Springer 2005

Plant Cell Tiss Organ Cult (2005) 81:109112

DOI 10.1007/s11240-004-3017-x

Research note

In vitro regeneration of Bambusa balcooa Roxb.: Factors aecting changes of

morphogenetic competence in the axillary buds
Malay Das & Amita Pal*
Plant Molecular & Cellular Genetics, Bose Institute, Kolkata 700054, India (*requests for oprints; Fax:
+91-033-2334-3886; E-mail: amita@bic.boseinst.ernet.in)
Received 18 August 2004; accepted in revised form 4 September 2004

Key words: axillary bud, bambusa balcooa, morphogenesis, nodal explant, rainfall, temperature

Abstract
Axillary buds from eld-grown culms of Bambusa balcooa were used as explants to induce multiple shoots
in liquid Murashige and Skoogs (MS) medium supplemented with 11.25 lM of 6-benzylaminopurine
(BAP) and 4.5 lM kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength
MS medium with 1.0 lM of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds
varied widely in dierent months of two consecutive calendar years. The highest morphogenetic potentials
were observed in October. The major problem encountered was presence of systemic fungal contaminants.
Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic
content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands.
To the best of our knowledge this is the rst report on in vitro regeneration of B. balcooa from mature eldgrown axillary buds.
Abbreviations: BAP 6-benzylaminopurine; IBA 3-indolebutyric acid; Kn kinetin; MS Murashige
and Skoogs (1962) medium; TPC total phenol contents
Bambusa balcooa is accredited as a priority bamboo species by FAO (www.unep-wcmc. org., www.
inbar.int.). To fulll the demand for large scale
commercial uses, induction of adventitious morphogenesis from the axillary buds that are available
in large numbers in the nodes of adult bamboos can
be widely used. However, the success of clonal
multiplication from adult culm is restricted by
many factors as demonstrated by Lin and Chang
(1998) and maturation of the tree species adversely
aects the morphogenetic potential of the axillary
buds was reported by Pierik (1990). Consequently,
reports on clonal multiplication from adult bamboos are limited (Chaturvedi et al., 1993; Saxena
and Bhojwani, 1993; Ramanayake and Yakandawala, 1997). In this study an attempt was made
to investigate the factors aecting higher morphogenetic competence of the axillary buds from mature eld grown stand of B. balcooa.

The nodal segments of B. balcooa Roxb. (1.5

2.0 cm in length) were collected from a natural
bamboo stands (ca. 30 years old). After removing
the leaf sheaths carefully, axillary buds were
thoroughly sterilized and inoculated in liquid
Murashige and skoogs (1962) (MS) basal medium supplemented with 11.25 lM 6-benzyl amino
purine (BAP) and 4.5 lM kinetin (Kn) for initiation of culture and subsequent shoot proliferation (Figure 1ad). Shoots were subcultured to
fresh medium at regular interval of 15 days or
otherwise whenever the medium turned brown.
Clusters of 34 shoots were used as propagule for
root induction and multiplication in half strength
MS basal medium with 1.0 lM indole butyric
acid (IBA). Profuse root development was noticed within 45 days of inoculation (Figure 1e).
The rooted plantlets were successfully transplanted to the soil. Monthly variations in the

110

Figure 1. Dierent stages of in vitro shoot regeneration from

nodal explants of Bambusa balcooa Roxb. in MS medium
supplemented with 11.25 lM BAP and 4.5 lM Kn. (a) Swelling
of axillary bud after 7 days of inoculation; (b) multiple bud
dierentiation after 12 days of inoculation (c) sprouting of
axillary buds after 20 days of inoculation; (d) proliferation of
shoots after 45 days of inoculation; (e) regenerated shoot with
roots (indicated by arrow) in half strength MS medium with
1.0 lM IBA.

in vitro bud-break eciency were initially observed in the year 2000. To study the environmental inuence, experiments were conducted in
the following two consecutive years, i.e. from
January 2001 to December 2002. Explants were
collected at the middle of every month from the
same donor plant. A set of 50 nodal explants was
inoculated (one explant/culture vessel) each
month into the shoot induction and proliferation
MS medium containing 11.25 lM BAP and
4.5 lM Kn. Numbers of nodal explants showing
bud-break and/or fungal-contamination were recorded after 28 days of inoculation. Maximum,
minimum atmospheric temperatures and rainfall
data of the bamboo collection site were furnished
by the Regional Meteorological Department,
Kolkata, India.

Fungal contaminants and exudates release

from the inoculated explants were encountered as
major obstacles to establish successful culture regenerants. These fungal contaminants are systemic
in nature and sometimes appeared even after 3rd
or 4th subcultures when the shoots had prolic
growth. The released brown coloured exudates
with stringent odour was tested and identied as
phenolic compounds, which led us to estimate the
total phenol contents of the explants every month
at the time of culture initiation, of the two calendar years (12 2 observations, 3 replicates). The
bud-break frequency of the comparable nodal
explants was scored. About 3.5 g of nodal explant
was reuxed in 50 ml absolute alcohol at 98 C in
a water bath for 6 h. Total extract was dried
completely and dissolved in 50 ml distilled water
following the method of Anbazhagan et al. (1990).
The phenolic content of 0.5 ml of the aqueous
extract was measured at 765 nm in a spectrophotometer after 1 h incubation with 4.5 ml of reaction mixture. The reaction mixture contained
3.5 ml double distilled water + 0.25 ml Folin
phenol reagent + 0.75 ml saturated Na2CO3
solution. The TPC was calibrated from the standard curve prepared using chlorogenic acid as
standard and represented as gm%.
Bud-break frequency of the axillary buds was
not uniform during the period under study. The
variation was more apparent in the year 2001
than in 2002 (Figure 2a). The lowest morphogenetic competence of the axillary buds was observed from January to May in both the years.
While, morphogenetic competence was more in
the following months i.e. from June to October
and the highest frequency 48.7 and 50.0 was recorded in October 2001 and 2002, respectively.
Likewise the frequency of fungal contaminants
that appeared from the sterilized cultured explants uctuated in the dierent months of the
years under study (Figure 2b). An inverse relationship was observed between the morphogenetic
competence of the axillary buds and frequency of
the fungal contaminants during the months when
relatively high (September December) or low
frequency (February May) of adventitious budbreak was recorded (Figure 2a, b).
The gradual decrease in the frequency of fungal
contaminants from August to December in both
the years (Figure 2b) prompted us to check the
rainfall intensity during these months of the 2

