Biology project on plant tissue culture of brahmi leaves, and a gist of the general calyx induction.

1. PLANT TISSUE CULTURE
AND ITS
APPLICATIONS
BIOLOGY
PROJECT
SUBMITTED BY
NAME: VAISHNOVI SEKAR
CLASS: XII C
ROLL NO:

2. INDEX
# AIM
# CERTIFICATE
# ACKNOWLEDGEMENT
# DECLARATION
# OBJECTIVE
# INTRODUCTION TO PLANT TISSUE CULTURE
# APPLICATION OF PLANT TISSUE CULTURE
# PLANT TISSSUE CULTURE FOR MASS PRODUCTION OF HIGH VALUE
SECONDARY METABOLITIES
# THEORY
# EXERCISE 1: PREPARATION OF STOCK SOLUTION




MACRONUTRIENTS STOCK SOLUTIONS
MICRONUTRIENTS STOCK SOLUTION
VITAMIN STOCK SOLUTION
IRON STOCK SOLUTION
# EXERCISE 2: PREPARATION OF MURASHIGE AND SKOOG(MS)
MEDIUM
# EXERCISE 3: INOCULATION OF EXPLANTS NODAL SEGMENT
# EXERCISE 4: PREPARATION OF MS MEDIUM SUPPLEMENTED WITH
PLANT GROWTH HORMONES FOR REGENERATION OF
EXPLANTS
# RESULT AND OBSERVATION
# BIBLIOGRAPHY

3. AIM:
Micro propagation of Brahmi (BACOPA
MONNIERI L.PENNELL) BY INDIRECT
ORGANOGENESIS

4. CERTIFICATE
This is to certify that this project is submitted by
VAISHNOVI SEKAR to the biology department,
CHINMAYA VIDYALAYA, NEW DELHI, was carried out by
her under the guidance and supervision of
Mrs Anubha Srivastav during academic session 20102011.
Date: 03 Nov. 10
Mrs Anubha Srivastav
(Biology teacher)

5. ACKNOWLEDGEMENT
I wish to express my deep gratitude and sincere
thanks to Mrs Anubha Srivastav for her
encouragement and for all the facilities that
she provided for this project work. I sincerely
appreciate this magnanimity by taking me into her
fold for which I shall remain indebted to her. I also
thank our lab assistant, Mrs. Nikhila Nair.
I take this opportunity to express my deep sense of
gratitude for her invaluable guidance, constant
encouragement, constructive comments,
sympathetic attitude and immense motivation in
all stages of this project work.
VAISHNOVI SEKAR

6. DECLARATION
I do hereby declare that this project work has
been originally carried under the guidance
and supervision of
Mrs Anubha Srivastav
Biology teacher
Chinmaya Vidyalaya
VAISHNOVI SEKAR

7. OBJECTIVE
The purpose of the experiment is – to grow new
plant segment with the help of invitro culturing of
the plant nodal segment. I got interested to this
project after learning about various technical
advancements in the field of growth and metabolic
engineering of plants.
Micro propagation of plants of medical importance
and multiplication of high yielding plants is done by
use of plant tissue culture and they are adapted to
local atmospheric conditions by biotechnological
approaches.

8. Introduction: plant tissue culture
Plant tissue culture or the aseptic culture of cells, tissues,
organs and their components under defined physical and
chemical conditions in vitro, has become am important
tool in both basic and applied research and in commercial
applications. It is an outcome of the genesis of an idea of
the German scientist, Haberlandt, at the beginning of the
20th century who believed in the concept of Toti potency.
It relies on maintaining plant cells in aseptic conditions on
a suitable nutrient medium. The culture can be sustained
as a mass of undifferentiated cells for an extended period
of time, or regeneration into whole plants. Designing a
strategy to culture cells from a plant for the first time can
still seem like a matter of trial and error, and luck.
However, the commercial production of valuable
horticulture crops by micro propagation shows that it
exists in the routine, as well as experimental world.

9. Applications: Plant tissue culture
Plant tissue culture now has direct commercial applications
as well as value in basic research into cell biology, genetics
and biochemistry. The techniques include culture of cells,
anthers, ovules and embryos on experimental to industrial
scales, protoplast isolation and fusion, cell selection,
meristem and bud culture.
Application include
 Micro propagation using meristem and shoot culture to
produce large numbers of identical individuals
 Large scale growth of plant cells in liquid culture as a
source of secondary products
 Crossing distantly related species by protoplast fusion and
regeneration of the novel hybrid
 Production of dihaploid plants through pollen or anther
culture to achieve homozygous lines more rapidly for
breeding programmes
 As a tissue for transformation for regeneration of
transgenic plants
 Removal of viruses by propagation from meristematic
tissues
 Secondary metabolites production
 Triploid through endosperm culture
Tissue culture offers an additional or alternative way of
propagation of selective genotypes. This technique is being
commercially exploited for mass propagation of
herbaceous, ornamental and fruit trees.

