteinaceous material. Fluorescence staining of the with cell walls so thick that the lumen is almost absent.
same region (fig. 2B, 2D) shows the seed coat layers in more These cells are long and comparatively narrow with rounded
detail, staining b-glucan-type polysaccharides in cell walls ends. The outer palisade layer stains deeper blue green than
blue green and protein, red. The seed coat of the kabuli the rest of the palisade layer, suggesting it contains more
chickpea (fig. 2A, 2B) is distinctly different from the desi b-glucan-type polysaccharides and may therefore be the most

Table 1
Thickness of Seed Coat and Constituent Tissues in desi and kabuli Genotypes
Thickness (mm)
Palisade/sclerenchyma Hypodermal Parenchyma Total seed
Genotype Type region region region coat
Bumper kabuli 110 (palisade) 28 113 251
Amethyst desi 141 (sclerenchyma) 23 179 343
90101-57Q desi 144 (sclerenchyma) 33a 246 423
a
Heterogeneous.
 WOOD ET AL.—MORPHOLOGY OF CHICKPEA SEEDS 635

Fig. 2 Cross sections (320) of the seed coat, showing cellular structure. A, Kabuli (cv. Bumper), bright field. B, Kabuli (cv. Bumper),
fluorescence. C, Desi (cv. Amethyst), bright field. D, Desi (cv. Amethyst), fluorescence. cot, cotyledon; hyp, hourglass cells of the hypodermis;
ic, inner cuticle; ip, inner palisade; oc, cuticle; op, outer palisade; par, parenchyma cells. Bar ¼ 100 mm.

rigid part of the seed coat. The inner palisade region in desi tein appear to lie along many of the inner surfaces of cell
seeds is also composed of thickened sclerenchyma cells (mac- walls in the hypodermal and palisade regions. A cuticle or
rosclerids) but they are wider, have visible lumina, and have similar structure is located on the inner surface of the seed
weaker b-glucan staining with some internal protein staining coat, and both staining methods indicate the presence of
(fig. 2C, 2D). b-glucan-type polysaccharides as well as a proteinaceous ma-
A hypodermal layer comprising a single layer of hourglass terial (fig. 2).
cells (osteosclereids) with thick cell walls is located beneath
the palisade/sclerenchyma region in both desi and kabuli seeds.
Cotyledons
These cells have more thickening and stronger b-glucan stain-
ing on their distal region compared with their proximal re- Figure 4 illustrates a typical peripheral cotyledonary profile
gion. Cells in this hypodermal region are homogeneous for of chickpea. Each cotyledon has a complete single layer of
the Cicer arietinum genotypes, having a typical hourglass epidermal cells. These are protein filled and void of starch
shape. However, figure 3 shows the corresponding cells in granules, and their peripheral walls are thickened. However,
the interspecific line 90101-57Q, which are variable in ap- the cells differ depending on whether they are located on the
pearance, with many deviating from the typical hourglass abaxial (adjacent to seed coat) or adaxial (adjacent to its
shape. The inner region of the seed coat (beneath the hypo- paired cotyledon) side. Abaxial epidermal cells are spherical
dermal layer) is composed of irregularly shaped parenchyma and smaller than adaxial epidermal cells, which are spherical,
cells that become more compressed closer to the cotyledon tending to ellipsoid. Subepidermal cells on the abaxial face
interface in both desi and kabuli seeds. Fluorescence in the are similar to the epidermal cells (i.e., protein filled, with no
most compressed area indicates the presence of b-glucan-type starch granules) but are larger and irregularly arranged in
polysaccharides (fig. 2B, 2D). layers from one to three cells deep (figs. 2, 4). On the adaxial
Protein-type material occurs sporadically throughout the face, the cells immediately below the epidermal cells are
seed coat of all genotypes (fig. 2). In particular, traces of pro- larger than the epidermal cells (but not as large as inner coty-
636 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Hilar Region
Figure 8 shows the hilar region in the chickpea beak (con-
taining the hilum and micropyle) and is where the seed was
attached to the pod during development. Sclerenchyma cells
form the palisade layer and, with hourglass cells, are readily
distinguishable on the outer edges of the seed beak. Paren-
chyma cells are observed inside the hypodermal layer, while
the proximal ends of sclerenchyma cells are visible in the cen-
tral region of the hilum.
Fig. 3 Fluorescence micrograph (310) of seed coat cross section of
the interspecific desi genotype (90101-57Q), showing heterogeneous
hourglass cells. Bar ¼ 100 mm. Discussion

