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Journal of Biotechnology

& Crop Science


5(6): 62-68, 2016

Rapid protocol for micropropagation of rose (Rosa indica L.)


Pankaj Singh, SP Singh
Received: 13 January 2016 Revised Accepted: 04 March 2016

ABSTRACT
In an attempt to find out a standard and rapid protocol of micropropagation of Rose (Rosa indica L.) an experiment was
conducted with different type of explants with various combination of Murashige Stock culture media was used in the present
investigation. It was observed that rose explant with nodal segment was more responsive than shoot tip explants. One of the
problems with nodal explants was phenolics secretion, which retarded the growth of plant in cultured media and caused
browning of media. The problem was overcome by supplementing the some antioxidant and antiphenolic compounds like
Charcoal, PVP, AgNO3, etc. Increasing the concentration of cytokinine exerted positive effect on shoot proliferation and
addition of growth hormone also exerted positive effect on shoot proliferation. Some other factors such as pH, temperature
and light influenced the plant growth in media.

Key Words: Micropropagation, MS cultural media, Plant tissue culture, Rose

INTRODUCTION

Rose is one of the most important commercial crops the scientists around the world for multiplication and
and came to us very early as a source of fragrance, propagation of rose. In the present study an attempt
as a cure for illness and as a symbol of beauty but has been made to standardize a rapid protocol for
not a popular bush for the garden until recently. It is micropropagation of Rose (Rosa indica L.)
important not only for its outstanding aesthetic
beauty but also for its potentiality marketing as cut MATERIALS AND METHODS
flowers and potted plants to many countries of the
world. Roses are generally multiplied vegetatively The plants selected for the present study belong to the
by grafting buds on stems of wild rose and by group Rosa indica grown in the nursery of
cuttings. This conventional met hod of Department of Horticulture Banaras Hindu
p r o p a g a t i o n i s v er y s l o w . Moreover, University. The various explants used were shoot tip,
d i s ea s e a n d environmental hazards make the nodal segment, and immature flower bud. The
cultivar to degenerate gradually. Therefore, this explants were first washed with double distilled water
conventional process is not satisfactory in two to three times to removes dust particles, then
multiplication of Rosa spp. Micropropagation method explants were treated with antibiotics viz citramide
is especially applicable to species in which clonal Tween 20 solution. After that explant were subjected
propagation is needed (Gamborg and Phillips 1995). to surface sterilization using 0.1% solution of HgCl2
Since the rapid clonal multiplication of plants in vitro under laminar air flow for 5 minutes. After
is quicker and cheaper than in vivo, the tissue culture discarding sodium hypochlorite, the explants were
technique has become the first major attraction to washed three times with sterilized distilled water to
remove all the traces of sodium hypochlorite. Each
Pankaj Singh ( ), SP Singh explant was inoculated to MS medium with different
Department of Horticulture Institute of Agricultural Sciences,
Banaras Hindu University Varanasi concentrations of hormones. Care was taken not to
Email: singh.pankaj009@gmail.com dip explants completely in the medium and also tips
of forceps were not allowed to touch the agar

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J of Biotech & Crop Sci (2016) 5(6): 62-68

medium. The culture tubes were sealed immediately the most efficient auxin was NAA followed by IBA
with cotton plug. The same procedure was repeated and IAA. In all the treatments, level beyond 0.5mgl -1
for multiple shoot as well as root formation. Forceps, gave the callusing.
scalpels, needles and other instruments were grown
under 16 hours light and 8 hours dipped in alcohol Effect of cytokinine: Cytokinines viz, BAP, Kinetin
(70%v/v) and were flamed before use. All culture (0.5 to10mgl-1) were used to note their efficiency in
were dark period in air-conditioned culture room, inducing multiple shoot of both the explants and
illuminated by 40W (watts) white fluorescent lights. results have been presented in Table 2. Maximum
The intensity of light was regulated between 2500- shoot proliferation of 55.6 percent observed in the
3000 lux. The temperature of culture room was shoot tip at BAP 2.0mgl-1 after 30.4+3.4 days with
maintained at 250C. For shoot formation from apical Kn 2.0mgl-1. Similarly for nodal segment maximum
meristems and nodal explants different concentration of 65.3 percent sprouting was noted after 24.6+1.8
of IAA, IBA, BAP, Kn and combination of growth days with the treatment BAP 4.0mgl-1, followed by
regulators viz, IAA+BAP, IBA+BAP were used. For 62.4 percent after proliferation in the treatment of
root formation using half strength of MS medium and BAP 1.0mgl-1 after 23.9+3.3 days. In general both the
different concentration of growth regulators like IAA cytokinines were effective in inducing shoot
IBA was used. proliferation but BAP proved better over Kn. Further,
the higher levels of kinetin i.e., 6.0, 10.0mgl-1 gave
RESULTS AND DISCUSSION poor response.

