In Vitro Propagation of an Edible Bamboo Bam-
busa Bambos and Assessment of Clonal Fidelity
through Molecular Markers
Manju Anand and Jasmine Brar
Thapar University, Department of Biotechnology, Patiala (PB), India
Email: {manandtiet, jasminethapar83}@gmail.com
Anil Sood
CSIR- Institute of Himalayan Bioresource Technology, Palampur (HP), India
Email: asood@ihbt.res.in
Abstract—An efficient and reproducible protocol has been
established through the technique of forced axillary branch-
ing for the propagation of an important edible bamboo spe-
cies namely Bambusa bambos. High frequency multiple
shoot induction was achieved from nodal segments collected
from elite genotype on Murashige and Skoog’s (MS) medi-
um supplemented with 4.4 µM Benzylaminopurine (BAP)
and 1.16 µM Kinetin (Kn). The size of explant and season
greatly influenced the frequency of bud break. Rooting
posed a major problem to be worked out in this particular
species. Best rooting response was observed on 9.80 µM of
Indole- 3 Butyric acid (IBA) with 60 ± 14.1 % rooting. In
vitro raised plants were successfully acclimatized and estab-
lished in the field conditions where they exhibited normal
growth. In a bid to ascertain genetic fidelity, DNA was ex-
tracted by CTAB method and samples were analysed in
1.8% agarose gel electrophoresis. In the present study no
variation was reported among the in vitro raised progeny
and the mother plant in the banding profiles generated by
the total of fifteen Random Amplified polymorphic DNA
(RAPD) and Inter Simple Sequence Repeats (ISSR) mark-
ers. Hence, molecular analysis confirmed that these plants
were genetically similar and can be used as elite plants.
Index Terms—Bambusa bambos, in vitro, micropropagation,
RAPD, ISSR
I. INTRODUCTION
Bamboos are the most important forest species of
Southeast Asian countries where they form the backbone
of rural economy. Next to China, India is the second
largest producer of bamboos in the world producing 4-6
million tones of bamboos annually, out of which 1.9
million tonnes are used in paper industry. Besides their
application in paper industry, they are extensively used
for house building, furniture making, floor tiles, for soil
conservation, eating purposes and their leaves make an
excellent cattle fodder. With renewed interest in bamboo
propagation in India through Bamboo Mission
Manuscript received June 2, 2013; revised August 2, 2013.
Programme, the emphasis is on producing quality
bamboo material on large scale and to introduce other
economically important bamboo species. Among these,
Bambusa bambos attains the greater significance.
Bambusa bambos called as ‘Giant Thorny’ is a densely
tufted bamboo growing up to a height of 25 m having
multifarious uses [1]. Currently, International Union for
Conservation of Nature (IUCN) Red list of endangered
bamboo species has included Bambusa bambos as a
priority species and hence needs conservation [2]
Limitations in traditional propagation methods such as
the use of offsets, branch cuttings together with
unpredictable and long flowering cycle (40-60 years)
warrants an urgent need for an alternative approach for
developing efficient and reproducible protocol for its
mass propagation. In vitro propagation or tissue culture
technique holds tremendous potential for the production
of high-quality planting material [3]. In this report we
describe an efficient and reproducible micropropagation
protocol for generating uniform clones through axillary
branch proliferation using vegetative tissue derived from
mature field-grown clumps of B. bambos and
establishing the genetic fidelity through RAPD and ISSR
markers. Earlier, [4] reported somatic embryogenesis in
B. bambos which carries a higher risk of genetic
instability due to somaclonal variations, thereby
defeating the purpose of micropropagation. [5] reported
multiple shoot proliferation from nodal explants but no
rooting could be induced. None of these studies
attempted to analyse the genetic fidelity of in vitro raised
plants of B. bambos, which is of utmost importance in
determining the practicality of any micropropagation
protocol.
