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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
- 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
- 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
- 51. Ti Plasmids monocots resist agrobacterium infection researchers are working to overcome this
- 52. Luciferase an enzyme put into tobacco using Ti plasmid
- 53. Luciferase when transformed tobacco plants are watered with solution containing Luciferin they break it down and emit light
- 54. Luciferase glowing in the dark like a fire fly
- 55. Screening techniques used to identify if culture has taken on desired new trait
- 56. Examples sensitivity to antibiotics color sensitivity to excess deficiencies of substances in growth media
- 57. Conventional plant breeding egg cell gives half the chromosomes and almost all of the cytoplasm male only gives its chromosomes
- 58. Cont……. This condition is called maternal cytoplasmic inheritance
- 59. Microinjection single cells from culture are held stationary with gentle suction injected with a tiny syringe loaded with DNA
- 60. Microinjection done under electron microscope
- 61. Electroporation desired DNA in solution outside cell high energy pulses - 50,000 volts for a millisecond
- 62. Electroporation cause tiny pores to open allows DNA to enter the cell