Eur J Forest Res (2011) 130:729736

DOI 10.1007/s10342-010-0462-4

ORIGINAL PAPER

Induction of somatic embryogenesis and analysis of genetic

fidelity of in vitro-derived plantlets of Bambusa nutans Wall.,
using AFLP markers
Rupali Mehta Vikas Sharma Anil Sood
Madhu Sharma Ram Kumar Sharma

Received: 29 October 2009 / Revised: 7 October 2010 / Accepted: 23 November 2010 / Published online: 29 December 2010
Springer-Verlag 2010

Abstract Bambusa nutans Wall., is an evergreen,

perennial, and multipurpose bamboo having strong culms,
which are largely used for construction, scaffolding, craft
purposes, pulp, and paper industry. Multiple shoots from
nodal segments (34 cm) of young branches of mature
culms were established in Murashige and Skoog (1962)
(MS) medium supplemented with various concentrations
of 6-benzylaminopurine (BAP) (1.06.0 mg l-1) or in
combination with a-naphthaleneacetic acid (NAA)
(0.51.0 mg l-1) or kinetin (Kn) (1.02.0 mg l-1).
FebruaryMarch and December were found to be the best
seasons for culture establishments. Maximum shoots were
achieved on MS medium fortified with BAP (2.0 mg l-1).
Embryogenic callus (slightly greenish compact, globular,
and slow growing) was initiated from the base of severed
sprouted buds in 23 subsequent subcultures on MS medium supplemented with 2,4-dichlorophenoxy acetic acid
(2,4-D) (5.0 mg l-1) under dark incubations. Maturation
and germination of well-organized somatic embryos was
achieved on MS medium containing BAP and 2,4-D
(1.0 mg l-1 each) with 20.0 mg l-1 ascorbic acid. Fullstrength MS medium supplemented with 2% glucose
favored further development of proliferated somatic
embryos into plantlets. Genetic variations of fieldestablished B. nutans plants regenerated through tissue
Communicated by R. Matyssek.
R. Mehta
V. Sharma
A. Sood (&)
M. Sharma

R. K. Sharma (&)
Biotechnology Division, Institute of Himalayan Bioresource
Technology, Council of Scientific & Industrial Research,
Palampur 176061, Himachal Pradesh, India
e-mail: asood@ihbt.res.in
R. K. Sharma
e-mail: ramsharma@ihbt.res.in

cultures were assessed by amplified fragment length

polymorphism (AFLP) analysis using 6 primer combinations. Four hundred and seven scorable fragments were
amplified, of which 402 (98.8%) have recorded conservation at various morphogenetic stages leading to plantlets
regeneration, therefore, revealed a high level of genetic
stability.
Keywords AFLP
Auxins
Cytokinins
Embryogenic
callus
Excised bud segments
Shoot multiplication
Abbreviations
2,4-D 2,4-Dichlorophenoxy acetic acid
AFLP Amplified fragment length polymorphism
BAP
6-Benzylaminopurine
Kn
Kinetin
MS
Murashige & Skoog (1962) medium
NAA a-Naphthaleneacetic acid
PGR
Plant growth regulator
masl
Meter above sea level

Introduction
Bambusa nutans Wall., is an evergreen, perennial, and
multipurpose bamboo having strong culms (25 m in height
and 510 cm in diameter), which are largely used for
construction, scaffolding, handicrafts, and also as raw
material in paper and pulp industry. Distribution of this
species is confined to the states of Himachal Pradesh, Uttar
Pradesh, and northeastern states of India (Orissa, Sikkim,
and West Bengal). It grows well between an altitudinal
range of 5001500 meter above sea level (masl), on moist
hill slopes, flat uplands and sandy to clayey loam.

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Limitations in traditional propagation methods (NMBA

