International Journal of Biology Research

ISSN: 2455-6548
www.biotechjournals.com
Volume 1; Issue 1; March 2016; Page No. 23-27

Propagation of Rose (Rosa Hybrida L.) Under Tissue Culture Technique

1
Faiza Nizamani, 2 Ghualm Shah Nizamani, 3 Muhammad Rashid Nizamani, 4Saeed Ahmed, 5 Nazeer Ahmed
1
Department of Plant Breeding & Genetics, Sindh Agriculture University, Tando Jam-Pakistan
2
Nuclear Institute of Agriculture, Tando Jam-Pakistan
3
College of Forestry, Northwest A&F University, Yangling China.
4
State University of Londrina Centre of Agriculture Sciences Londrina, Parana (PR) Brazil.
5
Department of Entomology, The University of Agriculture,Peshawar-Pakistan.

Abstract
The rose is the most popular ornamental plant in the world, as well as the most important cut flower. Throughout history no other
plant has such wide appeal and been the center of so much attention than the Rose. Roses are one of the world's most important
ornamentals for a long time and are most often used for ornamental, medicinal and aromatic purposes. The experiment was
conducted at Tissue Culture Laboratory; Plant Breeding and Genetics Division, Tando Jam during 2014. The aim of present
investigation was to determine appropriate basal medium and growth regulators for in vitro propagation of Rosa hybrida from
nodal meristem explants. The basal medium of Murashige and Skoog (1962) containing with different concentrations of MS + 30 g
L-1 sugar, MS + BAP 0.5 mg L-1 + 30 g L-1 sugar, MS + IBA 0.1 mg L-1 + BAP 5 mg L-1 + 30 g L-1 sugar, MS + NAA 0.5 mg L-1 +
BAP 0.5 mg L-1 + 30 g L-1 sugar, MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30 g L-1 sugar for shoot induction and MS½ + 30 g L-1
sugar, MS½ + NAA 1 mg L-1 + 30 g L-1 sugar, MS½ + NAA 2 mg L-1 + 30 g L-1 sugar, MS½ + IBA 1 mg L-1 + 30 g L-1 sugar,
MS½ + IBA 2 mg L-1 + 30 g L-1 sugar for root induction were used in this study. The statistical analysis of variance showed that
days to initiation, number of shoots, shoot length, number of leaves, number of roots and root length were highly significant at 5%
probability level. The results showed that early days to initiation was recorded, maximum number of shoots bottle-1, shoot length
bottle-1 and number of leaves bottle-1 were recorded under the concentration of MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30 g L-1
sugar, followed by number of shoots bottle-1, shoot length bottle-1 and number of leaves bottle-1 were obtained under the
concentration of MS + NAA 0.5 mg L-1 + BAP 0.5 mg L-1 + 30 g L-1 sugar and minimum number of shoots bottle-1, shoot length
bottle-1 and number of leaves bottle-1 were recorded under the concentration of MS + 30 g L-1 sugar. The results indicated that
maximum number of roots and root length were achieved under MS½ + IBA 2 mg L-1 + 30 g L-1 sugar and minimum number of
roots and root length were recorded under MS½ + 30 g sugar L-1. IBA is an auxin plant growth regulator used to promote and
accelerate root formation of plant. It was concluded from this study that MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30 g L-1 sugar
for shoot induction and MS½ + IBA 2 mg L-1 + 30 g L-1 sugar proved best for root induction in rose.

Keywords: Rose, Tissue Culture Technique

Introduction special place in our culture as there is hardly any event where
Rose (Rosa hybrida L.) is the most popular of the flowers roses are not displayed. Rose production has great potential in
because of its beauty and fragrance that is why it is rightly Pakistan because it has an agricultural economy with diverse
called the queen of flowers. The genus Rosa contains more climatic conditions (Khan, 2005) [15].
than 1400 cultivars and 150 species, which are grown for There are more than 20,000 commercial cultivars, which
rootstocks, curiosity value and striking floral display. Apart collectively are based on only 8 wild species (Kim et al.,
from its ornamental value, it is also used for the production of 2003) [16]. They belong to the Rosaceae and are grown
essential oil and vitamin C and is rightly called the queen of worldwide as cut flowers and potted plants and in home
flowers Jafar et al, (2005) [12]. Roses are best known as gardens. The flowers vary greatly in size, shape and color.
ornamental plants grown for their flowers in the garden and They serve as rootstocks, onto which other species or
sometimes indoors. Rose is one of the most important cultivars are grafted to increase their rate of propagation; they
commercial flower crop used in the floriculture and cut supply the cut-flower market used in the extraction of attar as
flower industry throughout the world (Rajeshbabu et al, 2014) rose oil (Hameed et al., 2006) [9]. Conventionally, it is
[22]
. The rose is one of the leading cut flowers in the global propagated asexually through cuttings, budding or grafting
floriculture trade and is used at almost every event in both scion cultivars on specific rootstocks in the particular seasons.
local and international markets. The major rose producing These methods are laborious and time taking with very low
countries of the world include Netherlands, Colombia, Kenya, percentage of success. It has also been observed that plants
Israel, Italy, United States, and Japan (Evans, 2009) [7]. Rose raised from these methods are infected with different diseases
has always been the favorite flower in Pakistan and has a that affect flower production and quality, and ultimately their