10. High value secondary metabolites
Plant tissue culture forms the backbone for plant
biotechnology. It encompasses micro propagation,
induction of soma clones, somatic hybridization, cry
preservation and regeneration of transgenic plants.
Interest in medicinal plants as a re-emerging health
aid has been fuelled by the rising costs of
prescription drugs in the maintenance of personal
health and well being, besides the reported side
effects and inability to cure chronic diseases, coupled
with bio-prospecting of new plant derived drugs. In
order to have standardised formulations, the
chemical constituents from plants and their parts are
required to be uniform both qualitatively and
quantitatively.
Furthermore, an ever increasing demand of uniform
medicinal plant-based medicine warrants their mass
cloning through plant tissue culture strategy. A good
number of medical plants have been reported to
regenerate in vitro from their various parts.

11. Most secondary metabolites are important medicinal
compounds. Plants cells culture provides an excellent
system for large scale production of these
compounds. There are a wide variety of secondary
metabolites such as taxol, artemisinin, morphine,
quinine, vinblastine, atropine, scopolamine and
digoxin that can be produced from plants. This
technique represents an optimal solution that has
negated problems such as the slow growth cycle of
the plant source. Plant cell culture allows for the
synthesis of the bioactive substances present in
plants, and is often the only available source,
unhampered by quantitative limitations, of active
ingredients which are poorly available or difficult to
manufacture by chemical synthesis.
Tissue culture is an alternative way for the
production of photochemical of therapeutic
importance. The technique like hairy root culture,
biotransformation, immobilization and elicitations
are used for the increased production of secondary
metabolites. By this approach, therefore, an
increased production of secondary metabolites can
be obtained.

12. Theory:
Plant tissue cultures can be initiated from almost any
part, the explants, of a plant. The physiological state
of the plant does have an influence on its response.
The mother plant must be healthy and free from
obvious sign of disease or decay. Younger tissue
contains a higher proportion of actively dividing cells
and is more responsive to culture conditions.
The exact conditions required to initiate and sustain
plant cells in culture, or to regenerate plantlets from
cultured cells, are different for each plant species.
Each variety of a species will often have a particular
set of culture requirements. Despite all the
knowledge that has been obtained about plant tissue
culture during the 20 th century, there conditions have
to be identified for each variety through
experimentation.
In tissue culture, propagation involves five important
steps:
Step I: involves the initiation of culture which should
be infection free. The culture may be of shoot, root,
nodal and inter-nodal segments etc. care should be
taken to ensure the survival of explants after
sterilization process.
Step II: is the rapid multiplication of culture to
produce plantlets regularly. In a number of species,
this stage is easily carried out by the axillary shoot

13. initiation or adventitious organ formation which is
preferred because somatic embryogenesis has been
utilized to produce faster seedling production. In
case of axillary multiplication, plants have the same
genetic pool as the maternal source. The rate of
multiplication is not high.
Step III: prepare the plant propagules for transfer
from in vitro or in vivo condition. It requires the
induction of roots and hardening of plantlets. Both
the processes start when they are maintained in
cultural conditions and receive in vitro treatments.
Step IV: transfer of tissue culture raised plantlets to
the field to adapt to natural environmental condition.
A number of methods have been applied which
includes the use of mist or fog, carbon-dioxide
enrichment, optimization of light and temperature,
etc. the transferred plantlets grow slowly at initial
stages and restore normal growth later.
Step V: the selection of mother plants is very
important for commercial success of tissue culture
propagation. It may involve the process where the
mother plants are grown under strictly controlled
conditions-25°C temperature with humidity of 75.

14. EXERCISE 1: STOCK SOLUTION
AIM: PREPARATION OF STOCK SOLUTION
PROCEDURE: PREPARE SEPARATELY THE STOCK SOLUTION OF
MACRONUTRIENTS, MICRONUTRIENTS, VITAMIN AND IRON BY
DISSOLVING ALL THE CONSTITUENTS INDIVIDUALLY IN CONICAL FLASK
OVER A MAGNETIC STIRRER.
THE COMPOSITION OF EACH STOCK IS GIVEN BELOW:-
Nutrients
Major nutrients
concentration
NH4NO3
CaCL2
MgSO4
KNO3
KH2PO4
Minor nutrients
16.5g in 100ml
4.4g in 100ml
3.7g in 100ml
19.0g in 100ml
1.7g in 100ml
H3BO4
MnSO4
ZnSO4
CuSO4
CoCl2
KI
Na2MoO4
0.124g in 100ml
0.312g in 100ml
0.172g in 100ml
0.025g in 100ml
0.25g
0.83g
in 100ml
0.25g
Iron solution
FeSo4
0.895g
Na2Edta
Vitamin
0.932g
Glycine
Nicotinic acid
Pyridoxine HCl
Thiamine HCl
0.1g
0.025g
0.025g
0.01g
in 250ml
in 100ml
Precautions:
 Separately dissolve all salts to prevent any precipitation
 Use lukewarm water for easy dissolution

15. 