ledon cells), and most contain small starch granules sur- Cubero (1987) previously described the anatomy of the
rounded by a protein matrix (figs. 4, 5). chickpea seed. SEM has shown chickpea constituent parts
There is no evidence of endosperm or aleurone in any ge- such as the seed coat (Singh et al. 1984) and cotyledons
notype. Figure 5 shows the epidermal outer cell walls in the (Otto et al. 1997). More recently, Hassan (2000) described
intercotyledon region (adaxial), which are coated with a cuti- seed anatomy and seed coat structure in a range of Cicer spe-
cle. The cuticle stains similarly to the cotyledon cell walls. cies after SEM and light microscopy examination. Our study
Small globules of a substance not stained with either method employed staining techniques that revealed not only seed
are visible at the interface between the two cotyledons in the structural architecture but also the clear differentiation be-
kabuli seed only (fig. 5A). tween various components such as starch granules, cell walls,
Figure 6 shows the inner cotyledon structure; composed of protein bodies, and other fine features. For example, the
parenchyma cells containing numerous starch granules sur- structure and composition of the seed coat layers and cotyle-
rounded by protein. The parenchyma cells are round to ellip- dons for three genotypes differing significantly in morphol-
soid; when elongated, they tend to lie in the direction from ogy and internal structures are shown in this study.
the cotyledon center toward the abaxial side. Kabuli seeds Figures 9 and 10 have been constructed to show a more
show more blue fluorescence of the parenchyma cell walls, accurate and informative model of desi and kabuli seed coat
indicating a greater content of b-glucan-type polysaccharides structures, respectively. The different staining techniques
than in desi seeds (fig. 6B, 6D). Cell walls in the parenchyma used clearly demonstrate the weak cell structure of the kabuli
layer are surrounded by nonfluorescing material, probably seed coat compared with the stronger, thickened cell struc-
pectic polysaccharides present in the middle lamella and in- ture of the desi seed coat. Moreover, these constructions visu-
tracellular spaces. Cotyledonary cells of the interspecific line ally depict and highlight both structural and compositional
have a slightly thicker cell wall lining than the other chick- differences between chickpea types and between the tissues
peas (fig. 6). Cell wall thickening is more pronounced in cells within each seed coat. As such, they will provide useful refer-
that are closer to the edge of the cotyledons for the three ge- ences for future chickpea research.
notypes examined.
Bright-field staining of kabuli parenchyma cells shows very
small organelles overlapping protein-stained tissue (fig. 6A).
The presence of organelles is confirmed by nonfluorescing
areas in the fluorescence image (fig. 6B). The desi genotypes
have fewer of these organelles in their parenchyma cells (figs.
6C, 8D). They are generally very small in the interspecific
line, with a few large organelles similar in size to starch gran-
ules and even less abundant in the desi genotype Amethyst.
Figure 7 shows that the interior cotyledon cells of chick-
peas are protein packed, containing starch granules and trans-
parent organelles. The starch granules are elliptical, with a
central dimple (the hilum of the starch granule) and clearly
visible growth rings (concentric layers of crystalline and amor-
phous structures). While the clear organelles often make the
protein appear globular in nature, the protein actually streams
around the organelles (fig. 7A). The dimpled shape of the
starch granule is most pronounced in the fluorescence images,
whereas the growth rings are more evident using bright-field
staining (fig. 7B). Fig. 4 Fluorescence image (310) of a cotyledon corner from desi
A third cell type within cotyledons is smaller, protein filled, cv. Amethyst. The abaxial cotyledon face adjoins the seed coat (right
but devoid of starch granules. These cells congregate in irreg- side), and the adaxial face adjoins the other cotyledon (top side). A
ular bunches and may be vascular bundles within the cotyle- single continuous layer of epidermal cells extends around both sur-
dons (fig. 6C). faces of each cotyledon. Bar ¼ 100 mm.
 WOOD ET AL.—MORPHOLOGY OF CHICKPEA SEEDS 637

Fig. 5 Cross sections of the intercotyledon junction and adaxial surfaces. A, Kabuli (cv. Bumper), bright field (340). B, Kabuli (cv. Bumper),
fluorescence (320). C, Interspecific desi genotype (90101-57Q), bright field (340). D, Interspecific desi genotype (90101-57Q), fluorescence (320).
cw, cell wall; e, epidermis. Arrows point to organelles. Bar = 100 mm.