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market value is decreased (Norton and Boe, 1998). In general vitro flowering research system, it is necessary to develop a
cuttings of hybrid roses are difficult to root. Tissue culture reliable and rapid shoot organogenesis protocol. In the present
methods have been developed as a potential tool for rapid and study planned an efficient tissue culture technique to yield
mass propagation in number of plant species. The central large number of shoots from nodal explants of rose in
concept of tissue culture is totipotency i.e., every living cell controlled condition segmented with different hormonal
has the genetic information needed to develop into complete effects on in vitro propagation in rose (Rosa hybrida L.).
organism (Khan and Shaw, 1988) [14].
Micropropagation of plants through tissue culture has been Materials And Methods
considered as an important and very popular method to The experiment was conducted in Tissue Culture Laboratory,
produce plants which are very difficult to propagate Plant Breeding and Genetics Division at Nuclear Institute of
conventionally by seeds and other natural means. The great Agriculture, (NIA), Tando Jam. Fresh plant materials (lateral
benefit of in vitro propagation technique is the enormous buds) were collect from the rose plant grown in garden at
multiplicative capacity to produce disease free plants in a Nuclear Institute of Agriculture (NIA), Tando Jam. The
relative short period of time with independent of seasonal excised young and mature shoot tips were washed in running
factor in a cost effective manner. The plantlets developed water for ten minutes. The Murashige and Skoog (1962) [17]
through tissue culture reduce input costs, increase effective medium containing with different concentrations of MS + 30
management and enable market pricing because of g L-1 sugar, MS + BAP 0.5 mg L-1 + 30 g L-1 sugar, MS +
contamination and disease free products. Although vegetative IBA 0.1 mg L-1 + BAP 5 mg L-1 + 30 g L-1 sugar, MS + NAA
propagative method like cutting, layering, budding and 0.5 mg L-1 + BAP 0.5 mg L-1 + 30 g L-1 sugar, MS + NAA 0.1
grafting is a predominant technique in roses, yet it does not mg L-1 + BAP 2 mg L-1 + 30 g L-1 sugar for shoot induction,
ensure healthy and disease free plants (Dhawan and MS½ + 30 g L-1 sugar, MS½ + NAA 1 mg L-1 + 30 g L-1
Bhojwani, 1986) [6]. Various tissue culture techniques, such as sugar, MS½ + NAA 2 mg L-1 + 30 g L-1 sugar, MS½ + IBA 1
propagation, may decrease propagation time and could mg L-1 + 30 g L-1 sugar, MS½ + IBA 2 mg L-1 + 30 g L-1 sugar
virtually eliminate the need for grafting onto root stocks; were used for root induction. Rose explants excrete phenolic
propagation has been shown to be a highly effective method compounds, which caused browning of media and mortality
of rapidly propagating disease free, uniform rose plants of explants. Therefore, activated charcoal (0.5 mg L-1) was
(Wang et al., 2002) [24]. added to MS media to control browning. Nodal explants
Plant tissue culture is a propagation technique widely used in containing lateral buds of actively field grown rose were used
modern agriculture that allows a complete plant to be grown for multiplication in the experiment. They were cut in 3-4 cm
from a single plant cell. Tissue culture is considered an length segments and surface disinfested using 70% ethanol
asexual propagation technique since it only involves the cells for 30 seconds and then immersed in 10 % sodium
from a single parent plant. Asexual propagation techniques hypochlorite solution of commercial laundry bleach (5.25%
produce plants that are genetically identical to the parent plant NaOCl) containing 2 drops of Tween-20 emulsifier to aid
and to each other (Kane, 1991) [13]. wetting for 20 minutes. The pH of medium was adjusted at
Eventually, most of the new growth in the plant becomes 5.7-5.8 before autoclaving and media was autoclaved at 121
restricted to specific areas at the tips of the stems and roots °C and 1.05 kgcm2 (15-20 psi) for 20 minutes. Uniform culture
called meristems. The cells that compose the meristems conditions were maintained as 16-hour photoperiod at 25±2
o
(meristematic cells) are relatively unspecialized and retain the C for growth temperature. The experiments were laid out in
ability to become any of the specialized cells in the plant. completely randomized design (CRD) with three replications.
Meristematic cells are usually the preferred cell to initiate The days to initiation, number of shoots bottle-1, shoot length
new plants since they begin to develop into stems and leaves (cm) bottle-1, number of leaves bottle-1, number of roots
very quickly. The use meristematic cells from a part of the bottle-1, number of root length bottle-1 were recorded. The
plant called an axillary bud (Hasegawa, 1980) [10]. experimental data were recorded and subjected to factorial
All of the different cells in a plant must develop and work design of analysis of variance (ANOVA) under linear models
together in a coordinated manner in order to carry out the of statistics to observe statistical differences among different
various processes necessary for the plant to live. During traits of wheat using computer program, Student Edition of
normal development, the specialized cells within the plant are Statistix (SWX), Version 8.1 (Copyright, 2005, Analytical
produced at the proper times in response to growth Softwear-USA). Further least significant difference (LSD)
stimulating and regulating chemicals called hormones. During test was also applied to test the level of significance among
tissue culture, the hormones must be supplied artificially to different combination means (Gomez and Gomez, 1984) [8].
the plant at the proper time. Two important classes of
hormones used in tissue culture are cytokinins and auxins. Results And Discussions
These hormones promote division and specialization of cells, Shoot induction
and later development of stems, leaves, and roots. It is often
necessary to treat the developing plant with different The statistical analysis of variance showed that days to
hormones at different times because a hormone that promotes initiation, number of shoots, shoot length and number of
stem and leaf development may inhibit root formation leaves were highly significant at 5% probability level and
(Hyndman et al, 1982) [11]. Tissue culture techniques should data are presented in Appendix-I, Table 1. The results showed
minimize the time necessary for the introduction of new that early days to initiation was recorded 10.00 days, while,
cultivars into the commercial market and thus increase the maximum number of shoots bottle-1 (7.00), shoot length (6.79
availability of plants with improved horticultural cm) bottle-1 and number of leaves bottle-1 (11.00) were
characteristics (Previati et al., 2008) [21]. To establish an in recorded under the concentration of MS + NAA 0.1 mg L-1 +
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BAP 2 mg L-1 + 30 g L-1 sugar, followed by number of shoots that the influence and interaction of growth regulators (BA,
bottle-1 (5.00), shoot length (5.57 cm) bottle-1 and number of NAA) and carbohydrates (sucrose, glucose) in multiplication
leaves bottle-1 (9.66) were recorded under the concentration of of rose cultivars. The proliferation with BAP and NAA
MS + NAA 0.5 mg L-1 + BAP 0.5 mg L-1 + 30 g L-1 sugar and significantly increased number of new green leaves and
minimum number of shoots bottle-1 (1.66), shoot length (3.05 axillary shoots and leaves. The results supported by Skirvin et
cm) bottle-1 and number of leaves bottle-1 (2.66) were al, (1990) [23] Pati et al (2001) [20], Allahverdi et al, (2010) [1].
recorded under the concentration of MS + 30 g L-1 sugar. The results fully support by Asad et al. (2010) [4] that the
Yan et al., (1996) [25]; Ara et al., (1997) [3] reported that the treatment containing BAP was found to be the best one for
most important technique of micropropagation. The meristem shoot regeneration from nodal segments. The treatment with
proliferation in which apical buds or nodal segments having NAA in combination with BAP was found to be suitable
an axillary bud are cultured to regenerate multiple shoots treatments for production from leaf explants for
without any intervening callus phase achieved that in spite of micropropagation.
many improvements for some cultivars of roses and observed

