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 °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 umol•m-2•sec-1 at culture level (in a greenhouse the irradiance levels range from 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 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

51. Chionanthus virginicus embryo excision and placement in tissue culture
After 2 years
from TC
Growth after 2 years
from seed

52. Micrografting

53. Problems in tissue culture
Lack of epicuticular waxes
Phenolic build-up in medium

54. Difficulties in shoot production in Gymnocladus dioicus
Problems in tissue culture
Difficulties in shoot production in Gymnocladus dioicus
“kentuky coffeetree”

55. Sources for supplies/info.
Storage of culture in refrigeration