The seed coat and cotyledons of kabuli seeds did not sepa- vars grown in India in 1982–1983. Environmental factors are
rate completely during the staining and fixing procedure, un- likely to have small contributions to differing seed coat thick-
like the complete separation that occurred in desi seeds. Ravi ness, similar to seed coat content (Wood et al. 2008).
and Harte (2009) also showed that kabuli seed coats were The inner and outer palisade regions of desi seeds color
more difficult to remove than those of desi seeds. This is one differently after fluorescence staining, indicating differences
main reason that kabuli types are not decorticated to pro- in carbohydrate composition. These two regions are actually
duce dhal as is traditionally done with desi types. two separate cell layers of palisade/sclerenchyma cells. The
The kabuli chickpea (cv. Bumper) has a thinner seed coat line dividing these regions is not a light line (lina lucida), an
with thinner cell walls (no secondary thickening of palisade optical phenomenon that often results from the presence of
cells) compared with the desi genotypes, whose seed coats compact cellulose bundles at the ends of palisade cells in the
contain thickened sclerenchyma cells (macrosclerids) with outer palisade region (Jin et al. 1997). Rather, it is two rows
small or absent lumen. Desi seed coats were 1.2–1.7 times of palisade/sclerenchyma cells, consistent with the findings of
thicker than those of kabuli, much less than the threefold dif- Hassan (2000).
ference observed by Singh et al. (1984). Seed coat content is Webb (2003) characterized palisade/sclerenchyma cells of
mainly under genetic control and is negatively correlated mung bean (Vigna radiata) as having thick secondary walls
with seed size (Wood et al. 2008). This implies that the seed composed of highly organized cellulose microfibrils that are
coat thickness would also be primarily under genetic control. often lignified. Rangaswamy and Nandakumar (1985) found
Hence, the thinner desi seed coats observed in this study that the secondary thickening of palisade cells in the seed
probably result from genetic differences between our modern coat of snout bean (Rhynchosia minima) was compartmen-
Australian cultivars and the older Indian cultivars examined talized and stained for pentosan, while the outer region of
by Singh et al. (1984). Modern Australian desi cultivars prob- the wall was homogenous and cellulosic. A predominantly
ably have thinner seed coats than the more traditional culti- polysaccharide-cellulose composition of chickpea palisade/
638 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 6 Cross sections (320) of the inner cotyledon region, showing cellular structure. A, Kabuli (cv. Bumper), bright field. B, Kabuli (cv.
Bumper), fluorescence. C, Interspecific desi genotype (90101-57Q), bright field. D, Interspecific desi genotype (90101-57Q), fluorescence. cw, cell
walls; is, intercellular spaces; nf, nonfluorescing material in middle lamella (pectic polysaccharides); p, protein; s, starch granules; vb, vascular
bundle. Arrows point to organelles. Bar = 100 mm.

sclerenchyma cells, similar to other legume seeds, is consis- Beneath the palisade/sclerenchyma region is the hypoder-
tent with the staining patterns observed in this study. These mal layer, comprising a single layer of thick-walled osteo-
suggest that the outer palisade layer of desi seed coats con- sclereids (hourglass cells) that are divided by air spaces
tain more b-glucan-type polysaccharides than the inner pali- (McKee et al. 1977; Hassan 2000). Hassan (2000) found
sade layer, while the inner layer also has a protein content these cells to be uniformly thickened in chickpea, but we ob-
that is absent in the outer layer. Polyphenolic compounds served more thickening and stronger b-glucan staining on the
such as anthocyanins, proanthocyanidins, and certain flavo- distal portion of the cell compared with the proximal por-
nol glycosides and isoflavonoids are known to contribute to tion. Overall, the seed coat of the interspecific line differed
color of legume seed coats (Beninger et al. 2000). In desi little from the other desi chickpea genotype examined, except
chickpea, the palisade/sclerenchyma cells are responsible for for heterogeneity in hourglass cell shape. Hassan (2000) re-
seed coat color and have been shown to contain polyphenolic ported heterogeneity of hypodermal cells to be a signature
compounds (Singh et al. 1984). Moreover, desi types are trait of Cicer echinospermum, and this appears to have been
likely to have some lignification of these cells as shown by inherited by the interspecific line. Any role that this unique
the thickening. A comparable situation occurs in lima beans, trait may portray in seed dormancy or, more generally, in
where lignin contents of dark-colored varieties are higher seed protection is unclear, as is its effect on industrial seed
than those of light-colored varieties (Kannenburg and Allard processing.
1964). However, the seed coat of pea (Pisum sativum) is an The parenchyma region of the seed coat lies beneath the
example of secondary cell wall thickening without lignifica- hourglass cells (or osteosclerids). Its layer consists of large
tion (Harris 1984). Determination of chemical composition open parenchyma (or mesophyll) cells that became more
will be needed to confirm the presence or absence of lignin in compressed toward the inner boundary. The most com-
the seed coat of chickpea. pressed area fluoresces strongly, suggesting the presence of
 WOOD ET AL.—MORPHOLOGY OF CHICKPEA SEEDS 639

