Micropropagation

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• Micropropagation

the art and science of multiplying plants in
vitro.
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Rapid clonal in vitro propagation of plants from
cells, tissues or organs cultured aseptically on
defined media contained in culture vessels
maintained under controlled conditions of light
and temperature
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Toward Commercial Micropropagation 1950s Morel
Martin 1952 Meristem-tip culture for disease
elimination
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Commercialization of Micropropagation 1970s
1980s Murashige 1974 Broad commercial
application
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Clone Genetically identical assemblage of
individuals propagated entirely by vegetative
means from a single plant.
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Conventional Propagation

• Cuttings
• Budding, grafting
• Layering

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Conventional Propagation Advantages

• Equipment costs minimal
• Little experience or technical expertise needed
• Inexpensive
• Specialized techniques for growth control (e.g.
  grafting onto dwarfing rootstocks)

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Micropropagation Advantages

• From one to many propagules rapidly
• Multiplication in controlled lab conditions
• Continuous propagation year round
• Potential for disease-free propagules
• Inexpensive per plant once established

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Micropropagation Advantages

• Precise crop production scheduling
• Reduce stock plant space
• Long-term germplasm storage
• Production of difficult-to-propagate species

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• Micropropagation Production in the United States
• Foliage Plants 63,695,000
• Greenhouse Flowers 11,297,000
• Perennials 9,448,000
• Trees shrubs 15,294,000
• Vegetables 12,862,000
• Fruits 3,721,000
• Miscellaneous 4,545,000
• Total 120,862,000

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Micropropagation Disadvantages

• