basic principles and protocol in plant tissue culture

1. Basic Principles and Protocol in
Plant Tissue Culture
Chapter 9
Siti Norazura Jamal

2. Objective
Be familiar with the protocol in plant tissue culture
Get know the application of aseptic technique in plant
tissue culture

3. 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).

4. 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.

5. What is needed?
Tissue culture, both plant and animal has several critical
requirements:
1)Appropriate tissue
-Some tissue culture better than others

6. 2)A suitable growth medium
-Containing energy sources and inorganic salts to supply
cell growth needs. This can be liquid or semisolid.

7. 3)Aseptic conditions
- Microorganisms grow much more quickly than plant and
animal tissue and can over run a culture

8. 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.

9. 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.

10. 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.

11. 2)Sterilization
-This tissue must be sterilized to remove microbial
contamination

12. Culture type

13. 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.

14. 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

15. Dividing shoots
Warmth and good light are
essential

16. 5)Root formation
-The shoots are transfered to a growth medium with
relatively higher auxin: cytokinin ratios.

17. 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.