2002) associated with poor seed set and long flowering
cycle of *35 years restrict the availability of seeds in
B. nutans, and tissue culture technologies therefore can be
an alternative approach developing efficient and reproducible protocols for its mass propagation. Despite the
successful efficient multiplication system through somatic
embryogenesis in selected bamboo species (Mehta et al.
1982; Yeh and Chang 1986; Godbole et al. 2002; Gillis
et al. 2007), there is no report on somatic embryogenesis of
economically important B. nutans. Further, no efforts have
been done for generating plus bamboos for large-scale
plantations using explants with known age. However,
except few fragmentary reports on micropropagation using
various explants and strategy (Kalia et al. 2004; Nurul
Islam and Rahman 2005), there is no report on somatic
embryogenesis in B. nutans as employed in the present
study. Further, considering the importance of genetic uniformity during somatic embryogenesis to characterize somaclonal variation with greater precision as reported in
earlier tissue culture studies in different plant species
(Cloutier and Landry 1994; Hashmi et al. 1997), amplified
fragment length polymorphism (AFLP) fingerprinting was
employed to confirm the genetic stability. High levels of
reproducibility coupled with high multiplex ratio over the
other molecular markers techniques (Powell et al. 1996) are
the reasons for selecting AFLP markers for evaluating
genetic fidelity of tissue culture-raised plants in this study.
To our knowledge, this is the first report on somatic
embryogenesis and analysis of tissue culture-raised plants
using AFLP fingerprinting in B. nutans.

Materials and methods

Eur J Forest Res (2011) 130:729736

liquid detergent for 56 min and again washed thoroughly

34 times with sterilized distilled water. Explants were
dried on sterile filter paper by dabbing. Small portions
(12 mm) on both sides exposed to the sterilants were
removed with a pre-sterilized sharp secateur and inoculated
vertically in test tubes (Borosil, India; 200 9 25 mm and
capped with polypropylene caps; Tarsons, 25 mm d) containing basal Murashige and Skoog (1962) (MS) medium,
supplemented with sucrose 3% (w/v) and agar (0.8%; w/v,
Qualigens, Mumbai).
Multiplication of cultures
The explants were taken from a single mother plant. For
the establishment of cultures, hundred explants in 3 replicates were used at monthly intervals. After bud break,
different concentrations of 6-benzylaminopurine (BAP
1.06.0 mg l-1) either alone or in combination with
a-naphthaleneacetic acid (NAA 0.51.0 mg l-1) or kinetin
(Kn 1.02.0 mg l-1) were evaluated for multiple shoot
formation (see Table 1). From these, 5 explants in 5 replicates were taken in Erlenmeyer conical flasks (250 ml;
Qualigens, Mumbai, India) containing 100 ml of the corresponding culture medium. The pH was adjusted to
5.65.8 prior to autoclaving. Cultures were incubated at a
Photosynthetic Photon Flux Density (PPFD) of 70
5 lmol m-2 s-1 provided by cool, white, fluorescent
lamps (PHILIPS, TRULITE, India) at 25 2C. Day
length was maintained at 14-h light/10-h dark cycles.
Both single and bunches of 34 shoots were used to
attempt root induction in MS medium supplemented with
indole-3-butyric acid (IBA) and NAA (1.06.0 mg l-1
each) alone or in combination with BAP (1.0 mg l-1), and
the cultures were kept in total darkness as well as under
normal light conditions as described above.

Initiation of aseptic cultures

Statistics
Nodal segments (34 cm in length) of young branches of
mature field-selected plants of B. nutans growing in the
germplasm block at IHBT Palampur (32oN: 76oE at
1300 masl) in Kangra district of Himachal Pradesh, India,
were collected during different months (JanuaryDecember, 2006) and used for initiating aseptic cultures. Outer
leaf sheaths were removed to expose the tender buds,
which were surface-cleaned with sable-hair brush and a
liquid detergent (Tween 20; Hi Media, India) followed by
continuous rinsing with water. Thereafter, the explants
were treated with Bavistin (0.1%; w/v, BASF, India) and
streptomycin sulfate (0.05%; w/v, Sigma, USA) for
2025 min with constant stirring. Finally, explants were
surface sterilized in a laminar hood with 70% ethanol for
12 min followed by treatment with an aqueous solution of
mercuric chloride (0.04%; w/v) containing 12 drops of

123

Data were analyzed by General Linear Model for Main

Effect ANOVA (Analyses of Variance) followed by
Duncans multiple range test (STATISTICA Release 7,
Statsoft Wipro).
Somatic embryogenesis
For initiation of embryogenic callus, 4- to 5-mm long
segments were taken from entire length of sprouted buds.
The severed buds were transferred to MS medium containing different concentrations of 2,4-D (1.0, 1.5, 2.0, 2.5,
5.0, and 7.0 mg l-1) as described earlier to induce callus
and somatic embryogenesis in Dendrocalamus hamiltonii
(Godbole et al. 2002). Somatic embryos initiated from the
compact nodular callus were maintained in MS medium

Eur J Forest Res (2011) 130:729736

731

Table 1 Multiple shoot formation on Murashige and Skoog (1962)

medium supplemented with different plant growth regulators
Plant growth
regulators
(mg l-1)

