Tissue culture


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Tissue culture

  1. 1. Plant Tissue Culture
  2. 2. T.C. Refers to technique of growing plant cells, tissues, organs, seedsor other plant parts in a sterile environment on a nutrient medium
  3. 3. HistoryIn 1902 Haberlandt proposed that single plant cells could be cultured
  4. 4. Haberlandtdid not culture them himself
  5. 5. 1930’sWhite worked on T.C.discovery of plant growth regulators
  6. 6. 1930’simportance of vitamins was determined for shoot and root culturing
  7. 7. 1930’sIndole-Acetic AcidIAAdiscovered in 1937
  8. 8. IAA2,4-DDicambaNAAIBAall synthetic hormones
  9. 9. 1957-58Miller and SkoogUniversity of Wisconsin - Madisondiscovered Kinetin
  10. 10. Kinetina cytokininplays active role in organogenesis
  11. 11. 1958Steward developed somatic embryo from carrot cells
  12. 12. 1958-60Morel cultured orchids and dahliasfreed them from a viral disease
  13. 13. 1962Murashige and Skoogpublished recipe for MS Medium
  14. 14. 60’s 70’sMurashige cloned plants in vitropromoted development of commercial plant T.C. labs
  15. 15. 1966raised haploid plants from pollen grains
  16. 16. 1972used protoplast fusion to hybridize 2 species of tobacco into one plantcontained 4N
  17. 17. 4Nall chromosomes of both plants
  18. 18. 70’s 80’sdevelop techniques to introduce foreign DNA into plant cellsbeginning of genetic engineering
  19. 19. T.C. Mediafunctionsprovide H2Oprovide mineral nutritional needs
  20. 20. T.C. Mediaprovide growth regulatorsProvide vitaminsprovide organic compounds
  21. 21. T.C. Mediaprovide access to atmosphere for gas exchangeserve as a dumping ground for plant metabolites
  22. 22. T.C. MediaH2O is usually distilledminerals must provide 17 essential elementsenergy source and carbon skeletons - sucrose is preferred
  23. 23. Vitaminsthiaminepyridoxinnicotinic acidbiotin
  24. 24. Vitaminscitric acidascorbic acidinositol
  25. 25. Growth Regulatorsauxins and cytokininsgibberellic acidabscissic acid
  26. 26. pH of mediausually 5.0-5.7
  27. 27. Mediamust be sterileautoclave at 250 F at 15 psi for 15 minutes
  28. 28. T.C. StagesExplanting- Stage Iget plant material in sterile culture so it survivesprovide with nutritional and light needs for growth
  29. 29. Stage IIrapid multiplicationstabilized culturegoal for a commercial labdifficult and time consuming to maintain
  30. 30. Stage IIoccurs in different pathways in different plants
  31. 31. Rooting - Stage IIImay occur in Stage IIusually induced by changes in hormonal environmentlower cytokinin concentration and increase auxin
  32. 32. Rootingmay skip stage III and root in a greenhouse
  33. 33. Stage IVtransplantation and aftercareusually done in greenhousekeep RH high (relative humidity)
  34. 34. Stage IVgradually increase light intensity and lower RH after rooting occursallows plants to harden and helps plants form cuticle
  35. 35. Cuticlewaxy substance promotes development of stomatesplants in T.C. don’t have cuticle
  36. 36. Explantportion of plant removed and used for T.C.Important featuressizesource - some tissues are better than others
  37. 37. Explantspecies dependentphysiological age - young portions of plant are most successful
  38. 38. Explantdegree of contaminationexternal infestation - soak plant in sodium hypochlorite solution
  39. 39. Explantinternal infection - isolate cell that is not infectedroots - especially difficult because of soil contact
  40. 40. Explantherbaceous plantssoft stemeasier to culture than woody plants
  41. 41. Patterns of multiplicationstage II - light 100-300 foot candlescallus - shoots - rootsstage III - rooting - light intensity 1000-3000 foot candles
  42. 42. Genetic transformationpermanent incorporation of new or foreigh DNA into genome of cell
  43. 43. Transformation methodsprotoplast fusioncell wall is enzymatically removed from cell
  44. 44. Protoplastsnaked plant cellsfrom 2 different plants can be mixed together and forced to fuse
  45. 45. Protoplast fusionresults in heterokaryoncell containing two or more nuclei from different cellshomokaryon - from same cell
  46. 46. Protoplast fusionallowed to regenerate cell wall and then grow into calluscallus turns to shoots
  47. 47. Shotgun approachDNA coated micro bullets of gold or tungstonshot into growing cellsDuPont holds the patent
  48. 48. Shotgun approachinjures cellsrandom success rate
  49. 49. PEGPolyethylene glycolpores open similar to electroporation
  50. 50. Ti PlasmidsTumor inducingAgrobacterium temefasciensinfect cells with agrobacterium which contains desired DNA
  51. 51. Ti Plasmidsmonocots resist agrobacterium infectionresearchers are working to overcome this
  52. 52. Luciferasean enzymeput into tobacco using Ti plasmid
  53. 53. Luciferasewhen transformed tobacco plants are watered with solution containing Luciferinthey break it down and emit light
  54. 54. Luciferaseglowing in the darklike a fire fly
  55. 55. Screening techniquesused to identify if culture has taken on desired new trait
  56. 56. Examplessensitivity to antibioticscolorsensitivity to excess deficiencies of substances in growth media
  57. 57. Conventionalplant breedingegg cell gives half the chromosomes and almost all of the cytoplasmmale only gives its chromosomes
  58. 58. Cont…….This condition is called maternal cytoplasmic inheritance
  59. 59. Microinjectionsingle cells from culture are held stationary with gentle suctioninjected with a tiny syringe loaded with DNA
  60. 60. Microinjectiondone under electron microscope
  61. 61. Electroporationdesired DNA in solution outside cellhigh energy pulses - 50,000 voltsfor a millisecond
  62. 62. Electroporationcause tiny pores to openallows DNA to enter the cell