Principles of Tissue Culture for Micropropagation

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Principles of Tissue Culture for Micropropagation

• Chapter 17

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Principles of Micropropagation

• Terminology
• Tissue culture - procedures used to maintain and
  grow plant tissues (callus, cells, protoplasts)
  or organs (roots, stems, embryos) in aseptic
  culture
• Uses
• Propagation
• Plant breeding (genetic manipulation)
• Production of secondary products
• Germplasm preservation
• Scientific studies

Biotech.
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Gene gun
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Bombarded cells on plate and GUS transformed
cells
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Principles of Micropropagation

• Micropropagation in vitro propagation of plants
• Four stages
• Establishment
• Multiplication
• Rooting
• Acclimatization

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Principles of Micropropagation

• Explant a piece of the plant (propagule) used
  to initiate the micropropagation or tissue
  culture process

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Principles of Micropropagation

• Organogenesis the process of developing
  adventitious roots or shoots

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Principles of Micropropagation

• Somatic embryogenesis

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Principles of Micropropagation

• Somatic embryogenesis development of embryos
  from vegetative cells rather than gametes

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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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Principles of Micropropagation

• Types of tissue culture (seeds/seedlings)
• Aseptic seed culture
• 1922
• Lewis Knudson
• Cultured orchid seed aseptically
• Approx. 30,000 seeds per cattleya seed capsule
• All orchid seed lack an endosperm.
• Normally require mycorrhizae to germinate
• Tissue culture eliminates this requirement.

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Cattleya orchid
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Principles of Micropropagation

• Embryo culture/Embryo rescue
• From interspecific hybrids that normally abort in
  nature
• From early ripening cultivars of many fruit trees
  (where the embryo has not fully developed)
• To remove dormancy problems (i.e. Peony)

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Hybridization between commercial tomato
cultivars of Lycopersecon esculentum and wild
species Lycopersicon peruvianum
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Habitat of Lycopersicon peruvianum in Peru
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Peony embryo excision and placement in tissue
culture
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Principles of Micropropagation

• Ovary and ovule culture
• Unfertilized ovules cultured, pollen added, the
  egg is fertilized and a zygote forms

cotton
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Principles of Micropropagation

• Plantlets from Tissue Culture (Developmental
  Stages)
• Stage I Establishment and stabilization of
  explants in culture
• Get explant clean and put into aseptic culture
• Provide in vitro environment that yields stable
  shoot production

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Principles of Micropropagation

• Explant selection
• Genotype correctly IDed
• True-to-type
• Juvenility of explant (for woody plants)
• Physiologically older explants will produce
  shoots but may still be difficult-to-root

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Principles of Micropropagation

• Explant disinfestation
• Remove fungi, yeast, bacteria, viruses, etc.
• Clean surface to reduce internal contamination
• Clean tools and work area
• Grow stock plants in a greenhouse or in pots

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Principles of Micropropagation

• Explant disinfestation (continued)
• Avoid overhead irrigation or high humidity
• Keep plant parts off the ground
• Control insects (esp. mites and thrips)

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Principles of Micropropagation

• Explant disinfestation (continued)
• Outside, cover shoots with paper bags
• Use systemic fungicides

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Principles of Micropropagation

• Explant disinfestation (continued)
• Clean surface with calcium hypochlorite or sodium
  hypochlorite
• Virus-index stock plants

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6.0
0.5- 2.0
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Principles of Micropropagation

• Culture medium
• Semisolid support (agar/Gelrite)
• Basal medium (BM) inorganic elements including
  macro- and micronutrients
• Energy source (generally sucrose)
• Vitamins
• Growth regulators/hormones cytokinins and auxin

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Membrane rafts on liquid nutrient medium
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Banana on membrane raft Potato
on membrane raft
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Principles of Micropropagation

• Problems in culture medium
• Exudation
• Esp. from woody plants and orchids
• Phenolic compounds (oxidized brown/black)
• Treat with antioxidants (citric or ascorbic acid)
• Treat with absorbent material (activated
  charcoal)
• Stabilization of cultures
• Through repeated subcultures

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Phenolic build-up in medium
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Stabilization of cultures
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Principles of Micropropagation

• Stage II Multiplication
• Maintain the microculture in a stabilized state
  and multiply the microshoots to the required
  for rooting
• Factorial approach best if you do not know the
  amount of cytokinin to use for a specific species
  (or any other factors in the medium)
• i.e. 0, 0.01, 0.1, 1.0 10, 100 mg/L ...

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Principles of Micropropagation

• Stage II Multiplication (continued)
• Generally, the minimum concentration of cytokinin
  that stimulates lateral shoot development is
  selected for multiplication
• Too high a level of cytokinin inhibits shoot
  elongation and produces adventitious shoots
  (which may not have the desired characteristics ,
  i.e. chimeric plants)

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Determining the proper amounts of cytokinins
4.4 µM BA 8.8 µM BA
Pulmonaria lungwort
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Principles of Micropropagation

• Stage II Multiplication (continued)
• Auxin is seldom used during multiplication
• Subculturing - every 2 - 8 weeks (a 1 month
  schedule is typical)
• Seasonal rhythmic patterns often occur therefore
  you get greater shoot proliferation in
  spring/summer than in winter (even under
  completely controlled conditions!)

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Principles of Micropropagation

• Stage III Root formation
• Rooting microcuttings and preparing them for
  transplanting to ex vitro conditions
• In vitro rooting
• Subculture clumps onto reduced cytokinin and
  increased auxin medium also containing 1/2 conc.
  basal salts
• Transfer individual shoots to inducing medium
  (often liquid)
• Then transfer to auxin-free medium and reduced
  basal salts for rooting

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Principles of Micropropagation

• Stage III Root formation(continued)
• In vitro rooting (continued)
• Problems roots produced in vitro may not
  survive. They have a reduced vascular system,
  thin cell walls, contain excess water, and are
  brittle

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Principles of Micropropagation

• Stage III Root formation(continued)
• Ex vitro rooting
• Common as a commercial practice
• Remove individual microcuttings from proliferated
  clumps
• Quick-dip in auxin
• -OR-
• Treat microcuttings by sticking in liquid or
  agar-solidified medium containing auxin for 5 -
  15 days
• Remove, rinse and stick in conventional medium ex
  vitro

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Principles of Micropropagation

• Note Difficult-to-root plants can often be
  rooted as microcuttings
• The more you subculture, the more the plant
  material becomes physiologically juvenile
• The reversion is temporary

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Principles of Micropropagation

• Stage IV Acclimatization
• Preparation for establishment of the microplant
  outdoor environment
• Shift from a heterotrophic (sugar-requiring) to
  an autotrophic (free-living) condition
• Transition from high humidity to reduced humidity
  should be gradual

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Principles of Micropropagation

• Why gradual?
• Microplants often lack epicuticular waxes on
  leaves
• Stomata often are non-functional and fail to
  close after removal from the in vitro environment

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Lack of epicuticular waxes
Leaf with waxes
Leaf without waxes
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Principles of Micropropagation

• Ways to condition the microshoot
• Reduce humidity in the culture vessel
• Remove Parafilm and crack lid for 5-7 days
• Increase the osmotic potential in the medium
  (inc. sugar or agar concentration)
• Use mist, fog, a tent or dome to keep humidity
  high around microplant during transition

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