Number of shoots formed (response)

4 weeks
(SEM = 0.22)

8 weeks
(SEM = 0.29)

Control*

2.0a

2.4b

BAP 1.0

3.2

6.2c

BAP 2.0

5.4e

11.0f

BAP 4.0

4.2e

3.0b (stunted shoots)

BAP 6.0

ab

2.8
3.0

BAP 1.0 ? NAA 0.5

3.0 (leaves
turned yellow)

0.0a (necrosis)

BAP 1.0 ? NAA 1.0

3.2ab (leaves
turned yellow)

0.0a (necrosis)

BAP 1.0 ? Kn1.0

3.8

BAP 1.0 ? Kn 2.0

4.0e

cd

7.2d
6.6

cd

No. of shoots taken initiallyone node having 2 sprouted buds. Mean

values are of 5 replicates. Different lower-case letters within columns
indicate significant differences among treatments. SEM represent
standard error of mean
Duncans multiple range test P B (0.05)
Control* MS without plant growth regulators

containing BAP, 2,4-D (1.0 mg l-1 each), various concentrations of ascorbic acid (20.0, 40.0, 80.0 mg l-1) with
sucrose at 3% (w/v), and agar (0.8% w/v) and incubated at
the same culture conditions for maturation and germination. To avoid browning of somatic embryos, various
concentrations of sucrose (18%) and glucose (18% w/v;
filter sterilized with 0.22-lm filter membrane, Millipore;
added in medium after autoclaving) were tested. All the
cultures were subcultured at 5- to 6-week intervals.
For the callus induction, 5 severed segments in 3 replicates were taken in 250 ml Erlenmeyer conical flasks.
Cultures were incubated at 25C 2 under total darkness
until germination initiations. Germinated somatic embryos
were subsequently shifted to light for further proliferations
(Photosynthetic Photon Flux Density of 70 5 lmol m-2
s-1, 14-h light/10-h dark cycles). Healthy plantlets derived
from somatic embryos were transferred to plastic pots
containing sand in poly tunnels and covered with glass jars
to maintain a higher relative humidity. After 1 month,
plants were transferred to pots containing soil and sand
(1:1) mixture.

AFLP analyses
AFLP analyses were conducted to establish the genetic
uniformity of tissue culture-raised plants after the callus
phase. Fresh tissue samples were collected after callus
phase from different morphogenetic stages that included,
globular, mature somatic embryos (3035 weeks old), and
plantlets regenerated (leaf samples of ten randomly selected plantlets derived from germinating embryos). Mother
plant was also included in the test array to compare the
genetic stability. Total genomic DNA was extracted from
in vitro-maintained cultures by CTAB method (Doyle and
Doyle 1990) with minor modifications (4% b-mercaptoethanol and 2 M NaCl). The quality and concentrations
of the extracted DNA were estimated on 0.8% agarose gel
using diluted uncut lambda DNA as standard. AFLP fingerprinting was done based on the protocol earlier reported
by Vos et al. (1995). Genomic DNA (250 ng) was
restricted with EcoRI and MseI (1.5 ll each) in a restriction
buffer (50 mM TrisHCl, pH 7.5, 50 mM magnesium
acetate, 250 mM potassium acetate) in a total volume of
25 ll. MseI and EcoRI adapters were subsequently ligated
to digested DNA fragments. The adapter-ligated DNA was
pre-amplified using the following cycling parameters: 20
cycles of 30 s at 94C, 60 s at 56C, and 60 s at 72C. The
pre-amplified DNA was diluted in a ratio of 1:50 and was
used as a template for the selective amplification using six
EcoRI and MseI primer combinations (E-AGG/M-CAA,
E-AGG/M-CTA,
E-AGG/M-CTG,
E-AGC/M-CAA,
E-AGC/M-CAC, E-AGC/M-CTG). The EcoRI primer was
labeled with c-33P-ATP (BRIT, India). The cycling
parameters were the following: 1 cycle of 30 s at 94C,
30 s at 65C, and 60 s at 72C. The annealing temperature
was lowered by 0.7C per cycle during the first 12 cycles
followed by 23 cycles at 94C for 30 s, 56C for 30 s, and
72C for 60 s.
The samples were resolved on 6% polyacrylamide gel, and
approximate size of the different fragments was estimated
using 20-bp DNA ladder (Cambrex Bio Science Rockland,
Inc). AFLP fragments in the size range of 100600 bp were
scored for their presence (1) and absence (0).