Fig. 7 Bright-field close-up (3100) of cotyledon cell. A, Kabuli (cv. Bumper). B, Interspecific desi genotype (90101-57Q). Crystalline structure
of starch granules is visible as concentric rings. cw, cell wall; is, intracellular space; p, protein matrix; s, starch granules. Arrows point to
organelles. Bar ¼ 10 mm.

pectic polysaccharides. This class of polysaccharide, such ied that support the exalbuminous nature of chickpea. The
as rhamnogalacturonans (with galactan or arabinan side aleurone and endosperm present in the developing seed ap-
chains), has been found in the cell walls of pea seed coat, pear to have been sequestered early for seed growth and
with concentrations varying by location and developmental development, a process observed in pea (P. sativum; Mari-
stage (McCartney and Knox 2002). Similarly, during seed nos 1970; Hardham 1976) and bean (Phaseolus vulgaris;
development in pea and lima bean, Reeve (1946a, 1946b) Yeung 1990). b-glucans are most concentrated in the sub-
found a pentosan-cellulose complex in the secondary wall aleurone layers of the endosperm in barley and other cereal
thickenings of the palisade/sclerenchyma and hourglass cells grains (Fulcher et al. 1972, 1977), and it is possible that
and pectins in the middle lamellae. All cellular regions the compressed parenchymatous and cuticular area of
(palisade/sclerenchyma, hypodermis, and parenchyma) of the the chickpea seed coat contains remnants of aleurone and
seed coat have the potential to contain tannin and pigments endosperm that were present in the early stages of seed de-
(McKee et al. 1977); however, in chickpea seed coats, we velopment. Since the endosperm of endospermic legumes,
have confirmed that the color resides only in the palisade/ such as guar, is composed mostly of galactomannan (Mc-
sclerenchyma region. Clendon et al. 1976), the presence of endospermic remnants
There appears to be a cuticle beneath the parenchyma in the innermost area of the chickpea seed coat cannot be
region, with staining indicating the presence of pectic discounted.
polysaccharides and proteaceous material. No endosperm or The periphery of chickpea cotyledons has a continuous
aleurone layers were observed in any of the genotypes stud- outer layer of single epidermal cells that have a cuticle molded

Fig. 8 Images (310) of the hilar region of the seed coat of desi cultivar Amethyst. A, Bright field. B, Fluorescence. hi, hilar area (aerial
cross section of palisade cells); hyp, hourglass cells of the hypodermis; ip, inner palisade; mp, micropyle; op, outer palisade; par, parenchyma cells.
Bar ¼ 10 mm.
640 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 9 Longitudinal section of Cicer arietinum L. (cv. Amethyst), showing the structural anatomy of the desi seed coat (320).

neatly over their outer surface. These cells are protein filled, lipid, hemicellulose, or pectinaceous compounds (Singh and
devoid of starch granules, and slightly larger and more ellip- Mathur 2004).
soid in shape on the adaxial face of the cotyledon. This is Subepidermal cells are present on the abaxial side of the
similar to seeds of P. vulgaris, in which epidermal cells are cotyledon. They are protein filled but without starch gran-
also much smaller than the cotyledon parenchyma cells ules, larger than the epidermal cells (but not as large as the
(Öpik 1968). The cuticle exhibited similar staining to the cell inner cotyledon cells), and irregularly arranged in layers one
walls, possibly containing pectic polysaccharides. No men- to three cells deep. The adaxial side of the cotyledon does
tion of the composition of cuticles on the inner seed coat and not have the same subepidermal profile, but it does contain
cotyledon surfaces could be found in the literature. However, small cells filled with protein as well as some small starch
the external cuticle of the seed coat is composed of waxy, granules. Öpik (1968) noted a subepidermal layer of small
 WOOD ET AL.—MORPHOLOGY OF CHICKPEA SEEDS 641

Fig. 10 Longitudinal section of Cicer arietinum L. (cv. Bumper), showing structural anatomy of the kabuli seed coat (320).