16. Exercise 2: MS medium
Aim: Preparation of Murashige and Skoog medium
Procedure:
1. Required amount of major, minor, iron and vitamin stocks for preparation
of 1L of MS medium
Major stock
Minor stock
Iron stock
Vitamin
2.
100ml
50ml
10ml
2ml
Take 1L beaker
Dissolve 3% sucrose in 300ml double distil water
Add required amount of major, minor, iron and vitamin stocks
Make up the volume to 1L
Adjust pH to 5.70
(With the help of 1N HCL/1N NaOH)
Supplement medium with 0.63% agar
Boil the medium till agar gets dissolved

17. Pour the medium in culture vessels
(Test tubes/ flasks)
Plug the vessels, cover them with aluminium foil
Sterilize the medium by autoclaving at 121°C for 15 minutes
After autoclaving, the medium is kept inside the culture room.
Precautions:
 the vessels should be sterilized in order to prevent the growth of
micro organism like fungus, etc.

18. Exercise 3: Inoculation
Aim: inoculation of explant, nodal segment
Procedure:
1. sterilization of explant
The nodal segments have to be treated before inoculation
a. under tap water for 15 mins
b. Tween 20 (2ml/100ml) for 20mins
c. Fungicide (Bavistin) (0.01g/L) for 45min
d. HgCl2(0.1%) for 10min
2. Preparation of laminar air flow chamber(LAFC)
Wipe the laminar air flow chamber with 70% alcohol.
Keep:
a. Spirit lamp (filled with spirit)
b. 1 flask containing absolute alcohol filled up to neck
c. Another flask containing 50ml (70% alcohol)
d. Match box
e. Sterile blades
f. Sterile water
g. Vessels containing medium
h. Sterile instrument pair
i. Sterile pertiplate
In the laminar air flow chamber
Switch on UV light in the LAFC for 15 minutes

19. Switch on Air flow after 15 minutes
Can start inoculation
3. Inoculation of explants
Wipe the hands with 70% alcohol
Fix the blade in the scalpel, flame the forceps and scalpel,
After flaming, keep them back in absolute alcohol
With the help of forceps, drop the explants into sterile water containing
flask
Flame the sterile Petri plate with 70% alcohol
Wait for 1-2 minutes so that the surface of pertiplate gets cooled
Take out the explants (nodal segment) from the sterile water
Cut from the top and bottom so that the sterilant at the two ends doesn’t
affect the result
Care should be taken to maintain polarity
Hence cut the lower end can be cut in slanting way so that it can be
demarcated

20. Open the vessel containing medium in front of the flame
Pick the nodal segments and insert the lower cut end into the medium
Close the vessel by putting back the plug in front of the flame
Keep the culture in the culture room
Take observation periodically
Culture room conditions
Temperature: 25±2°C
Humidity: 55±5%
Photoperiod (light/dark):16hr/8hr

22. Exercise 4: MS medium
supplemented with plant growth
hormones
1.
Preparation of stock solution
Weight 50mg BAP and put it in sterile flask
Dissolve BAP in 1N HCL (2ml)
Make up the volume up to 50ml with the help of double distilled water
Store the stock in transparent glass bottle and label the content
(1mg/ml)
Keep it in refrigerator at 20°C
2.
PREPARATION OF MEDIA (500ml)
 2.5mg/l
Take 2L beaker
Dissolve 15g sucrose in 500ml double distilled water
(3% sucrose is used)
Add required amount of major, minor, iron and vitamin stocks

23. Make up the volume to 500ml
Supplement medium with 0.63% agar
Pour the medium in culture vessels, label them A-F
Plug the vessels, cover them with Aluminium foil
Sterilize the medium by autoclaving at 121°C for 15 minutes
After autoclaving, the medium is kept inside the culture room
Inoculate the explants on above medium after 48 hours of media
autoclaving
Study regeneration process periodically

25. RESULT AND OBSERVATIONS
THE ORGANOGENESIS WAS OBSERVED AS SHOWN IN
PICTURES

26. Conclusion:
Micro-propagation is the development of new plants in
an artificial medium under aseptic conditions. This
method is employed for rapid multiplication of elite
plant species.
Discussion:
The most frequently used micro propagation method for
commercial production utilizes enhanced axillary shoot
proliferation from cultured meristem.
Micro propagation process supports in achieving mass
production of healthy plants in low or minimum space
requirement.
On the other hand, micro propagation process comprises
of high labour costs, danger of variation and loss by
contamination.
Multiplication rates are tend to be slow at first, but
later, if the cultural conditions are satisfactory (temp:
25 ± 2 °; humidity 55± 5%; photoperiod – light/dark:
16 hrs/8 hrs), a rapid multiplication can be achieved.
Generally, the plant taken for micro propagation are the
ones which are high in demand in terms of number, are
in demand round the year and have to be multiplied
irrespective of season, are amongst the endangered
ones, have to be cloned, or have to be preserved for
their special qualities.

27. BIBLOGRAPHY
 Wikipedia – the free encyclopedia
 Website: www.icbse.com
 Biology manual