Results and discussion

Histological studies

Initiation of aseptic cultures

For histological studies, green nodular structures were

fixed in FAA (formaldehyde: acetic acid: 50% ethanol:
1:1:18) and embedded in paraffin wax for microtomy.
Sectioned material (12 lm thick) was stained in safranin
and fast green combination and mounted in DPX mountant
(Pati 2001).

Selecting appropriate season for raising aseptic cultures has

its own merits. With the use of explants from mature tissue,
the chances of endogenous contamination are enhanced.
Best seasons for establishing aseptic cultures were found to
be February, March, and December, where rainfall data of
these months were at par with each other and low

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Eur J Forest Res (2011) 130:729736

2006), B. tulda, B. vulgaris (Saxena 1990), D. hamiltonii

(Sood et al. 2002), and D. giganteus (Ramanayake and
Yakandawala 1997). The multiple shoots thus obtained
were used for root induction. Due to limited success (data
not shown) in rooting of microshoots, an alternative route
of somatic embryogenesis was successfully deployed.
Somatic embryogenesis

Fig. 1 Effect of month of collection on establishment of aseptic

cultures from the nodal segments (numbers in parenthesis indicate
rainfall data in mm). {Different lower-case letters within columns
indicate significant differences among treatments n = 3 (Duncans
multiple range test P B 0.05), SEM = 1.29}

contamination rates 2030% were obtained (Fig. 1). While

maximum contamination rate 6070% were found in the
month of August, having maximum rainfall over all other
months. Earlier Kumari and Ramanayake (1996) and
Ramanayake and Yakandawala (1997) had reported that
microbial culture contaminants were affected by rainfall
pattern in Bambusa vulgaris and Dendrocalamus giganteus,
respectively.
Multiplication of cultures
In B. nutans, bud break took place within 1 week on MS
medium (Fig. 2a), and further sprouting could be achieved
with 10- to 15-day interval (Fig. 2b). Sprouted buds of
23 cm length were excised at the base and transferred to
shoot multiplication medium containing BAP alone or in
combination with Kn or NAA. For shoot multiplication,
nodes preferably having two sprouts were selected. In the
present study, maximum shoots (11 Nos.) was noticed on
MS medium containing BAP (2.0 mg l-1) over a period of
8 week incubation (Table 1; Fig. 2c). Higher concentration
of BAP (6.0 mg l-1), however, induced stunted shoot
growth as was observed earlier in B. vulgaris (Ramanayake
et al. 2006). MS medium supplemented with BAP ? Kn
(1.0 mg l-1 each) produced an average of 7.2 shoots that
were significantly at par with BAP (1.0 mg l-1) ? Kn
(2.0 mg l-1), which produced an average of 6.8 shoots.
BAP in combination with NAA, however, produced
necrotic shoots after 8 weeks. Success in shoot multiplication from nodal segments has also been reported in other
bamboo species namely Guadua angustifolia (Jimenez
et al. 2006), Bambusa bambos var. gigantea (Kapoor and
Rao 2006), B. vulgaris var. Striata (Ramanayake et al.

123

Tissues excised from the base of the sprouted buds were

proved to be a promising starting material for callus induction. Tissue segments (45 mm) taken from entire length of
sprouted buds were utilized for initiation of embryogenic
callus. The severed buds were transferred to MS containing
different concentrations of 2,4-D (1.0, 1.5, 2.0, 2.5, 5.0, and
7.0 mg l-1). After 1012 weeks, 3040% of inoculated
segments responded for callus induction at the cut ends with
2,4-D (5.0 mg l-1). However, no response was recorded at
lower or higher concentrations of 2,4-D. Earlier reports
suggest that 2,4-D was the most promising among the other
auxins used for embryogenic callus induction. The suggested
concentrations of 2,4-D in earlier reports were ranging from
1.0 to 25.0 mg l-1, and more frequently at 2.0 and
3.0 mg l-1 were utilized (Mehta et al. 1982; Rout and Das
1994; Sood et al. 1994). After 1012 weeks, such callus
lumps were then transferred to MS medium supplemented
with BAP and 2,4-D (1.0 mg l-1each) with sucrose at 3%
(w/v) and agar (0.8% w/v). Compact and slightly green callus
lumps (Fig. 2d) were formed after 23 subcultures with
4 week intervals on similar medium. These lumps were
subsequently transformed into somatic embryos. Further
incubation on the same media composition containing
additional ascorbic acid (20.0 mg l-1) with sucrose at 3%
(w/v) resulted maturation and germination (Fig. 2e). Longterm maintenance of these cultures on similar medium supplemented with 3% sucrose resulted in browning of somatic
embryos after 35 subculturing. This adverse effect was
overcome by replacing 3% sucrose with glucose 2%. Further,
maturation and germination of somatic embryos was
achieved on glucose containing MS medium. Earlier, Woods
et al. (1992) reported better germination of somatic embryos
of Otatea accuminata on medium supplemented with 2%
sucrose than 5% sucrose concentration. As in B. nutans,
prolonged culturing in sucrose was responsible for the
browning of somatic embryos; the successful role of glucose
has been highlighted for the first time in bamboo for the
embryo conversion. Further, glucose is found to have growth
hormone-like activities and is one of the signaling molecules
for gene expression, cell proliferation, root, inflorescence
growth, and leaf expansion (Cho et al. 2006). The role of
carbohydrates as an important source of energy and osmoticum was reported by Cuenca and Vieitez (2000), Romano
et al. (1995), and Custodio et al. (2004).