II. MATERIAL AND METHODS
The nodal explants were collected from the precocious
branches of 5 years old healthy field grown plant of
B.bambos. After washing with teepol solution and
bavistin (0.1% w/v), explants were surface sterilized with
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of Medical and Bioengineering Vol. 2, No. 4, December 2013
257
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doi: 10.12720/jomb.2.4.257-261
0.1% (w/v) aqueous solution of HgCl
2
and were cultured
on MS [6] medium augmented with different growth
regulators, 2% sucrose and 0.8% agar or 0.2% phytagel
with pH adjusted to 5.8 before the addition solidifying
agent. All the inoculated cultures were incubated in
growth room at a temperature of 25 ± 2ºC with a
photoperiod of 16 hours per day with an illumination of
50 µmol/m
-2
/s
-1
at the level of cultures. Various factors
like initial response of explant, contamination rate, bud
break and survival percentage were considered for
initiation of cultures. Basal MS medium supplemented
with a combination of two cytokinins viz BAP (1.1 to
13.2 µM) and Kn (1.16 to 9.3 µM) with sucrose (1 to 3 %)
was tested for standardization of axillary proliferation.
For root induction, shoots were excised in clumps of 3-4
from multiple shoot bunches and transferred onto MS
media in full, half and quarter strengths supplemented
with IBA (4.90 to 24.5 µM) and NAA (5.37 to 24.16 µM)
with 0.8% agar or 0.2% phytagel. The rooted plantlets
were hardened on sand, soil and farmyard manure (1:1:1)
under greenhouse conditions and finally transferred to
full sunlight. To test the clonal fidelity, the DNA of the
mother plant and in vitro raised hardened plants was
extracted from young leaves by using modified Cetyl
Trimethyl Ammonium Bromide (CTAB) method [7]. A
total of Twenty five RAPD and ISSR primers at a
concentration of 1 µM / reaction were scanned in the
present study. PCR amplification was carried out in total
volume of 25 μl containing 2 μl (20 to 25 ng) of genomic
DNA. The reaction buffer for ISSR consisted of 2.5 μl
Taq buffer, 1 μl MgCl
2
, 0.15 μl dNTPs (10 mM each of
dATP, dGTP, dTTP and dCTP), 1.5 μl primer, 0.17 μl
Taq polymerase (Bangalore Genei Pvt. Ltd, Bangalore,
India) and 17.68 μl water. PCR amplification was
performed in a DNA thermal cycler which was
programmed for initial DNA denaturation at 94°C for 4
min, followed by 44 cycles of 1 minute denaturation at
94°C, 1 minute annealing (temperature specific to the
primer) and 1 minute extension at 72°C, with a final
extension at 72°C for 7 minutes. For RAPD, reaction
buffer consisted of 2.5 μl Taq buffer, 0.5 μl MgCl
2
, 0.2 μl
dNTPs, 1.5 μl primer, 0.17 μl Taq polymerase and 18.13
μl water. PCR amplification consisted of initial
denaturation at 94°C for 5 minutes, followed by 45
cycles of 1 minute denaturation at 94°C, 1 minute
annealing at 37°C and 2 minutes extension at 72°C, with
a final extension at 72°C for 7 minutes. The amplified
products were resolved by electrophoresis on 1.8%
agarose gel in tris-borate EDTA (TBE) buffer stained
with ethidium bromide. The fragment sizes were
estimated with 100 and 500 bp DNA ladders (Bangalore
Genei Pvt. Ltd, Bangalore, India).
III. RESULTS AND DISCUSSION
A. Initiation of Aseptic Cultures and Factors Affecting
Percentage Bud Break
Due to the considerable variations in the
environmental conditions during different periods of the
year, maturity status of the explant varied with season,
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hence response of explant to culture initiation also varied.
The best period for raising aseptic cultures was spring
(February and March) when 93% bud break was
observed. Rainy season had almost an equal frequency of
bud break but the rate of contamination was very high.
