1 / 62

Plant Tissue Culture

Plant Tissue Culture. Original by Linda Rist Modified by Georgia Agricultural Education Curriculum Office July, 2002. T.C. Refers to technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium. History.

arlen
Download Presentation

Plant Tissue Culture

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Plant Tissue Culture Original by Linda Rist Modified by Georgia Agricultural Education Curriculum Office July, 2002

  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 foreign DNA into genome of cell

  43. Transformation methods • protoplast fusion • cell wall is removed by enzymes 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

More Related