Research Article (Open access) |
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Int. J. Life. Sci. Scienti. Res., 3(3):
1052-1054,
May 2017
A Rapid in vitro Micro
Propagation of Bambusa Vulgaris Using
Inter- Node Explant
DSVGK Kaladhar*, Poonam Tiwari, Santosh Kumari Duppala
Department of Microbiology and Bioinformatics,
Bilaspur University, Bilaspur (CG), India
*Address for Correspondence: Dr.
DSVGK
Kaladhar,
Associate Professor and HOD, Department of Microbiology and Bioinformatics,
Bilaspur University, Bilaspur (CG), India
ABSTRACT- B. vulgaris (Bambusa vulgaris Schrad. ex Wendl) has been promoted in order to
solve the deforested environments and economic problems. The present
experimentation was conducted on a rapid
in vitro propagation of Bambusa
vulgaris commonly called as Buddha bamboo, with internode as explant. The
growth had a significant effect on development of the plants with three
cytokinins tested (IAA, NAA, and 2,4 D) along with 0.3 mg/l BAP was found to be
most effective in inducing bud break and multiple shoot formation. The growth
hormones NAA, IAA, 2,4-D, and BAP shown effective on root and shoot formation.
Key-words- B. vulgaris, Plant growth hormones, Internode, NAA, IAA, 2,4-D, BAP
INTRODUCTION- Plant micropropagation is one of the most promising methods in
plant biotechnology for the development of large-scale production of crops,
such as bamboo species [1-2]. Bamboo is one of the fastest growing
renewable resources in the world with great scope in reforestation [3].
Liquid media in the micropropagation processes was considered as an ideal solution
for reducing cost of plantlet production.
Bambusa vulgaris var. wamin, commonly
called as Buddha bamboo, is a native of China [4]. The plant is 4-8
m tall, ornamental bamboo with no reports on flowering [5]. Culms
are usually dark green in color, have short with much swollen (pitcher shaped)
internodes. Some internodes of bamboos remain in vegetative state for
indefinite periods. The rate of over exploitation of various economic trees
like bamboo, in the world is yielding to a bleak future of various tree plants
of significant important [6].
Production by ex-situ conservation is
not yet a viable option and conservation of bamboo diversity depends upon the
protection of natural habits [6-7]. Propagation through macro proliferation
technique is a major breakthrough but is again the limitation of requirement of
seeds. Hence the modern method of
conservation like Micropropagation provides an alternative for regeneration of
new plants rapidly in plants like bamboo [8-9].
Bambusa
vulgaris var. Striata (Yellow bamboo) is a moderate sized bamboo with culms
reaching a height of 8-20 m and a diameter of 5-10 cm. Branching is usually
from mid-culm to top; nodes prominent,
internodes up to 45 cm long. It is easy to propagate by culm and branch
cuttings [10-11]. Cuttings taken from1-2-year-old culms, planted in summer
months may give maximum rooting. Multiple shoot production has also been
reported from mature shoots in MS medium supplemented with coconut milk,
kinetin and BAP. Pre-rooted rhizome and culm cuttings can also be used. Ground
layering and air layering are also found successful. Bambusa vulgaris is used
for paper-making, scaffolding, poles, fencing, curios, handicraft, edible
shoots, medicine, etc [12]. Rings prepared from the split culms are put into
ear perforations by the Naga tribes of Manipur. Pulp made from this species is
used for mixing with hardwood pulps.
Fig 1: Bambusa vulgaris var striata (Yellow variety)
MATERIALS AND METHODS
Collection of Plant
Material- Healthy plant yellow bamboo spp. (Bambusa vulgaris) is collected from the
Raja nursery
Jarhabhata chowk, Bilaspur (CG), India at the green
to brownish stage and the experimentation was done in Department of
Microbiology and Bioinformatics, Bilaspur University, Bilaspur (CG).
Preparation of Explant- Inter-nodal region of stem were cut upto 3 inches Bambusa vulgaris Schrad. ex Wendl) with
sterilized blade. The upper layers of explant were scrubbed off to remove the
dust and wax. The internode explant was then washed in running tap water for 10
minutes. (The explant was washed with distilled water containing 1% of
detergent (Tween 20) for 5 min and rinsed 2–3 times with sterile distilled
water and then soaked in fungicide (Bavistin 1%) for 10 min followed by rinsing
with sterile distilled water. Thereafter, the explants were surface disinfected
with 70% ethanol for 1min and rinsed 2–3 times with sterile distilled water,
treated with 0.1% aqueous mercuric chloride (HgCl2) for 5 min and
thoroughly washed 4–5 times with sterile distilled water under aseptic
condition.