Summers and winters were the least preferred seasons for
the initiation of cultures as percentage of bud break was
very low being 37 and 50% respectively. The dependence
of bud break on external factors like the condition and the
health of the mother plant has been reported earlier by [8],
[9] and [10]. Size of explant also influenced the time
taken for the bud break. Small sized explants (5-7 mm)
did not respond well in the culture whereas explants of
25 mm took least number of days for initiation of
sprouting probably due to greater concentration of
endogenous hormones supporting bud break.
B. Shoot Multiplication
For inducing sprouting, nodal explants were inoculated
on MS medium with or without cytokinins. Nodal
explants cultured on MS basal medium without any
cytokinin took more time to sprout (25 days) where as
the nodal explants sprouted within 15 days of inoculation
on MS medium supplemented cytokinin (Fig. 1a). The
sprouted buds were excised from the nodal segment and
transferred onto MS medium containing different
concentrations and combinations of cytokinins like BAP
(1.1 to 13.2 µM) and Kn (1.16 to 9.3 µM). Axillary shoot
proliferation occurred on MS medium supplemented with
different concentrations of BAP but 4.4 μM of BAP gave
maximum multiplication rate. A significant increase in
the shoot number was observed when BAP (4.4 µM) was
used in conjunction with Kn (1.16 µM) forming 6-8
shoots after 3 weeks (Fig. 1b). The shoots multiplied
further forming 21.70
a
± 2.40 shoots after 6 weeks (Fig.
1c).
C. Rooting and Acclimatization
In general, rooting is a main bottleneck while carrying
out in vitro multiplication of bamboos and has been a
major problem to be worked out in this particular species.
It was observed that rooting was more effectively
induced when clusters of shoots rather than individual
shoot were used. The clumps of 3 to 4 shoots were
inoculated on MS medium supplemented with different
concentrations and combinations of auxins. Out of all the
auxins tried, initiation and growth of roots was observed
on IBA (9.80 µM) supplemented medium where rooting
occurred in 60% of cultures (Fig. 1d & e). The role of
IBA and NAA in root induction had been reported earlier
in other bamboos like Bambusa vulgaris by [11]; in B.
oldhamii by [12] and Dendrocalamus asper by [13].
Complete plantlets were hardened on soil: sand: farmyard
manure (1:1:1) by gradually lowering the relative
humidity and were successfully transferred from in vitro
to natural environmental conditions with 80% survival
rate. The plants showed well developed root and shoot
systems and all the plants are thriving very well in field
conditions with no apparent phenotypic variations (Fig.
1f).
Figure 1. a. Bud break on MS+ BAP (4.4 µM) b. Initiation of multiple shoots on MS + 4.4 µM BAP and 1.16 µM Kn after 3 weeks. c. Proliferation
of numerous shoots after 6 weeks. d. Root induction on 9.80 µM IBA. e. A complete plantlet formed f. 2- months-old hardened plant in field condi-
tion
Figure 2. Amplifications produced by using RAPD marker OPA 19. Amplifications produced by using ISSR marker UBC 818. Lane M represent
ladder, lane 1 is the mother plant and lane 2-16 represent the tissue culture (TC) – raised plants.
D. Clonal Fidelity Using RAPD and ISSR as Molecular
Markers
The scarcity of reports on ascertaining the genetic
fidelity of tissue culture raised plantlets can jeopardise
the quality of micropropogated plants, especially in
perennials like bamboos where any undesirable variant
would last for several years [14]. Therefore, it is pertinent
to screen the regenerants for the occurrence of any
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somaclonal variations. In the present investigation, for
ascertaining the clonal fidelity, 15 randomly selected
plants were subjected to RAPD and ISSR analyses. Out
of 15 RAPD markers scanned only 10 primers produced
amplicons as shown in Table I. In case of ISSR analyses
using 10 markers only, 5 markers gave scorable bands as
depicted in Table II. Out of total 25 scanned markers,
amplified products in the range of 100 to 1500 bp were
obtained as shown in Fig. 2. Optimum T
m
for RAPD
markers falls near 37º C and that for ISSR markers the
range falls between 45.4 to 54.3 ºC. For RAPD analysis,
OP series gave the best amplification. For ISSR analysis,
UBC 818 gave the maximum amplified products in the
range of 200 to 1400 bp. We found that all banding
profiles from the micropropagated plants were
monomorphic and similar to those of mother plant.