111
2001

2002

Fungal contamination (%)

Bud break (%)

50

(a)

40
30
20
10
0
Jan Feb Mar

50

(b)

40
30
20
10
0

Apr May Jun Jul Aug sep Oct Nov Dec

Jan

Feb Mar Apr

May Jun Jul Aug sep Oct Nov Dec

Months

Months
2002

Temp2001

Temp2002

Rainfall2001

35

(c)
Temperature ( c)

TPC (gm%)

2001

0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0

2002

60

30

(d)

300

25

250

20

200

15

150

10

100

50

0
Jan Feb Mar Apr May Jun

Jul Aug sep Oct

Nov Dec

Months

Rainfall2002
350

Rainfall (mm)

2001

60

Jan Feb Mar Apr May Jun Jul Aug sep Oct Nov Dec

Months

Figure 2. Frequency of in vitro bud break (a) and fungal contamination (b) in each month (from 50 replicates) of two consecutive
years. (c) mean total phenol contents of 3 replicates SE for each month of two consecutive years. (d) monthly variations of average
day and night temperatures (c) and rainfall (mm) of two consecutive years.

years. Records of the Meteorological Department

indicate that the rainy season starts from June,
due to the advent of South-West monsoon in this
part of the globe and persisted upto September or
October. A sudden rise in rainfall in May was
recorded that attained highest level in JuneAugust (Figure 2d). In nature, sprouting of the axillary buds is concomitant with heavy monsoon
showers. It was also reported earlier that the
morphogenetic potentials of the adult tree
increases in the course of growing season due to
changed proles of the endogenous growth regulators (Civinova and Sladky, 1990). Our contention generated from the present ndings is that in
addition to plant growth regulators some metabolites are also synthesized/released during this
sprouting season in B. balcooa, which encourage
the growth of the quiescent systemic fungi it harbours. Perhaps due to this break in dormancy
under natural environment, the systemic contaminants surfaced out and the nodal explants became
free of quiescent contaminants during sterilization. Thus, frequency of contamination reduced in
the sprouting season as reected under in vitro
condition (Figure 2b, follow AugustDecember in
the line graph). The inuence of seasonal rainfall

pattern on the rate of axillary bud-break and

fungal contamination in bamboos was reported
previously (Ramanayake and Yakandawala, 1997;
Saxena and Bhojwani, 1993).
The other concern of the present study stemmed from the nding that the morphogenetic
competence was adversely aected by the exudates
release from the excised explants, which caused
browning of the medium and ultimately resulted in
necrotic appearance of the shoots. The browning
phenomenon of cultured tissues was attributed to
oxidized phenolic compounds (Huang et al.,
2002). Phenolic oxidation in in vitro culture occurs
when cells are ruptured during excision of the
explants and compartmentalized enzymes (in this
case plastidial) and substrates released in the
culture medium. Presumably, after introduction
into in vitro culture, axillary-buds with a reduced
capacity for oxidative reaction had less browning
and as a consequence had better morphogenetic
competence than the one showed high oxidative
reaction.
The TPC increased gradually from January to
June followed by a sudden decrease in the
subsequent months until December (Figure 2c).
The morphogenetic competence of B. balcooa was

112
moderately high from August to October, when
TPC was comparatively low. Although TPC was
considerably low in November and December, yet
the morphogenetic competence was not substantial
in these months of both the years, when in nature,
the axillary buds undergo in the resting phase.
Due to the onset of winter season (November
to February) and low temperature the bud-break
frequency decreased substantially under in vitro
condition (Figure 2a, d). While, in subsequent
months, i.e. MarchMay, the bud-break frequency was low due to the browning of the in
vitro sprouted axillary buds. Seasonal eect on
browning of mature explants of few other species
viz., Bauhinia vahlii (Dhar and Upreti, 1999);
Chrysanthemum morifolium (Prasad and Chaturvedi, 1986); pear and apple (Wang et al.,
1994) have been reported previously. Andersone
and Ievinsh (2002) have reported that the axillary-buds remained dormant in vitro when excised from the eld, while prevailing temperature
was low. However, so far no critical study has
been undertaken to understand the cause of tissue browning which hinders the in vitro growth
leading to death of bamboo regenerants. In this
study, we have enunciated that an increase in
TPC level of the explant is detrimental for successful in vitro regeneration in B. balcooa. To the
best of our knowledge this is the rst report on
in vitro regeneration of B. balcooa from mature
eld-grown axillary buds.

Acknowledgements
This investigation was supported by a research
grant from the Council of Scientic & Industrial
Research [Grant No.38 (0966)/99/EMR-II], New
Delhi, India. The authors are thankful to the

anonymous reviewer for his comments and suggestions for the improvement of the manuscript.
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