The results of the present study have been presented Effect of BAP and IAA interaction on shoot
in Table 1 to Table 5. The result and discussion proliferation: The interaction effect of the BAP and
related to effect of different auxin on shoot NAA on the day to shoot proliferation, number of
proliferation of shoot tip and nodal segment of Rosa shootlets per explant and average length of shootlets
indica, Effect of various cytokinine on shoot tip and have been given in Table 3 and 4. For the nodal
nodal explants, Effect of some growth regulator on explants segment earlier sprouting was after 20.4+0.9
nodal explants proliferation, Effect of some growth days followed by 22.1+1.1days in the treatment of
regulator on shoot proliferation from shoot tip and BAP 1.0mgl-1 +NAA 1.0mgl-1. High number of
Effect of various auxin singly and in combination on shootlets (5.8+0.1) and 4.4+0.2 shootlets were
rooting in vitro regeneration shootlets of Rosa indica discovered in the treatments of BAP 2.0mgl -
1
of half MS strength are deliberated and discussed +NAA0.1mgl-1and BAP 4.0mgl-1+0.01mgl-1
below. respectively. Longest shoot after 45 days in culture
was reported to be 2.4+0.0 cm in BAP1.0mgl-1+NAA
Effect of auxin: The results of supplemented three 0.1mgl-1 followed by 2.1+0.1cm in BAP 1.0mgl-
1
auxins IAA, NAA, and IBA (0.01to 1.0) on shoot +NAA 0.25mgl-1. However for shoot tip explants
proliferation has been presented in Table 1. The shoot earlier sprouting was recorded after 31.7+2.6 days in
tip explant proliferated earliest on the auxin at the treatment of BAP 4.0mgl-1 +NAA 0.01mgl-1.
0.1mgl-1after 33+2.8 days with the frequency of 50.6 Maximum (2.9+0.3) number of shoots were obtained
percent followed by 46.7 percent after 35.6+1.9 days in the treatment of BAP 2.0mgl-1, and 0.3 shoots were
at NAA 0.25mgl-1. Likewise, for the nodal segment obtained in the treatment of BAP 2.0mgl -1
NAA 0.1mg-1gave rise to shoot proliferation earliest +NAA0.1mgl followed by 2.7 shootlets in BAP
with maximum frequency of 68.2 percent, also the 1.0mgl-1+NAA0.1mgl-1.
earlier proliferation was noted after 28.1+1.4 days,
when NAA was used at 0.01mgl-1. Of the three auxins

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J of Biotech & Crop Sci (2016) 5(6): 62-68

