Techniques of Micropropagation

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Techniques of Micropropagation

• Chapter 18

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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)

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Carnation meristem
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Nodal shoot production at cotyledonary stage
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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.)

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Bulblet formation in tissue culture
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Hosta culture
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Hosta culture
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Hosta culture
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Types of micropropagation
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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

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Direct shoot production (organogenesis)
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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)

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Cell cultures on a shaker
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Bioreactors for cells or protoplasts
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Protoplast culture
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Protoplast culture
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Sanguinaria canadensisbloodroot
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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)

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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

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Somatic embryogenesis (soybean)
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Somatic embryogenesis (soybean)
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Somatic embryogenesis (sitka spruce)
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Systems used to regenerate plantlets by
micropropagation

• Environmental conditions during tissue culture
• Temperature
• 68 - 81F
• Often held constant to reduce condensation but
  bulb crops prefer alternating temperatures
• Cultures can be refrigerated to slow growth and
  reduce subculture frequency

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Systems used to regenerate plantlets by
micropropagation

• Light
• Irradiance 40 - 80 umolm-2sec-1 at culture
  level (in a greenhouse the irradiance levels
  range from 600 - 1200 umolm-2sec-1 )
• Remember cultures are heterotrophic, therefore
  high light for photosynthesis is not critical.
  High sucrose levels and low CO2 levels inhibit
  photosynthesis

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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

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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

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

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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

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Stabilization of cultures
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Determining the proper amounts of cytokinins
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Determining the proper amounts of cytokinins
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Peony embryo excision and placement in tissue
culture
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Chionanthus virginicus embryo excision and
placement in tissue culture
After 2 years from TC
Growth after 2 years from seed
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Micrografting
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Problems in tissue culture
Lack of epicuticular waxes
Phenolic build-up in medium
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Problems in tissue culture
Difficulties in shoot production in Gymnocladus
dioicus kentuky coffeetree
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Sources for supplies/info.

• http//aggie-horticulture.tamu.edu/tisscult/microp
  rop/microprop.html

Storage of culture in refrigeration