basic principles and protocol in plant tissue culture


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basic principles and protocol in plant tissue culture

  1. 1. Basic Principles and Protocol in Plant Tissue Culture Chapter 9 Siti Norazura Jamal
  2. 2. Objective   Be familiar with the protocol in plant tissue culture Get know the application of aseptic technique in plant tissue culture
  3. 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. 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. 5. What is needed? Tissue culture, both plant and animal has several critical requirements: 1) Appropriate tissue Some tissue culture better than others 
  6. 6. 2) - A suitable growth medium Containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid.
  7. 7. Aseptic conditions - Microorganisms grow much more quickly than plant and animal tissue and can over run a culture 3)
  8. 8. 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. 4)
  9. 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. 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. 11. 2) - Sterilization This tissue must be sterilized to remove microbial contamination
  12. 12. Culture type
  13. 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. 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. 15. Dividing shoots Warmth and good light are essential
  16. 16. 5) - Root formation The shoots are transfered to a growth medium with relatively higher auxin: cytokinin ratios.
  17. 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.