Table 1: Days to initiation, number of shoots bottle-1, shoot length (cm) bottle-1, and number of leaves bottle-1 as affected under different
concentrations of phytohormones in rose (Rosa hybrida L.)
Days to Number of shoots Shoot length (cm) Number of leaves
MS + Concentrations
initiation bottle-1 bottle-1 bottle-1
MS + 30 g L-1 sugar 18.00 a 1.66 d 3.05 c 2.66 c
MS + BAP 0.5 mg L-1 + 30 g L-1 sugar 15.00 b 2.66 c 3.61 c 4.66 b
MS + IBA 0.1 mg L-1 + BAP 5 mg L-1 + 30 g
12.00 c 4.33 b 4.97 b 5.66 b
L-1 sugar
MS + NAA 0.5 mg L-1 + BAP 0.5 mg L-1 + 30
12.66 c 5.00 b 5.57 b 9.66 a
g L-1 sugar
MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30 g
10.00 d 7.00 a 6.79 a 11.00 a
L-1 sugar
Days to initiation SE (0.7601) LSD (5%) (1.7528)
Number of shoots bottle-1 SE (0.3944) LSD (5%) (0.9095)
Shoot length (cm) bottle-1 SE (0.46951) LSD (5%) (1.0827)
Number of leaves bottle-1 SE (0.7601) LSD (5%) (1.7528)

