Techniques of Micropropagation

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

12. 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

16. Direct shoot production (organogenesis)

17. 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)

18. Cell cultures on a shaker

19. Bioreactors for cells or protoplasts

20. Protoplast culture

21. Protoplast culture

24. Sanguinaria canadensis“bloodroot”

25. 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)

26. 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

27. Somatic embryogenesis (soybean)

28. Somatic embryogenesis (soybean)

29. Somatic embryogenesis (sitka spruce)

30. 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

31. 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

32. 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

33. 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

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

37. 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)

45. 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

47. Stabilization of cultures

48. Determining the proper amounts of cytokinins

49. Determining the proper amounts of cytokinins

50. Peony embryo excision and placement in tissue culture