Eur J Forest Res (2011) 130:729736

733

Fig. 2 ag Stages of multiple shoot formation and somatic embryogenesis in Bambusa nutans. a Nodal segment with sprouted bud.
b Bud sprouting within 10 days of inoculation (Arrow indicates the
basal portion of sprouted buds, which show maximum response).
c Multiple shoot formation on MS medium supplemented with BAP
(2.0 mg l-1) after 8 weeks in culture. d Slightly green embryogenic

compact callus on MS medium supplemented with BAP and 2,4-D

(1.0 mg l-1 each). e Somatic embryos germinating to form plantlets
after 810 weeks of culture. f Plantlet multiplication. g Plant with
fully developed shoot and root system (Bar line = 1 cm). (Color
figure online)

Further, spontaneous germinations in somatic embryos

were observed in few cases after 810 weeks on the same
medium, while others remained as such and proliferated
further to form secondary embryos. More than 50 somatic
embryo-derived plantlets (Fig. 2f and g) were transferred
to plastic pots containing sand in poly tunnels and covered
with jars to maintain the relative humidity high (8085%).
After 1 month of hardening, plants were transferred to pots
containing soil and sand (1:1) mixture in the green house
with 90% survival. These plants have so far shown normal
growth and produced 46 tillers.

Histological studies
Histological studies of embryogenic callus revealed the
presence of meristemoids with denser cytoplasmic cells
(Fig. 3a). Somatic embryos showing coleorhiza (cr), coleoptile (cl), and scutellum (sc) (Fig. 3b) were developed
after 1820 weeks of culture. Embryonal clumps, which
were of common occurrence (Fig. 3c), showed two
embryos with one root pole (coleorhiza; cr), fused
scutellum (sc), and two well-developed shoot poles
(coleptiles; cl). Such kind of abnormalities may give rise to

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Fig. 3 ac Histological studies. a Meristemoids showing cells with

denser cytoplasm (Bar = 50 lm). b Somatic embryo with differentiated coleorhiza (cr), coleoptile (cl), and scutellum (sc)
(Bar = 50 lm). c Abnormal embryo showing two shoots (cl) with
one root (cr) and fused scutellum (sc) (Bar = 50 lm)

two growing axes with one root. However, fate of such

embryos could not be traced. Similarly, histological studies
have also been reported earlier in other bamboo species
such as B. edulis (Lin et al. 2004) and B. glaucescens
Golden goddess (Jullien and Tran Thanh Van 1994).
AFLP analysis
Amplified fragment length polymorphism (AFLP) due to its
high multiplex ratio (Powell et al. 1996) and reproducibility
has proven to be a highly efficient tool for characterizing
somaclonal variation (Carolan et al. 2002; Popescu et al.
2002). Furthermore, various workers (Gagliardi et al. 2007;
Singh et al. 2002) have proved suitability of AFLP marker
technique for genetic fidelity studies. The present comprehensive analysis based on AFLP markers was conducted to
check the possible epigenetic changes in tissue cultureraised plantlets regenerated through somatic embryos.
Samples were collected after callus phase from different
morphogenetic stages that included globular, mature
somatic embryos (3035 weeks old), and regenerated
plantlets (ten randomly selected plantlets derived from
germinating embryos). Six EcoRI and MseI primer
combinations (E-AGG/M-CAA, E-AGG/M-CTA, E-AGG/