Hence, clonal fidelity was established with no apparent
genetic and epigenetic variations. Earlier, [9] established
the clonal fidelity of regenerants of Bambusa tulda and B.
balcooa using only four markers to assess the genetic
uniformity among the regenerants. [15] assessed the
clonal fidelity of in vitro raised plants of Dendrocalamus
hamiltonii using 18 RAPD markers. Later, [16] employed
30 RAPD and 27 ISSR markers to validate the clonal
fidelity of in vitro raised Guadua angustifolia plantlets
through the axillary bud proliferation. However, there is
no report available on the comparative genetic stability of
regenerants and mother plant of Bambusa bambos using
RAPD and ISSR markers. In the present study, we did
not find any polymorphism during the RAPD and ISSR
analysis of in vitro raised clones, thus confirming true to
type nature of in vitro raised plants.
TABLE I. RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) MARKERS UTILIZED TO VERIFY BAMBUSA BAMBOS CLONES
Primers
5’-3’ motif
Scorable bands
Monomorphic bands
Polymorphic bands
Range of Amplification
OPA 01
CAG GCC CTT C
3
3
0
1000, 1100, 1300
OPA 02
TGC CGA GCT C
3
3
0
400, 900, 1000
OPA 19
CAA ACG TCG G
3
3
0
450, 700, 1000
OPT 10
CCT TCG GAA G
3
3
0
300, 500, 1500
OPT 18
GAT GCC AGA C
2
2
0
800, 900
OPO 06
CCA CGG GAA G
1
1
0
1500
OPO 07
CAG CAC TGA C
2
2
0
450, 1400
OPO 08
CCT CCA GTG T
2
2
0
200, 1300
OPO 15
TGG CGT CCT T
2
2
0
800, 1500
OPO 18
CTC GCT ATC C
2
2
0
200, 1300
Total
10
23
23
0
200-1500
OP Series sequences of Operon Technologies – Alameda, USA
TABLE II: THE INTER SIMPLE SEQUENCE REPEATS (ISSR) MARKERS UTILIZED TO VERIFY BAMBUSA BAMBOS CLONES
Primers
5’-3’ motif
T
m
º C
T
m
º C
Scorable
Bands
Monomorphic
Bands
Polymorphic
bands
Range of
Amplification (bp)
UBC 810
(GA)
9
T
45.4
42.5
4
4
0
100, 400, 450,
1000
UBC 812
(GA)
8
A
45.7
41.5
2
2
0
100, 350
UBC 818
(CAC ACA)
2
CAC AG
51.0
48.5
3
3
0
200, 400, 1400
UBC 834
(GA)
9
T
49.2
46.0
3
3
0
350, 400, 700
UBC 857
(GA)
9
T
54.3
50.5
2
2
0
400, 1500
Total
5
-
-
14
14
0
100-1500
UBC Series sequences of University of British Columbia, Canada; Y = (C,T)
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Dr.
Manju Anand
was born on 12th May 1951
in
Nangal, India. She got her
M. Sc (Hons) in Botany
and Ph.D
in
Plant Tissue Culture
from Panjab
University, Chandigarh, India in 1981.
Her major
field of study
is
biotechnology.
Jasmine Brar
was born on 18th July 1983
in
Patiala, India.
She got her M. Sc in Botany
and
Ph.D Pursuing in Biotechnology
from Thapar
University,
Patiala, India.
Her major field of
study
is
biotechnology.
Dr. Anil Sood
was born on 20th April 1955
in
Palampur, India. He got his M. Sc (Hons) in
Botany
and Ph.D
in
Plant Tissue Culture,
from
Panjab University, Chandigarh, India
in
1985.
His major field of study
is
biotechnology.
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