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Techniques of Micropropagation

Techniques of Micropropagation. Chapter 18. Systems used to regenerate plantlets by micropropagation. I.) Axillary shoot formation Meristem tip culture Results in plantlets free from viruses, fungi and bacteria (esp. when coupled with heat treatment)

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Techniques of Micropropagation

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  1. Techniques of Micropropagation Chapter 18

  2. Systems used to regenerate plantlets by micropropagation • I.) Axillary shoot formation • Meristem tip culture • Results in plantlets free from viruses, fungi and bacteria (esp. when coupled with heat treatment) • Important for many herbaceous crops (carnations, mums, orchids, geraniums, banana, potato, sweetpotato) • With woody plants, meristems are often grafted • Axillary shoot culture • Reliably reproduces the genotype of the parent plant (expands existing buds)

  3. Carnation meristem

  4. Nodal shoot production at cotyledonary stage

  5. Systems used to regenerate plantlets by micropropagation • Adventitious shoot formation • Initiated directly on the explant or indirectly from callus • Results in high rates of multiplication • Results in increased aberrant (“off-type”) plants • Parts used: • Leaf pieces (ie: African violet) • Cotyledons (ie: conifers) • Immature inflorescence (ie: Hosta and daylily) • Bulb scales (ie: Easter lily, hyacinths, etc.)

  6. Bulblet formation in tissue culture

  7. Hosta culture

  8. Hosta culture

  9. Hosta culture

  10. Types of micropropagation

  11. Systems used to regenerate plantlets by micropropagation • III.) Callus, cell & protoplast culture systems • Can be subcultured and maintained indefinitely • Callus culture • Produced in response to wounding & hormones • Almost all plant parts can be induced to produce callus • Both auxins & cytokinins must be in the medium • Can be induced to form organs (Organogenesis). Parenchyma produces meristems (= meristmoids) • First done with tobacco & carrot

  12. Direct shoot production (organogenesis)

  13. Systems used to regenerate plantlets by micropropagation • Cell suspensions • Produced from “friable” callus (= loose) • Maintained in shaker cultures or bioreactors • Protoplast culture • Cell culture without cell walls (cellulase added to degrades the cell wall) • Only plasmamembrane remains • Osmotic pressure must be maintained to keep cells from rupturing (mannitol used) • Why done? Secondary plant products that leak from the protoplasts are collected (ex: taxol, sanguinaria)

  14. Cell cultures on a shaker

  15. Bioreactors for cells or protoplasts

  16. Protoplast culture

  17. Protoplast culture

  18. Sanguinaria canadensis“bloodroot”

  19. Systems used to regenerate plantlets by micropropagation • IV.) Somatic embryogenesis & Synthetic seed • Development of embryos without a zygote (i.e. from non-gamete cells) • Roots and shoots develop simultaneously to form embryoids (i.e.: carrots)

  20. Systems used to regenerate plantlets by micropropagation • Arise from: • Adventitious somatic embryogenesis (directly from cells = embryogenic cells). Usually arise near zygotic cells • Induced somatic embryogenesis. Callus must form first (often in suspension culture). Usually conditioned on high levels of auxin (2,4-D) • Uses: • Clonal propagation • Genetic manipulation -using Agrobacterium tumefasciens or a gene-gun

  21. Somatic embryogenesis (soybean)

  22. Somatic embryogenesis (soybean)

  23. Somatic embryogenesis (sitka spruce)

  24. Systems used to regenerate plantlets by micropropagation • Environmental conditions during tissue culture • Temperature • 68 - 81°F • Often held constant to reduce condensation but bulb crops prefer alternating temperatures • Cultures can be refrigerated to slow growth and reduce subculture frequency

  25. Systems used to regenerate plantlets by micropropagation • Light • Irradiance 40 - 80 umol•m-2•sec-1 at culture level (in a greenhouse the irradiance levels range from 600 - 1200 umol•m-2•sec-1 ) • Remember: cultures are heterotrophic, therefore high light for photosynthesis is not critical. High sucrose levels and low CO2 levels inhibit photosynthesis

  26. Systems used to regenerate plantlets by micropropagation • Photoperiod: typically 12 - 16 hours • Light quality: typically cool-white fluorescent lamps used • Vessel and lid effects light quality reaching the culture • Incandescent (red) light increases shoot elongation • Fluorescent (blue) light reduces shoot elongation

  27. Systems used to regenerate plantlets by micropropagation • Gases: • O2, CO2 and C2H2 all affect the culture • Problems in tissue culture • Hyperhydricity (vitrification) • Water-soaked appearance from excess cell water • Leads to culture deterioration • Remedy: change agar type and concentration, reduce condensation/free water

  28. Hepa filters over vents on lids reduce condensation and improve gas exchange

  29. Systems used to regenerate plantlets by micropropagation • Internal pathogens- especially bacteria (can culture on a medium containing an antibacterial agent) • Release of phenolics (causes blackening of the medium). Can be controlled by adding activated charcoal to the medium • Tissue proliferation (TP) • Gall-like growths on micropropagated plants (especially rhododendrons)

  30. Systems used to regenerate plantlets by micropropagation • Habituation • Cultures (shoots) continue to proliferate even when moved to a medium without growth regulators • Variation in micropropagated plants • Increased vigor - not known why • Increased branching - in herbaceous plants especially • Genetic variation - especially of chimeric plants like Hosta

  31. Stabilization of cultures

  32. Determining the proper amounts of cytokinins

  33. Determining the proper amounts of cytokinins

  34. Peony embryo excision and placement in tissue culture

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