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Eur J Forest Res (2011) 130:729736

M-CTG, E-AGC/M-CAA, E-AGC/M-CAC, E-AGC/M-CTG)

that were utilized earlier for phylogenetic and genetic
diversity analysis in bamboo by our group (unpublished
data) were selected for testing of genetic fidelity in the
present study. A total of 407 clear reproducible fragments
were amplified. Of these, 402 (98.8%) were remained
monomorphic. The number of fragments detected by each
primer combination ranged from 59 (E-AGG/M-CTA) to 78
(E-AGC/M-CAA) with an average of 67.8. An average
polymorphism frequency recorded between different morphogenetic events was 1.2% (Table 2). A high frequency of
morphological variants has been reported in sandalwood
regenerants derived through somatic embryogenesis (Rao
et al. 1984). A significant polymorphism rates were also
found in plants regenerated via somatic embryogenesis in
different plant species (Aronen et al. 1999; Al-Zahim et al.
1999; Vendrame et al. 1999; Hornero et al. 2001; Popescu
et al. 2001). However, recent methylation-sensitive AFLP
(MSAP) analysis of somatic embryogenesis-derived plantlets in Bambusa balcooa Roxburgh could not detect epigenetic changes (Gillis et al. 2007). The literature reveals
that the presence of various hormones in tissue culture
medium released cytotoxic by-products,which act as a
stress environment, which induces a programmed loss of
cellular controls (Phillips et al. 1994; Kaeppler et al. 1998).
In the present study, 2,4-D containing medium was used for
the induction of somatic embryogenesis. LoSchiavo et al.
(1989), however, showed that the hormone 2,4-D causes a
dramatic elevation of cytosine methylation in plant tissue
cultures. An increase in 2,4-D concentration in carrot
(Daucus carota) suspension cultures from 0.5 to 2 pg/ml
raises the percent of 5-methylcytosine from 16 to 40% in
only 5 days.
AFLP results of the present investigations demonstrate
that no major genetic variation occurred during in vitro
shoot regeneration in B. nutans through somatic embryogenesis via callus (Fig. 4). Due to stringent genetic control
in somatic embryo formation and high selection pressure
against the abnormal type, somatic embryogenesis is usually considered as one of the best modes of regeneration
next to axillary multiplication (Leroy et al. 2000). However, few off-type micropropagated plants (1.2%) as
revealed in the present case can be due to genetic variations
during tissue culture, which includes chromosomal rearrangements, single-gene mutations, and DNA methylations
(Kaeppler and Phillips 1993; Xu et al. 2004). Further, to
find out the possible explanation of polymorphism detected
after callus phase, mother plant was independently sampled, subsequently analyzed again with selected primers,
and found to be genetically uniform.
The present communication reports the use of new
sprouts of the mature nodal buds for induction of somatic
embryogenesis via callus. The protocol for somatic

Eur J Forest Res (2011) 130:729736

735

Table 2 AFLP analysis of the mother plant, globular embryo, mature somatic embryo, and regenerated plantlets of Bambusa nutans
Primer combinations

Total
fragments

Fragments details (morphogenetic events wise)

Mother
plant

Globular
embryo

Mature somatic
embryo (SE)

Plantlets derived
through SE

Polymorphic
bands

Percentage
polymorphism (%)

E-AGG/M-CAA

65

63

65

65

63

3.1

E-AGG/M-CTA

59

58

58

59

59

1.7

E-AGG/M-CTG
E-AGC/M-CAA

64
78

64
78

64
78

64
78

64
78

E-AGC/M-CAC

77

75

77

76

76

2.6

E-AGC/M-CTG
Total

64

64

64

64

64

407

402

406

406

404

1.2

1 2 3 4

5 6 7

8 9 10 11 12 13

500 bp

this important bamboo species. Genetic uniformity of the

micropropagated plants as reported in the present study
adds to the significance of this protocol.
Acknowledgments We are thankful to Director, IHBT for providing necessary facilities. Financial assistance from Department of
Biotechnology, Government of India, New Delhi, is gratefully
acknowledged. This is IHBT publication 0698.

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200 bp

Fig. 4 AFLP profiles of the field-grown mother plant (lane 1),

globular embryos (lane 2), mature somatic embryos (lane 3), and
regenerated plantlets derived from somatic embryos (lane 413) of B.
nutans as revealed by AFLP primer combinations E-AGC/M-CAA,
(M = DNA maker 20-bp ladder)

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