tissue culture hybridization


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tissue culture hybridization

  1. 1. jaisreenivasan PLANT TISSUE CULTURE
  2. 2. An Introduction Plant tissue culture is:  A tool in biotechnology, used in micropropagation, secondary metabolic production(taxol), hybridisation etc.  It refers to the growth of plant parts(organs, tissues etc.) in a sterile environment in a nutrient medium.
  3. 3. Why Plant T.C.??  Saves time & labour in comparison to conventional hybridization techniques.  Useful when other techniques are not useful. Eg.: Apical meristems which are free of viruses(present in shoot tips) can be harvested & nurtured to grow into virus-free plants in times of viral attack.
  4. 4. Uses • To save species from extinction • To isolate disease from plants • To produce plants with enhanced stress or pest resistance • To create new plant varieties • To make money
  5. 5. Speed of multiplication in T.C.
  6. 6. Totipotency  Plant tissue culture is based on totipotency  Plant cells have the ability to produce the whole plant from single cells. This is called totipotency.  Plants have the ability to reproduce asexually through a type of cell division- mitosis.
  7. 7. Mitosis • Daughter cells of identical chromosome numbers are produced.
  8. 8. Beginning of T.C.  In 1902, a German physiologist, Gottlieb Haberlandt proposed that single plant cells could be cultured.  He isolated single fully differentiated individual plant cells from different plant species and was first to culture them in Knop’s salt solution enriched with glucose.
  9. 9. History-1930s  In the 1930s, importance of vitamins was determined for shoot and root culturing  Indole-Acetic Acid(IAA), a natural auxin, and the most important, was discovered during the 1930s.  Many more synthetic auxins were discovered. Eg.: 2,4d & NAA(Napthaleneacetic acid)
  10. 10. History-1957-58  Miller and Skoog discovered kinetin, a synthetic cytokinin, in the University of Wisconsin – Madison.  Kinetin plays active role in organogenesis.  Steward developed somatic embryo from carrot cells
  11. 11. History-1958-60  Morel cultured orchids and dahlias & freed them from a viral disease History-1962 • Murashige and Skoog published recipe for MS Medium for T.C.
  12. 12. T.C. Medium  Functions: provide H2O, provide mineral & vitamin nutritional needs.  H2O is usually distilled  minerals must provide 17 essential elements  energy source - sucrose is preferred  provide access to atmosphere for gas exchange  serve as a dumping ground for plant metabolites
  13. 13. T.C. medium  Macronutrients-eg: K, Ca, Mg, N, P, O etc.  Micronutrients-eg: Fe, Mn, Mo, etc.  Vitamins & Amino acids:thiamine, pyridoxin, nicotinic acid, biotin, citric acid, ascorbic acid, Inositol, Glycine etc.  Growth Regulators: auxins and cytokinins  Carbon Source:Sucrose  pH usually 5.0-5.7
  14. 14. T.C. Steps 1. Explant collection 2. Callus generation 3. Organogenesis (shoot and root induction) 4. Hardening  Micropropagation does not involve the second step. There is direct organogenesis.
  15. 15. Explant collection  portion of plant removed and used for T.C.  Important features  size  source - some tissues are better than others  species dependent  physiological age - young portions of plant are most successful
  16. 16. Explant collection  degree of contamination  external infestation - soak plant in sodium hypochlorite solution  internal infection - isolate cell that is not infected  roots - especially difficult because of soil contact  herbaceous plants  soft stem  easier to culture than woody plants
  17. 17. Callus Generation • The dedifferentiation of a plant cell(explant) produces callus. • Callus is then expanded into a larger mass of undifferentiated cells. • Callus is then activated, by selective use of plant hormones to redifferentiate to produce, shoots, roots and ultimately, plantlets
  18. 18. Organogenesis  Usually induced by changes in hormonal environment  Shooting: Higher cytokinin concentration & lower auxin concentration(cytokinins promote shoot growth).  Rooting: lower cytokinin concentration and increase auxin(auxins promote shoot growth)
  19. 19. Hardening  Hardening involves the formation of the waxy cuticle on the leaves of the plant.  Plants in T.C. do not have cuticle  usually done in greenhouse with high relative humidity(RH).  gradually increase light intensity and lower RH after rooting occurs  allows plants to harden and helps plants form cuticle
  20. 20. CREDITS TO  The inventors of tissue culture  Google images  The internet’s information  Microsoft Office 2010