introduction on tissue culture

1. PLANT
TISSUE CULTURE & APPLICATIONS
BY U.SRINIVASA

2. WHAT IS IT ?
Tissue culture is the term used for “ the process of
growing cells artificially in the laboratory ”
Tissue culture involves both plant and animal cells
Tissue culture produces clones, in which all product
cells have the same genotype (unless affected by
mutation during culture)
OR

3. Tissue culture is the culture and maintenance of plant
cells, tissues or organs (explants) in sterile, nutritionally
(synthetic media) and environmentally, (controlled)
supportive conditions (in vitro)
What conditions do plant cells need to multiply in
vitro?
Freedom from competition
Nutrients and removal of waste products
A controlled environment

4. Explant - Definition
This means to simply cut-out a very small piece of leaf or stem
tissue, or even isolate individual cells, and place them in a tissue
culture container.
• The tissue has to be surface-sterilized so it will not have any
contaminating bacteria or fungus.
• It is then placed inside the tissue culture vessel (dish, jar,
etc.)containing a gel called agar. In the agar is dissolved all the
sugar, nutrients and plant growth regulators the explant needs.

5. WHAT IS NEEDED?
TISSUE CULTURE, BOTH PLANT AND ANIMAL HAS
SEVERAL CRITICAL REQUIREMENTS:
Appropriate tissue :(some tissues culture better than
others)
A suitable growth medium : containing energy sources
and inorganic salts to supply cell growth needs. This
can be liquid or semisolid
Aseptic (sterile) conditions, as microorganisms grow
much more quickly than plant and animal tissue and
can over run a culture

6. Growth regulators - in plants, both auxins &
cytokinins. In animals, this is not as well defined and
the growth substances are provided in serum from
the cell types of interest
Frequent subculturing to ensure adequate nutrition
and to avoid the build up of waste metabolites

7. APPLICATIONS OF PLANT TISSUE
CULTURE
A single explant can be multiplied into several thousand
plants in less than a year - this allows fast commercial
propagation of new cultivars
Taking an explant does not usually destroy the mother plant,
so rare and endangered plants can be cloned safely
Once established, a plant tissue culture line can give a
continuous supply of young plants throughout the year

8. In plants prone to virus diseases, virus free explants
(new meristem tissue is usually virus free) can be
cultivated to provide virus free plants
Plant ‘tissue banks’ can be frozen, then regenerated
through tissue culture
Plant cultures in approved media are easier to export
than are soil-grown plants, as they are pathogen free
and take up little space (most current plant export is now
done in this manner)

9. Tissue culture allows fast selection for crop
improvement - explants are chosen from superior
plants, then cloned
Tissue culture clones are ‘true to type’ as compared
with seedlings, which show greater variability

10. TYPES OF CULTURE
1.Cell culture
2.Organ culture
3.Embryo culture
4.Protoplast culture

11. CELL CULTURE
Cultivation of cells on a solid, semisolid or in a liquid
medium is called cell culture
Based on the type of medium used they are
classified into
1. Callus culture
2. Suspention culture

12. SUSPENSION CULTURE
Here cells are cultivated on liquid medium
Liquid suspension culture consists of mixtures of
cell aggregates, cell clusters and single cells

13. CALLUS CULTURE TECHNIQUE
Establish the callus culture from the seeds of T.Foenum-
Graecum seeds
Procedure:
1.Perform all the operations under aseptic
conditions.
2. Immerse the seeds in 70% ethanol for
2 minutes and rinse thrice with sterile distilled
water

14. 3.Carry the surface sterilization of seeds by submerging
for 5 minutes in 2% v/v bromine solution or 2% aqueous
solution of sodium hypochlorite. Wash the seeds three
times sterile water to totally remove the sterilizing agent
4.Germinate the seeds in dark for 2 to 3 days on sterile
filter paper or cotton wool, previously moistened with
sterile distilled water in Petri dishes at 26 + 2C

15. 5. Remove the cotyledon portion by cutting with sterile
scalpel and transfer the explant portion onto solid sterile
medium(25ml) in culture flasks
6. Incubate the culture at 26+ 1C in darkness for 3
weeks .Transfer the cultures aseptically on sterile fresh
medium at an interval of 4 weeks.

16. 7. Calculate the growth rate in terms of Growth
Index (G.I) as follows
G.I= Final weight of callus/Initial weight of callus
8. Use these static cultures for detection of plant
metabolites and precursor studies in
bioproduction of secondary products

17. IMPORTANCE OF CALLUS CULTURE
1. Production of plantlets through somatic embryogenesis or
organogenesis.
2. For obtaining virus-free plants.
3. As a source of protoplasts and suspension cultures.
4. Production of useful secondary metabolites
5. For biotransformation studies.
6.Selection of cell lines with valuable properties such
as resistance to disease, herbicides, overproduction
of secondary metabolites etc.
7. For mutagenetic studies.

18. SUSPENSION CULTURE
When cells or cell aggregates are cultured in liquid
medium, it is known as suspension culture
Types of suspension culture
1. Batch culture
2. Continuous culture

19. Batch culture:
This is the type of suspension culture in which cells grow
in a definite volume of nutrient medium is called Batch
culture
Continuous culture:
This is a type of culture where cells are separated
mechanically from outflowing medium and again
balanced by inflowing the fresh medium is called
Continuous culture

20. SUSPENSION CULTURE TECHNIQUE
1.The suspension medium is taken in the conical
flask ,autoclaved and used for this technique
2. A Pre-established callus culture is taken and it is
introduced inside the conical flask keeping all steps
aseptic culture.

21. 5.The filtrate ( i.e. free cells) is centrifuged and the
supernatant are poured off. The residue is the free
cells and cell aggregates
6.These cells are again cultured in a fresh liquid
medium and the flasks again agitated by a shaker so
that the cells are suspended equally in the flask

22. 3.The conical flask is closed with cotton plug and
placed with in the clamps of rotary shaker moving at
the 8-120 rpm
4.After considerable time (3-7 days depending on the
growth of the callus),the entire contents of the flask
taken out, filtered through sterilized sieve and
collected the filtrate in a presterilized container.

23. 7. From this the culture used as inoculum and a
subcultred in an another presterlized liquid medium
containing flask dispensing equally the cells or cell
aggregates
8.Cell aggregates are taken and kept in culture room
for the study of regeneration of plant

24. IMPORTANCE
1.The metabolic events of individual cells may
be studied
2.It forms important tools for the development
of organs such as embryo
3.Induction of polyploidy