Preparation of MS Media- Culture medium and growth conditions MS (Murashige and Skoog
1962) medium with 2% (w/v) sucrose was used for the present study. The medium was
further amended with BAP (0.3mg/L) in combination with 3 mg/l of IAA, NAA and
2,4-D respectively. The pH of the medium was adjusted to 5.6 before gelling
with 1% agar. The chemicals used in this study are prepared media (Hi-media,
Qualigens and SD fine chemicals, India). Murashige and Skoog (50ml) each was
dispensed into 150 ml sterilized conical flask (Borosil) and plugged with
non-absorbent cotton plug.
Storage of Prepared Media-
After preparation the
media were autoclaved and the
left for a while to reach an ambient temperature and
stored in the refrigerator
at 6°C.
Volume of Culture Media used in Culture Jar- For normal propagation plantlet regeneration experiment, 20 ml
of semi-solid culture media were dispensed in each conical flask.
Establishment of Shoot- Surface sterilized immature and semi-hard wood shoots were
cultured on MS media with and without 0.1 % activated charcoal and the survived
explants were transferred to regeneration media. Percentages of browning and
survivals as well as the number of shoot buds initiated, the new leaves formed
and callus formation were recorded over a period of 4 weeks. Then, the cultured
explants were maintained inside the plant tissue culture room at 25 ± 20C, and
16 h photoperiod were provided by cool white fluorescent tubes. The relative
humidity was 50- 55%.
RESULTS AND DISCUSSION
The present experimentation on a rapid in vitro propagation of Bambusa
vulgaris, commonly called as Buddha bamboo, with internode as explant was
conducted in lab conditions. Table 1 represents various culture conditions
taken for in vitro cultivation of Bambusa
vulgaris Schrad. ex Wendl by plant
tissue culture. Inter node explants of Bambusa vulgaris internode
survived on MS medium supplemented with IAA NAA and 2,4-D and shoot initiated
in 3 weeks.
Table 1. Culture
condition required for in vitro
cultivation of Bambusa vulgaris
Schrad. ex Wendl
Ex–plant |
Temp. |
Moisture |
Ligt period |
Time of regenretion |
Inter node |
25± 2 |
50-55 |
16 hours |
3 Week |
Table 2 represents survival
shoot initiation and regeneration of explant “inter node” in MS media. In the
present experimentation, B. vulgaris internode produced multiple shoots on MS medium
supplemented with different plant growth regulators in combination. Internode
explants took 25 days to initiate shoots. The type and concentration of
cytokinin influenced the average number of inter node produced per explant as
well as mean length of the shoots. The growth had a significant effect on
development of the plants with three cytokinins tested combined with 0.3 mg/l
BAP. The reports were found to be most effective in inducing bud break and
multiple shoot formation from the explants by producing maximum of (2 cm) shoot
lets/explant as an average.
Table 2. Culture of
explant (Internode region) on MS media in BAP (0.3mg/L) in combination with 3
mg/l of IAA (R1), NAA (R2) and 2,4-D (R3) respectively
Explant
In MS Agar
Media |
Percentage (%) of
Explant survival |
Average
No. of shoot initiation |
||||
Inter Node |
R1 |
R2 |
R3 |
R1 |
R2 |
R3 |
66% |
66% |
66% |
2/3 |
2/3 |
2/3 |
Note: IAA (R1), NAA (R2) and 2,4-D (R3)
Fig 2: Shoot initiation
and regeneration of internode of bamboo
Table 3: Effect of plant growth regulators on multiple shoot
induction
S. No. |
Plant growth regulator (mg/l) |
Shoot |
Root |
1 |
NAA (3) + BAP (0.3) |
++ |
+ |
2 |
IAA (3)+ BAP (0.3) |
+ + |
++ |
3 |
2,4-D (3) + BAP (0.3) |
+ + |
_ _ |
Table 3 shows the growth hormones NAA, IAA, 2,4-D 3mg/l
concentration respectively with BAP 0.3 mg/l and its effect on shoot. All the
plant growth regulators showed good results in shoot regeneration. Root
regeneration was found better in IAA combined with BAP. During the
acclimatization phase, the in vitro plants showed 75% survival.
CONCLUSIONS- The present report has
shown positive effect of growth in B. vulgaris var. Striata (Yellow bamboo) by in vitro propagation.
ACKNOWLEDGMENTS-
The authors would like to thank the management and staff of
Bilaspur University, India for their kind support in bringing out the above literature
and providing lab facilities.
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