Tc

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Tc

  1. 1. Plant Tissue Culture
  2. 2. T.C. <ul><li>Refers to technique of growing plant cells, tissues, organs, seeds </li></ul><ul><li>or other plant parts in a sterile environment on a nutrient medium </li></ul>
  3. 3. History <ul><li>In 1902 Haberlandt proposed that single plant cells could be cultured </li></ul>
  4. 4. Haberlandt <ul><li>did not culture them himself </li></ul>
  5. 5. 1930’s <ul><li>White worked on T.C. </li></ul><ul><li>discovery of plant growth regulators </li></ul>
  6. 6. 1930’s <ul><li>importance of vitamins was determined for shoot and root culturing </li></ul>A,D,E,K,C, B complex
  7. 7. 1930’s <ul><li>Indole-Acetic Acid </li></ul><ul><li>IAA </li></ul><ul><li>discovered in 1937 </li></ul>
  8. 8. IAA <ul><li>2,4-D </li></ul><ul><li>Dicamba </li></ul><ul><li>NAA </li></ul><ul><li>IBA </li></ul><ul><li>all synthetic hormones </li></ul>
  9. 9. 1957-58 <ul><li>Miller and Skoog </li></ul><ul><li>University of Wisconsin - Madison </li></ul><ul><li>discovered Kinetin </li></ul>
  10. 10. Kinetin <ul><li>a cytokinin </li></ul><ul><li>plays active role in organogenesis </li></ul>
  11. 11. 1958 <ul><li>Steward developed somatic embryo from carrot cells </li></ul>
  12. 12. 1958-60 <ul><li>Morel cultured orchids and dahlias </li></ul><ul><li>freed them from a viral disease </li></ul>
  13. 13. 1962 <ul><li>Murashige and Skoog </li></ul><ul><li>published recipe for MS Medium </li></ul>
  14. 14. 60’s & 70’s <ul><li>Murashige cloned plants in vitro </li></ul><ul><li>promoted development of commercial plant T.C. labs </li></ul>
  15. 15. 1966 <ul><li>raised haploid plants from pollen grains </li></ul>
  16. 16. 1972 <ul><li>used protoplast fusion to hybridize 2 species of tobacco into one plant </li></ul><ul><li>contained 4N </li></ul>
  17. 17. 4N <ul><li>all chromosomes of both plants </li></ul>
  18. 18. 70’s &80’s <ul><li>develop techniques to introduce foreign DNA into plant cells </li></ul><ul><li>beginning of genetic engineering </li></ul>
  19. 19. T.C. Media <ul><li>functions </li></ul><ul><li>provide H2O </li></ul><ul><li>provide mineral nutritional needs </li></ul>
  20. 20. T.C. Media <ul><li>provide growth regulators </li></ul><ul><li>Provide vitamins </li></ul><ul><li>provide organic compounds </li></ul>
  21. 21. T.C. Media <ul><li>provide access to atmosphere for gas exchange </li></ul><ul><li>serve as a dumping ground for plant metabolites </li></ul>
  22. 22. T.C. Media <ul><li>H2O is usually distilled </li></ul><ul><li>minerals must provide 17 essential elements </li></ul><ul><li>energy source and carbon skeletons - sucrose is preferred </li></ul>
  23. 23. Vitamins <ul><li>thiamine </li></ul><ul><li>pyridoxin </li></ul><ul><li>nicotinic acid </li></ul><ul><li>biotin </li></ul>
  24. 24. Vitamins <ul><li>citric acid </li></ul><ul><li>ascorbic acid </li></ul><ul><li>inositol </li></ul>
  25. 25. Growth Regulators <ul><li>auxins and cytokinins </li></ul><ul><li>gibberellic acid </li></ul><ul><li>abscissic acid </li></ul>
  26. 26. pH of media <ul><li>usually 5.0-5.7 </li></ul>
  27. 27. Media <ul><li>must be sterile </li></ul><ul><li>autoclave at 250 F at 15 psi for 15 minutes </li></ul>
  28. 28. T.C. Stages <ul><li>Explanting- Stage I </li></ul><ul><li>get plant material in sterile culture so it survives </li></ul><ul><li>provide with nutritional and light needs for growth </li></ul>
  29. 29. Stage II <ul><li>rapid multiplication </li></ul><ul><li>stabilized culture </li></ul><ul><li>goal for a commercial lab </li></ul><ul><li>difficult and time consuming to maintain </li></ul>
  30. 30. Stage II <ul><li>occurs in different pathways in different plants </li></ul>
  31. 31. Rooting - Stage III <ul><li>may occur in Stage II </li></ul><ul><li>usually induced by changes in hormonal environment </li></ul><ul><li>lower cytokinin concentration and increase auxin </li></ul>
  32. 32. Rooting <ul><li>may skip stage III and root in a greenhouse </li></ul>
  33. 33. Stage IV <ul><li>transplantation and aftercare </li></ul><ul><li>usually done in greenhouse </li></ul><ul><li>keep RH high (relative humidity) </li></ul>
