PLANT TISSUE CULTURE & APPLICATIONS

BY U.SRINIVASA

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

WHAT IS IT ?

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 competitionNutrients and removal of waste productsA controlled environment

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.

Explant - Definition 

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

WHAT IS NEEDED?TISSUE CULTURE, BOTH PLANT AND ANIMAL HAS

SEVERAL CRITICAL REQUIREMENTS:

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

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

APPLICATIONS OF PLANT TISSUE CULTURE

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

1.Cell culture

2.Organ culture

3.Embryo culture

4.Protoplast culture

TYPES OF 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

CELL CULTURE

Here cells are cultivated on liquid medium

Liquid suspension culture consists of mixtures of

cell aggregates, cell clusters and single cells

SUSPENSION CULTURE

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

CALLUS CULTURE TECHNIQUE

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

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.

IMPORTANCE OF CALLUS 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

SUSPENSION 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

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.

SUSPENSION CULTURE TECHNIQUE

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

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

IMPORTANCE