Effect of auxin: IBA (0.1 to 1.0mgl-1) NAA (0.1 to combination treatment of IBA 0.1mgl-1+NAA 0.1mgl-
1.0mgl-1) and their interactions this were applied to 1
respectively. In the absence of growth regulator in
observe this efficiency on rooting. It is clearly evident strength ms medium, rooting was delayed up to
from the data that maximum of 70.3 percent of 45.3+5.2 days. Maximum 6.2+2.4 numbers of roots
rooting was observed in the treatment of IBA 0.2mgl- were produced per shootlet in the treatment of IBA
1
+NAA 0.2mgl-1 followed by 67.7 and 66.5 percent 0.2 mgl-1+0.2mgl-1 followed by 5.8+1.4 and 5.7+3.3
for the treatment of IBA 0.5mgl-1+NAA 0.2mgl-1 and roots in the treatment combination of IBA 0.1mgl -
0.1 IBA +0.1mgl-1NAA respectively (Table 5). As 1
+NAA 0.2 mgl-1. Comparing the individual auxin
compared to single auxin treatment, IBA was slightly treatment, it was found that IBA was better over
better than NAA. Earlier root striking (26.4+2.7days) NAA in regenerating higher number of roots. In the
in the treatment of IBA 0.2mgl-1+NAA 0.2mgl-1, control only 1.3+0.4 roots per shootlets were formed.
followed by 28.4+1.7 days and 28.4+3.1 days for the

Table 1 Effect of different auxin on shoot proliferation of shoot tip and nodal segment of Rosa indica.
Auxin Shoot tip Nodal segment
Shoot (%) day to shoot shoot (%) day to shoot
proliferation proliferation proliferation proliferation
IAA
0.01 34.6 35.2+2.7 34.8 32.4+1.2
0.1 42.3 31.2+4.3 35.4 30.8+1.1
0.25 25.7 28.2+1.5 32.3 31.0+1.8
0.5 28.3 30.0+1.8 33.5 28.0+1.7
1.0 C __ C __
IBA
0.01 38.9 34.8+1.4 49.5 29.8+3.2
0.1 50.6 33.4+0.0 60.2 21.1+1.4
0.25 46.7 35.6+1.9 46.1 32.5+3.1
0.5 15.3 39.3+2.1 48.1 31.2+3.1
1.0 C __ C __
NAA
0.01 28.8 41.4+5.3 42.7 34.5+4.3
0.1 36.6 38.1+2.8 44.4 38.2+2.1
0.25 39.3 43.6+4.6 39.6 37.3+1.4
0.5 C __ C __
1.0 C __ C __

Table 2 Effect of various cytokinine on shoot tip and nodal explants.


Cytokinine Shoot tip Nodal segment
Shoot (%) day to shoot shoot (%) day to shoot
BAP proliferation proliferation proliferation proliferation
0.5 52.1 37.9+2.3 58.3 27.8+4.3
1.0 50.8 34.8+1.7 62.4 25.9+3.3
2.0 55.6 30.4+1.6 57.3 23.8+4.6
4.0 53.2 31.3+2.7 65.3 24.5+1.8
Kn
0.5 41.6 32.8+2.6 38.6 28.4+2.1
1.0 48.3 36.7+1.9 45.5 30.2+2.2
2.0 54.6 34.6+3.4 52.6 27.6+3.4
4.0 43.8 35.8+4.2 30.8 34.5+4.8
6.0 __ __ 15.1 45.8+4.8
10.0 __ __ __ __

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J of Biotech & Crop Sci (2016) 5(6): 62-68

Table 3 Effect of some growth regulator on nodal explants proliferation.


Growth regulator Nodal explant
BAP NAA Days to shoot No. of shoot per Av.length
Proliferation plant of shoot (cm)
1.0 0.01 32.4+2.1 2.2+0.3 0.8+0.1
2.0 0.01 24.0+2.1 4.4+0.2 1.4+0.3
5.0 0.01 27.0+1.9 3.6+0.8 1.7+0.2
1.0 0.1 22.1+1.1 4.2+0.1 2.4+0.0
2.0 0.1 20.4+0.9 5.3+0.3 1.8+0.1
4.0 0.1 27.3+0.7 3.3+0.2 1.7+0.3
5.0 0.2 C __ __
1.0 0.2 C __ __
2.0 0.2 26.7+3.8 2.6+0.7 2.1+0.3
4.0 0.2 27.4+4.7 2.1+0.1 0.8+0.6
BAP IAA
1.0 0.01 29.2+3.4 2.4+0.2 0.5+0.2
2.0 0.0 28.6+2.6 3.8+0.6 1.1+0.3
1.0 0.1 24.4+1.8 2.4+0.3 1.3+0.6
2.0 0.1 25.3+2.4 2.7+0.2 0.9+0.1

