PLANT TISSUE CULTURE & APPLICATIONS BY U.

SRINIVASA

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

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

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.

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

 Growth regulators - in plants, both auxins & cytokinins. In animals, this is not as well

defined
interest Frequent

and

the

growth

substances

are

provided in serum from the cell types of

subculturing

to

ensure

adequate

nutrition and to avoid the build up of waste

metabolites

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

 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)

 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

TYPES OF CULTURE 1.Cell culture 2.Organ culture

3.Embryo culture
4.Protoplast culture

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

SUSPENSION CULTURE

Here cells are cultivated on liquid medium

Liquid suspension culture consists of mixtures of cell aggregates, cell clusters and single cells

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

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

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.

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

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 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. cultures.

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

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

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.

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

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.

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

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