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

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.

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

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market value is decreased (Norton and Boe, 1998). In general
cuttings of hybrid roses are difficult to root. Tissue culture
methods have been developed as a potential tool for rapid and
mass propagation in number of plant species. The central
concept of tissue culture is totipotency i.e., every living cell
has the genetic information needed to develop into complete
organism (Khan and Shaw, 1988) [14].
Micropropagation of plants through tissue culture has been
considered as an important and very popular method to
produce plants which are very difficult to propagate
conventionally by seeds and other natural means. The great
benefit of in vitro propagation technique is the enormous
multiplicative capacity to produce disease free plants in a
relative short period of time with independent of seasonal
factor in a cost effective manner. The plantlets developed
through tissue culture reduce input costs, increase effective
management and enable market pricing because of
contamination and disease free products. Although vegetative
propagative method like cutting, layering, budding and
grafting is a predominant technique in roses, yet it does not
ensure healthy and disease free plants (Dhawan and
Bhojwani, 1986) [6]. Various tissue culture techniques, such as
propagation, may decrease propagation time and could
virtually eliminate the need for grafting onto root stocks;
propagation has been shown to be a highly effective method
of rapidly propagating disease free, uniform rose plants
(Wang et al., 2002) [24].
Plant tissue culture is a propagation technique widely used in
modern agriculture that allows a complete plant to be grown
from a single plant cell. Tissue culture is considered an
asexual propagation technique since it only involves the cells
from a single parent plant. Asexual propagation techniques
produce plants that are genetically identical to the parent plant
and to each other (Kane, 1991) [13].
Eventually, most of the new growth in the plant becomes
restricted to specific areas at the tips of the stems and roots
called meristems. The cells that compose the meristems
(meristematic cells) are relatively unspecialized and retain the
ability to become any of the specialized cells in the plant.
Meristematic cells are usually the preferred cell to initiate
new plants since they begin to develop into stems and leaves
very quickly. The use meristematic cells from a part of the
plant called an axillary bud (Hasegawa, 1980) [10].
All of the different cells in a plant must develop and work
together in a coordinated manner in order to carry out the
various processes necessary for the plant to live. During
normal development, the specialized cells within the plant are
produced at the proper times in response to growth
stimulating and regulating chemicals called hormones. During
tissue culture, the hormones must be supplied artificially to
the plant at the proper time. Two important classes of
hormones used in tissue culture are cytokinins and auxins.
These hormones promote division and specialization of cells,
and later development of stems, leaves, and roots. It is often
necessary to treat the developing plant with different
hormones at different times because a hormone that promotes
stem and leaf development may inhibit root formation
(Hyndman et al, 1982) [11]. Tissue culture techniques should
minimize the time necessary for the introduction of new
cultivars into the commercial market and thus increase the
availability of plants with improved horticultural
characteristics (Previati et al., 2008) [21]. To establish an in
vitro flowering research system, it is necessary to develop a
reliable and rapid shoot organogenesis protocol. In the present
study planned an efficient tissue culture technique to yield
large number of shoots from nodal explants of rose in
controlled condition segmented with different hormonal
effects on in vitro propagation in rose (Rosa hybrida L.).
Materials And Methods
The experiment was conducted in Tissue Culture Laboratory,
Plant Breeding and Genetics Division at Nuclear Institute of
Agriculture, (NIA), Tando Jam. Fresh plant materials (lateral
buds) were collect from the rose plant grown in garden at
Nuclear Institute of Agriculture (NIA), Tando Jam. The
excised young and mature shoot tips were washed in running
water for ten minutes. The Murashige and Skoog (1962) [17]
medium 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,
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
were used for root induction. Rose explants excrete phenolic
compounds, which caused browning of media and mortality
of explants. Therefore, activated charcoal (0.5 mg L-1) was
added to MS media to control browning. Nodal explants
containing lateral buds of actively field grown rose were used
for multiplication in the experiment. They were cut in 3-4 cm
length segments and surface disinfested using 70% ethanol
for 30 seconds and then immersed in 10 % sodium