  34. 34. Stage IV <ul><li>gradually increase light intensity and lower RH after rooting occurs </li></ul><ul><li>allows plants to harden and helps plants form cuticle </li></ul>
  35. 35. Cuticle <ul><li>waxy substance promotes development of stomates </li></ul><ul><li>plants in T.C. don’t have cuticle </li></ul>
  36. 36. Explant <ul><li>portion of plant removed and used for T.C. </li></ul><ul><li>Important features </li></ul><ul><li>size </li></ul><ul><li>source - some tissues are better than others </li></ul>
  37. 37. Explant <ul><li>species dependent </li></ul><ul><li>physiological age - young portions of plant are most successful </li></ul>
  38. 38. Explant <ul><li>degree of contamination </li></ul><ul><li>external infestation - soak plant in sodium hypochlorite solution </li></ul>
  39. 39. Explant <ul><li>internal infection - isolate cell that is not infected </li></ul><ul><li>roots - especially difficult because of soil contact </li></ul>
  40. 40. Explant <ul><li>herbaceous plants </li></ul><ul><li>soft stem </li></ul><ul><li>easier to culture than woody plants </li></ul>
  41. 41. Patterns of multiplication <ul><li>stage II - light 100-300 foot candles </li></ul><ul><li>callus - shoots - roots </li></ul><ul><li>stage III - rooting - light intensity 1000-3000 foot candles </li></ul>
  42. 42. Genetic transformation <ul><li>permanent incorporation of new or foreigh DNA into genome of cell </li></ul>
  43. 43. Transformation methods <ul><li>protoplast fusion </li></ul><ul><li>cell wall is enzymatically removed from cell </li></ul>
  44. 44. Protoplasts <ul><li>naked plant cells </li></ul><ul><li>from 2 different plants can be mixed together and forced to fuse </li></ul>
  45. 45. Protoplast fusion <ul><li>results in heterokaryon </li></ul><ul><li>cell containing two or more nuclei from different cells </li></ul><ul><li>homokaryon - from same cell </li></ul>
  46. 46. Protoplast fusion <ul><li>allowed to regenerate cell wall and then grow into callus </li></ul><ul><li>callus turns to shoots </li></ul>
  47. 47. Shotgun approach <ul><li>DNA coated micro bullets of gold or tungston </li></ul><ul><li>shot into growing cells </li></ul><ul><li>DuPont holds the patent </li></ul>
  48. 48. Shotgun approach <ul><li>injures cells </li></ul><ul><li>random success rate </li></ul>
  49. 49. PEG <ul><li>Polyethylene glycol </li></ul><ul><li>pores open similar to electroporation </li></ul>
  50. 50. Ti Plasmids <ul><li>Tumor inducing </li></ul><ul><li>Agrobacterium temefasciens </li></ul><ul><li>infect cells with agrobacterium which contains desired DNA </li></ul>
  51. 51. Ti Plasmids <ul><li>monocots resist agrobacterium infection </li></ul><ul><li>researchers are working to overcome this </li></ul>
  52. 52. Luciferase <ul><li>an enzyme </li></ul><ul><li>put into tobacco using Ti plasmid </li></ul>
  53. 53. Luciferase <ul><li>when transformed tobacco plants are watered with solution containing Luciferin </li></ul><ul><li>they break it down and emit light </li></ul>
  54. 54. Luciferase <ul><li>glowing in the dark </li></ul><ul><li>like a fire fly </li></ul>
  55. 55. Screening techniques <ul><li>used to identify if culture has taken on desired new trait </li></ul>
  56. 56. Examples <ul><li>sensitivity to antibiotics </li></ul><ul><li>color </li></ul><ul><li>sensitivity to excess deficiencies of substances in growth media </li></ul>
  57. 57. Conventional <ul><li>plant breeding </li></ul><ul><li>egg cell gives half the chromosomes and almost all of the cytoplasm </li></ul><ul><li>male only gives its chromosomes </li></ul>
  58. 58. Cont……. <ul><li>This condition is called maternal cytoplasmic inheritance </li></ul>
  59. 59. Microinjection <ul><li>single cells from culture are held stationary with gentle suction </li></ul><ul><li>injected with a tiny syringe loaded with DNA </li></ul>
  60. 60. Microinjection <ul><li>done under electron microscope </li></ul>
  61. 61. Electroporation <ul><li>desired DNA in solution outside cell </li></ul><ul><li>high energy pulses - 50,000 volts </li></ul><ul><li>for a millisecond </li></ul>
  62. 62. Electroporation <ul><li>cause tiny pores to open </li></ul><ul><li>allows DNA to enter the cell </li></ul>

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