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Plant Tissue Culture

Plant Tissue Culture

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Plant Tissue Culture

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  1. Plant Tissue Culture

  2. T.C. • Refers to technique of growing plant cells, tissues, organs, seeds • or other plant parts in a sterile environment on a nutrient medium

  3. History • In 1902 Haberlandt proposed that single plant cells could be cultured

  4. Haberlandt • did not culture them himself

  5. 1930’s • White worked on T.C. • discovery of plant growth regulators

  6. 1930’s • importance of vitamins was determined for shoot and root culturing A,D,E,K,C, B complex

  7. 1930’s • Indole-Acetic Acid • IAA • discovered in 1937

  8. IAA • 2,4-D • Dicamba • NAA • IBA • all synthetic hormones

  9. 1957-58 • Miller and Skoog • University of Wisconsin - Madison • discovered Kinetin

  10. Kinetin • a cytokinin • plays active role in organogenesis

  11. 1958 • Steward developed somatic embryo from carrot cells

  12. 1958-60 • Morel cultured orchids and dahlias • freed them from a viral disease

  13. 1962 • Murashige and Skoog • published recipe for MS Medium

  14. 60’s & 70’s • Murashige cloned plants in vitro • promoted development of commercial plant T.C. labs

  15. 1966 • raised haploid plants from pollen grains

  16. 1972 • used protoplast fusion to hybridize 2 species of tobacco into one plant • contained 4N

  17. 4N • all chromosomes of both plants

  18. 70’s &80’s • develop techniques to introduce foreign DNA into plant cells • beginning of genetic engineering

  19. T.C. Media • functions • provide H2O • provide mineral nutritional needs

  20. T.C. Media • provide growth regulators • Provide vitamins • provide organic compounds

  21. T.C. Media • provide access to atmosphere for gas exchange • serve as a dumping ground for plant metabolites

  22. T.C. Media • H2O is usually distilled • minerals must provide 17 essential elements • energy source and carbon skeletons - sucrose is preferred

  23. Vitamins • thiamine • pyridoxin • nicotinic acid • biotin

  24. Vitamins • citric acid • ascorbic acid • inositol

  25. Growth Regulators • auxins and cytokinins • gibberellic acid • abscissic acid

  26. pH of media • usually 5.0-5.7

  27. Media • must be sterile • autoclave at 250 F at 15 psi for 15 minutes

  28. T.C. Stages • Explanting- Stage I • get plant material in sterile culture so it survives • provide with nutritional and light needs for growth

  29. Stage II • rapid multiplication • stabilized culture • goal for a commercial lab • difficult and time consuming to maintain

  30. Stage II • occurs in different pathways in different plants

  31. Rooting - Stage III • may occur in Stage II • usually induced by changes in hormonal environment • lower cytokinin concentration and increase auxin

  32. Rooting • may skip stage III and root in a greenhouse

  33. Stage IV • transplantation and aftercare • usually done in greenhouse • keep RH high (relative humidity)

  34. Stage IV • gradually increase light intensity and lower RH after rooting occurs • allows plants to harden and helps plants form cuticle

  35. Cuticle • waxy substance promotes development of stomates • plants in T.C. don’t have cuticle

  36. Explant • portion of plant removed and used for T.C. • Important features • size • source - some tissues are better than others

  37. Explant • species dependent • physiological age - young portions of plant are most successful

  38. Explant • degree of contamination • external infestation - soak plant in sodium hypochlorite solution

  39. Explant • internal infection - isolate cell that is not infected • roots - especially difficult because of soil contact

  40. Explant • herbaceous plants • soft stem • easier to culture than woody plants

  41. Patterns of multiplication • stage II - light 100-300 foot candles • callus - shoots - roots • stage III - rooting - light intensity 1000-3000 foot candles

  42. Genetic transformation • permanent incorporation of new or foreigh DNA into genome of cell

  43. Transformation methods • protoplast fusion • cell wall is enzymatically removed from cell

  44. Protoplasts • naked plant cells • from 2 different plants can be mixed together and forced to fuse

  45. Protoplast fusion • results in heterokaryon • cell containing two or more nuclei from different cells • homokaryon - from same cell

  46. Protoplast fusion • allowed to regenerate cell wall and then grow into callus • callus turns to shoots

  47. Shotgun approach • DNA coated micro bullets of gold or tungston • shot into growing cells • DuPont holds the patent

  48. Shotgun approach • injures cells • random success rate

  49. PEG • Polyethylene glycol • pores open similar to electroporation

  50. Ti Plasmids • Tumor inducing • Agrobacterium temefasciens • infect cells with agrobacterium which contains desired DNA