hypochlorite solution of commercial laundry bleach (5.25%
NaOCl) containing 2 drops of Tween-20 emulsifier to aid
wetting for 20 minutes. The pH of medium was adjusted at
5.7-5.8 before autoclaving and media was autoclaved at 121
°C and 1.05 kgcm2 (15-20 psi) for 20 minutes. Uniform culture
conditions were maintained as 16-hour photoperiod at 25±2
oC for growth temperature. The experiments were laid out in
completely randomized design (CRD) with three replications.
The days to initiation, number of shoots bottle-1, shoot length
(cm) bottle-1, number of leaves bottle-1, number of roots
bottle-1, number of root length bottle-1 were recorded. The
experimental data were recorded and subjected to factorial
design of analysis of variance (ANOVA) under linear models
of statistics to observe statistical differences among different
traits of wheat using computer program, Student Edition of
Statistix (SWX), Version 8.1 (Copyright, 2005, Analytical
Softwear-USA). Further least significant difference (LSD)
test was also applied to test the level of significance among
different combination means (Gomez and Gomez, 1984) [8].
Results And Discussions
Shoot induction
The statistical analysis of variance showed that days to
initiation, number of shoots, shoot length and number of
leaves were highly significant at 5% probability level and
data are presented in Appendix-I, Table 1. The results showed
that early days to initiation was recorded 10.00 days, while,
maximum number of shoots bottle-1 (7.00), shoot length (6.79
cm) bottle-1 and number of leaves bottle-1 (11.00) were
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
bottle-1 (5.00), shoot length (5.57 cm) bottle-1 and number of
leaves bottle-1 (9.66) were recorded 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 (1.66), shoot length (3.05
cm) bottle-1 and number of leaves bottle-1 (2.66) were
recorded under the concentration of MS + 30 g L-1 sugar.
Yan et al., (1996) [25]; Ara et al., (1997) [3] reported that the
most important technique of micropropagation. The meristem
proliferation in which apical buds or nodal segments having
an axillary bud are cultured to regenerate multiple shoots
without any intervening callus phase achieved that in spite of
many improvements for some cultivars of roses and observed
that the influence and interaction of growth regulators (BA,
NAA) and carbohydrates (sucrose, glucose) in multiplication
of rose cultivars. The proliferation with BAP and NAA
significantly increased number of new green leaves and
axillary shoots and leaves. The results supported by Skirvin et
al, (1990) [23] Pati et al (2001) [20], Allahverdi et al, (2010) [1].
The results fully support by Asad et al. (2010) [4] that the
treatment containing BAP was found to be the best one for
shoot regeneration from nodal segments. The treatment with
NAA in combination with BAP was found to be suitable
treatments for production from leaf explants for
micropropagation.
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.)
MS + Concentrations Days to
initiation Number of shoots
bottle-1 Shoot length (cm)
bottle-1 Number of leaves
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
L-1 sugar 12.00 c 4.33 b 4.97 b 5.66 b
MS + NAA 0.5 mg L-1 + BAP 0.5 mg L-1 + 30
g L-1 sugar 12.66 c 5.00 b 5.57 b 9.66 a
MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30 g
L-1 sugar 10.00 d 7.00 a 6.79 a 11.00 a
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
The statistical analysis of variance showed that number of
roots and root length were highly significant at 5%
probability level and data are presented in Appendix I, Table
2. The results showed that maximum number of roots and
root length were obtained (3.00 and 3.75 cm) under MS½ +
IBA 2 mg L-1 + 30 g L-1 sugar, followed by (2.00 and 2.55
cm) with concentration of MS½ + IBA 1 mg L-1 + 30 g L-1
sugar, while minimum number of roots and root length were
obtained (0.33 and 0.90 cm) under MS½ + 30 g L-1 sugar.
IBA is an auxin plant growth regulator used to promote and
accelerate root formation of plant. IBA is also used on
ornamental turf to promote growth development of flowers
and fruit to increase crop yields. It is the most effective and
widely used for rooting of plantlets. IBA is an auxin plant
growth regulator to promote and accelerate root formation of
plant. IBA is also to improve growth and development for
rooting in flowers. It is the most effective and widely used for
rooting of plantlets and these results supported by Ozel and
Arsalan (2006) [19] and Chakrabarty et al., (2000) [5]. The
results agreed with Asad et al. (2010) [4] that medium
containing MS½ prepared with 1.0 mg L-1 IBA proved best
root induction in rose.
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
MS½ + 30 g L-1 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
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
Conclusion
The results of the experiment for the conclusion that the
concentration of MS + NAA 0.1 mg L-1 + BAP 2 mg L-1 + 30
g sugar L-1 performed very well for shoot induction, while
MS½ + IBA 2 mg L-1 + 30 g L-1 sugar proved best for root
induction under in vitro condition in rose.
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