basic principles and protocol in plant tissue culture

Basic Principles and Protocol in

Plant Tissue Culture

Chapter 9

Siti Norazura Jamal

Objective

Be familiar with the protocol in plant tissue culture

Get know the application of aseptic technique in plant

tissue culture

Tissue Culture

The term “ tissue culture” is commonly used in a very

wide sense to include in vitro aseptic culture of plant

cells, tissue and organs.

Is the term for “ the process of growing cells artifically in

the laboratory”.

Involves both plant and animal cells

Tissue culture produces clones, in which all product cells

have the same genotypes (unless affected by mutation

during culture).

Plant Tissue Culture

Is a practice used to propagate clones of a plant

There are various reasons this may be done:

1) To create exact copies of plants that produces

particularly good flowers or fruits.

2) To quickly produce mature plants.

3) To produce multiple of plants in the absence of seeds or

necessary pollination to produce seeds.

4) Used to regenerate the whole plants from plant cells

that have been genetically modified.

What is needed?

Tissue culture, both plant and animal has several critical

requirements:

1) Appropriate tissue

- Some tissue culture better than others

2) A suitable growth medium

- Containing energy sources and inorganic salts to supply

cell growth needs. This can be liquid or semisolid.

3) Aseptic conditions

- Microorganisms grow much more quickly than plant and

animal tissue and can over run a culture

4) Growth regulators

- In plants, both auxins and cytokins.

- In animal, this is not as well defined and the growth

substances are provided in serum from the cell types of

interest

5) Frequent subculturing

- To ensure adequate nutrition and to avoid the build up of

waste conditions.

Aseptic Technique

Is the exclusion of invading microorganisms during

experimental procedures

Using sterile instruments and culture media

Media and apparatus are sterile by autoclaving (121C for

15 minutes)

Aseptic transfer performed in a transfer chamber such as

laminar flow hood which also preferably equipped with a

bunsen burner.

Common sterilants are ethyl alcohol an clorox with an

added surfactants.

Culturing (micropropagating) plant Tissue –

the steps.

1) Selection of the plant tissue

- Plant tissue (explant) from a healthy vigorous “mother

plant”

- Often the apical bud, but can be other tissue.

2) Sterilization

- This tissue must be sterilized to remove microbial

contamination

Culture type

3) Establishment of the explant

- Establishment in a culture medium. The medium sustain

the plant cells and encourage cell division. It can be

solid or liquid.

- Each plant species has particular medium requirements

that must be established by trial and error.

4) Multiplication

- The explant gives rise to a callus ( a mass of loosely

arranged cells) which is manipulated by varying sugar

concentrations and the auxin (low): cytokinin (high)

ratios to form multiple shoots.

- The callus may be subdivided a number of times

Dividing shoots Warmth and good light are

essential

5) Root formation

- The shoots are transfered to a growth medium with

relatively higher auxin: cytokinin ratios.

Benefits 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, the regenerated through tissue culture.

Plant culture 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 selections for crop improvement- explants are chosen from superior plants, then cloned.