It's a six month dissertation work

1. In vitro STUDIES IN Baliospermum
montanum (Willd) Mull. Arg.
Midhun M Nair
School of Environmental Sciences
MG University,Kottayam,Kerala,India

2. INTRODUCTION
• Biotechnology is an advanced branch of biology having
many applications in different field of Biology such as
agriculture, medicine, forestry, food science,
environmental science etc
• Plant cell and tissue culture is one at the potential
aspects of biotechnology. Plant tissue culture is the
technique of growing plant cells, tissue and organ in an
artificially prepared nutrient medium under aseptic
conditions

3. MICROPROPOGATION
 The most popular application of plant tissue culture is
micropropagation which is an alternation to vegetative plant
propagation.
1.Propagation using shoot tip/nodal segment culture
2.Propagation by direct shoot organogenesion
3.Propagation by indirect shoot organogenesis
4.Propagation through somatic embryogenesis

4. HABIT
Baliospermum montanum (Willd) Mull. Arg.

5. Baliospermum montanum
 Baliospermum montanum is a stout monoecious shrub, 1-2m
in height, branchlets cylindrical with leaves alternate.
 Flowers are small numerous, unisexual in axillary racemes
with male flower above, female flower below.
 The flowers are minute , about 3mm across, greenish yellow
in colour arranged in axillary and terminal racemes, spike or
fascicles.
 Seeds egg shaped,flowering and fruiting takes place from
November to February.
 Distribute throughout the most hilly regions from sub
Himalayan tract to Deccan peninsula fairly common as
undergrowth in the dismoist forests of Karnataka and
Kerala.
 It is used as a medical shrub.

6.  Baliospermum montanum is an important medicinal plant
which become endangered due to the pharmaceutical over
exploitation.
 Roots of Baliospermum montanum is used to treat
jaundice, anemiaetc .
 The roots are sold under the name Dantimool or
chotidanti.
 The drugs forms an important con-stituents of preparation
like Dantyaristam, Kaisoragulgula etc .
 For getting large quantity of roots the entire plant is
destroyed or disturbed.
 Micro propagation techniques have often been successful
in
plant propagation.
 The quick multiplication is essential due to high economic
demand of the genus.
 Become constraints invitro technique can be adopted.

7. OBJECTIVES OF THE WORK
• Axillary bud proliferation from
nodal explants
• Callus induction
• Shoot tip culture for shoot
elongation

8. MATERIALS AND METHODS
• Several methods have been adapted for the in vitro
culture at explants tissue.
• The general protocol for the plant tissue culture is as
follows.
1. Aseptic technique
a) Sterilizing the culture vessels and instruments
b) Preparation and sterilization of nutrient medium
c) Sterilization of explant
2. Inoculation of explants
3. Incubation

9.  In the present study shoot tip, nodal
segment, stem segments were taken as
the explants.
 Explants were inoculated and cultured on
MS medium with different concentration
and combination at hormones.
 In different condition explants show
different response.

10.  Shoot tip were inoculated on the MS medium
containing hormones in different
concentration and combination.
 Shoot tips were cultured in BA alone and
along with, IAA or IBA showered different
response.
 Maximum shoot tip elongation from shoot tip
explants only in combination at BA(3mg/l)
and IBA(.4 mg/l)

11. • Nodes were cultured in different in different concentration
and combination of BA and Kin alone or along with NAA,
IBA, IAA and 2, 4-D.
• Medium containing BA along with IBA (0.1 mg/l) from
nodal region low leaf axillary bud development and callus
formation was observed.
• Concentration of IAA and BA was increased high degree
of axillary bud proliferation was observed.

12. Stem
 Stem was cultured on MS medium containing BA
(2mg/l) along with IBA (5mg/l) showed moderate
callusing.
 When stem was cultured BA (5mg/l) along with 2,4-D
(5mg/l) in high concentration, stem shows maximum
rate of callus formation.

13. Callus sub culturing
• Callus obtained from stem segments callus
on MS medium. IAA (2mg/l) along with KIN
(4mg/l) rooting was obtained.
• Callus cultured on MS medium containing
KIN (3mg/l) alone or KIN (2mg/l) along
with BA (3mg/l) recallusing was obtained.

14. Explants IAA IBA 2,4-D BA Response
Shoot tip
0.1mg/l
No response
0.4mg/l 3mg/l
Shoot tip elongation
Nodal
segment
0.5mg/l
Low rate axillary bud elongation
0.5mg/l 4mg/l
Maximum rate axillary bud elongation
Stem
5mg/l 2mg/l
Moderate callusing (++)
5mg/l 5mg/l
High callusing
(+++)
RESPONSETO VARIOUS HORMONES

15. DISCUSSION
 The present work was undertaken to induce regeneration in
Baliospermum montanum (Willd.) Mull. Arg.
 Even though reports are available ininvitro propagation of
explants in Euphorbaceae.
Shoot tip culture
 Maximum elongation of shoot tip was observed in BA(3mg/l)and
IBA(0.4mg/l) containing medium. BA has been reported to be a
good hormone for regeneration in many plants.
 KIN and NAA (0.2mg/l),no shoot formation.
 Only callusing was seen. This high callusing is due to the
presence of NAA.
 Auxins are known to promote high callusing and several cases
can let antogonostic to cytokinin.

16. Nodal segment culture
 KIN (4mg/l)and IAA(0.5mg/l) containing
medium show maximum axillary bud
elongation.
IAA(0.5mg/l) alone containing medium and
low rate of shoot tip elongation/axillary
elongation.
Even though axillury bud proliferation in
culture have been reported mostly in
cytokinincontainina medium.
 In the present study an auxin along with
cytokinin was needed for maximum shoot
multiplication .

17.  For stem segments moderate rate of
callusing was observed.
 Where stem was cultured in MS medium
containing BA (2mg/l) and IBA(5mg/l)showed
moderate rate of callusing.
 But when stem was cultured in BA (5mg/l)
along with 2,4-D(5mg/l) in high
concentration shows maximum rate of callus
formation.
 The maximum callus formation observed in
2,4-D containing medium.

18. Callus subculture
• Callus obtained from explants is subcultured
on MS medium containgIAA(2mg/l) alongs
with KIN(4mg/l).
• Auxin are known to promoterooting and
sexual cases let as antogonostic to cytokinin.
Recallusing was also observed.

19. SUMMARY AND CONCLUSION
 Baliospermum montanum is a valuable medicinal plant
 Roots are used against purgative, anti-inflammatory, anodyne, digestive, diuretic.
 Pharmaceutical over exploitation and habitat destruction makes it endangered.
 Tissue culture approaches helps in the rapid production of the plant and there by plants can be
protected from extinction and also may be use full for the extraction of valuable product from
the plant parts.
 Shoot tip cultures were used for plant regeneration. Maximum elongation of shoot tip was
resulted in BA (5mg/l) and IBA (0.4mg/l) containing medium.
 In nodal culture axillary bud proliferations was observed.
 Medium containing BA (4mg/l) and IAA (0.5mg/l) show maximum axillary bud elongation.
 IAA alone shows moderate callusing.
 Callus formation from the explant sources concentration and combination of the hormones for
callus induction explant such as stem were used.
 Stem shows maximum rate of callusing in medium containing BA (5mg/l) and alone with 2,4-D
(5mg/l)

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