Root induction ornamental turf to promote growth development of flowers

The statistical analysis of variance showed that number of and fruit to increase crop yields. It is the most effective and
roots and root length were highly significant at 5% widely used for rooting of plantlets. IBA is an auxin plant
probability level and data are presented in Appendix I, Table growth regulator to promote and accelerate root formation of
2. The results showed that maximum number of roots and plant. IBA is also to improve growth and development for
root length were obtained (3.00 and 3.75 cm) under MS½ + rooting in flowers. It is the most effective and widely used for
IBA 2 mg L-1 + 30 g L-1 sugar, followed by (2.00 and 2.55 rooting of plantlets and these results supported by Ozel and
cm) with concentration of MS½ + IBA 1 mg L-1 + 30 g L-1 Arsalan (2006) [19] and Chakrabarty et al., (2000) [5]. The
sugar, while minimum number of roots and root length were results agreed with Asad et al. (2010) [4] that medium
obtained (0.33 and 0.90 cm) under MS½ + 30 g L-1 sugar. containing MS½ prepared with 1.0 mg L-1 IBA proved best
IBA is an auxin plant growth regulator used to promote and root induction in rose.
accelerate root formation of plant. IBA is also used on

Table 2: Number of roots bottle-1, number of Root length (cm) bottle-1 as affected under different of phytohormones in rose (Rosa hybrida L.)
MS + Concentrations Number of roots bottle-1 Root length (cm) bottle-1
-1
MS½ + 30 g L sugar 0.33 c 0.90 c
MS½ + NAA 1 mg L-1 + 30 g L-1 sugar 0.66 c 1.66 bc
MS½ + NAA 2 mg L-1 + 30 g L-1 sugar 1.00 bc 2.53 b
MS½ + IBA 1 mg L-1 + 30 g L-1 sugar 2.00 ab 2.55 b
MS½ + IBA 2 mg L-1 + 30 g L-1 sugar 3.00 a 3.75 a
Number of roots bottle-1 SE (0.558) LSD (5%) (1.2862)
Root length (cm) bottle-1 SE (0.4447) LSD (5%) (1.0255)

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 Fig 1: Effect of different concentrations of growth hormones on rose (Rosa hybrida L.)

MS + 30 g L-1 MS + BAP 0.5 mg MS + IBA 0.1 mg L-1 + MS + NAA 0.5 mg MS + NAA 0.1 mg
sugar L-1 + 30 g L-1 BAP 5 mg L-1 + 30 g L-1 L-1 + BAP 0.5 mg L-1 + BAP 2 mg L-1
sugar sugar L-1 + 30 g L-1 sugar + 30 g L-1 sugar

Conclusion use of reduced concentrations of mineral salts. Hort. Sci.,

The results of the experiment for the conclusion that the 1982; 17(1):82-83.
concentration of MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30 12. Jafar Ali, Najma Yaqub Chaudry, Faheem Aftab. In vitro
g sugar L-1 performed very well for shoot induction, while development and improvement of chromium affected
MS½ + IBA 2 mg L-1 + 30 g L-1 sugar proved best for root adventitious root of Solaum of tuberosum L. with GA3
induction under in vitro condition in rose. and application. Pak. J Bot, 2005; 46(2):687-692.
13. Kane Michael. Rose flowers: The tissue culture
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