cells on the abaxial side of P. vulgaris cotyledons and also cell walls observed in images of cereal grains with similar
that both epidermal and subepidermal cells were less heavily staining (Andersson et al. 1999; Autio and Salmenkallio-Marttila
packed with reserves than were the inner cotyledon cells. Cu- 2001; Choct et al. 2001). We conclude that chickpea cotyle-
bero (1987) labeled the epidermal and subepidermal layers of don cell walls contain fewer b-glucan-pectic polysaccharides
cells in chickpea the ‘‘aleurone’’ layers, but we contend that than do cells of cereal endosperm.
these cells are essentially epidermal cells. The cell walls of the cotyledon parenchyma cells were thick-
The substantive part of the cotyledon, composed of paren- ened and surrounded by nonfluorescing material both inside
chyma cells, is slightly larger in the kabuli seeds than in desi the cell walls (middle lamella) and in the intracellular spaces.
seeds. These are round to ellipsoid and tend to be elongated The middle lamella are much thicker than those observed in
from the center of the cotyledons toward the abaxial side, cereal endosperms (Choct et al. 2001), and this thickening is
consistent with observations of other legume seeds by Irving more pronounced in cells closer to the cotyledon edge. The in-
(1984) and Otto et al. (1997). terspecific line appeared to have slightly more thickening than
The kabuli chickpea cotyledon cell walls fluoresced blue the other genotypes. Strong staining for cellulose in P. vulgaris
more strongly than those of the desi genotypes, indicating a cell walls and an absence of lignin (Öpik 1968) suggest that
higher content of pectin polysaccharides. None of the chick- the thickening observed in chickpea is due to pectic polysac-
pea cotyledon cell walls fluoresced blue green as strongly as charide accumulation rather than lignification.
642 INTERNATIONAL JOURNAL OF PLANT SCIENCES

The parenchyma cells in the cotyledons contained large, cotyledons, and all other parts of a plant, are linked to form
bean-shaped, starch granules enclosed by protein. Growth an integrated network of vascular supply (Carland et al. 1999;
rings (concentric layers of crystalline and amorphous struc- Endo et al. 2005).
ture) and a centralized hilum were clearly visible in the ellip- This is the first reported study examining chickpea seed tis-
tical starch granules. The size of the starch granules of the sues using light microscopy with differential staining for pro-
genotypes in this study were similar to those reported by tein, starch, b-glucan-type polysaccharides, and nonfluorescing
Hoover and Ratnayake (2002), who found a range from 14 compounds, and it has confirmed the basic morphology of
to 31 mm for one desi and one kabuli cultivar. Cicer arietinum seeds and highlighted differences between desi
Otto et al. (1997) also noted that flour fractions from the and kabuli types and a C. arietinum 3 Cicer echinospermum
cotyledon interior of chickpea and pea were higher in starch interspecific genotype. It has also provided additional informa-
and lower in protein, lipid, and fiber than were those of the tion about the specific locations and composition of tissues,
outer cotyledon. This is consistent with our observation that cells, and their components. Moreover, comprehensive visual
cells deeper within the cotyledon were more densely packed depiction of chickpea seed structure, including seed coat mor-
with starch granules than were those at the cotyledon periphery. phology of desi and kabuli types, has been compiled for future
Organelles that were nonfluorescing but clear in the bright- research purposes.
field images were observed in the cotyledon cells of all chick- Characterization of the microstructure and composition of
pea genotypes. They were generally very small in all genotypes components within legume seeds will aid in the understand-
and were much more abundant in the kabuli line and least ing of species and genotypic differences in processes such as
abundant in the desi genotype, Amethyst. The interspecific decortication, splitting (separating cotyledons), flour milling,
desi line also had these small organelles, and a few of them seed hydration, germination, and cooking. Further work is
approached the size of starch granules. The precise nature of required to investigate these associations.
these organelles is unclear, but they may be lipid globules
(elaioplasts), chromoplasts (containing carotenoids), or vacu- Acknowledgments
oles (possibly containing polyphenolic compounds). Staining for
lipids (lysochromy) and phenolic compounds would be necessary We thank Patrick Littlefield, manager of the electron mi-
to determine the exact nature of these structures. croscope unit at the University of New England, Armidale,
Areas of smaller cells that are bunched together and devoid New South Wales, and Barry Jensen, designer in the publish-
of starch granules are scattered within the cotyledon interior. ing unit at Industry and Investment NSW, Orange, New South
These structures may be related to vascular bundles, since Wales.

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