Table 4 Effect of some growth regulator on shoot proliferation from shoot tip.
Growth regulator Shoot tip
BAP NAA Days to shoot no. of shoot per av.length
Proliferation plant of shoot (cm)
1.0 0.01 32.0+1.0 2.1+0.3 1.1+0.4
1.0 0.5 22.0+0.4 2.2+0.3 1.2+0.8
2.0 0.01 34.3+2.3 2.4+0.2 1.2+0.1
4.0 0.01 31.7+2.6 1.5+0.4 1.2+0.1
1.0 0.1 34.5+3.4 2.7+0.1 1.3+0.6
2.0 0.1 32.6+1.7 2.9+0.3 1.5+0.3
0.4 0.1 36.8+4.2 2.3+0.1 0.9+0.2
0.5 0.2 C __ __
1.0 0.2 C __ __
2.0 0.2 37.4+3.1 2.3+0.5 1.2+0.4
4.0 0.2 35.0+2.5 2.4+0.6 1.4+0.9
BAP IAA
1.0 0.01 36.9+2.4 0.6+0.2 0.3+0.1
2.0 0.0 34.6+3.9 1.9+0.2 0.6+0.1
1.0 0.1 35.4+4.3 0.5+0.1 0.9+0.2
2.0 0.1 34.7+5.2 0.8+0.1 0.5+0.1

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Table 5 Effect of various auxins singly and in combination on rooting in vitro regeneration shootlets of Rosa indica of half
MS strength.
Growth Days to Percentage No. of Average
Regulator rhizogenesis rhizogenesis root per length of
mg/l Shoot root
IBA
0.0 45.3+5.2 12.5 1.3+0.4 3.2+0.4
0.1 32.4+2.4 47.6 2.8+0.7 3.8+0.5
0.2 29.6+2.8 0.4 4.3+0.8 4.1+0.2
1.5 31.1+3.4 55.1 3.1+1.7 3.6+0.3
1.0 32.2+2.4 48.1 4.2+1.2 2.1+0.3
NAA
0.1 39.8+5.1 42.7 2.9+0.8 1.4+0.2
0.2 34.4+5.6 44.5 2.3+1.0 2.1+0.5
0.5 38.9+3.3 46.1 1.8+0.4 1.7+0.1
1.5 36.4+2.6 32.9 2.3+0.6 2.1+0.5

Rooting of regenerated shootlets

Effect of auxin: For micropropagation, the best bud


position influences proliferation rate and best nodal
position was 8th to 12th node from terminal bud.
Similarly, in case of shoot tip the stage of growth
played a significant role. The best stage for efficient
proliferation was the elongated and color turning
stage of newly emerged shoots. February was the
most responsive month for shoot tip. While for nodal
segment two month i.e. March and December best
responses were obtained. Also the explant collected
Figure 1 Shoot proliferation of BAP- 2.0 mg/l + IAA-0.1mg/l.
during these month shows high proliferation, lower
phenolic problem and less microbial contamination.
The best season was noted from month of October-
March. Shoot tip optimum response on media
modified as MS (1/2 Macro+1/2 Micro), while nodal
segment half of MS strength have good proliferation.
NAA 0.1 mg/l was best auxin level for early shoot
proliferation for shoot tip, while nodal segment high
proliferation was obtained BAP 4.0mg/l by increasing
level of auxin beyond 0.2mg/l reduced proliferation,
well stunted growth was observed. In the interaction
treatment BAP 2.0mg/l and NAA 0.1 mg/l gave the
maximum responses. Similar studies were made by
Acker et al 1995 and Carelli and Echeverrigaray,
Figutr 2 Rooting of Regenerated Rose Shoots on one fourth 2009. In the present experiment, various factors were
strength of MS medium. studies for their influences on shoot proliferation. The
(a) IBA 0.2 mg/l (b) NAA 0.2 mg/l
pH of the media (5.85) proves to be best. However,

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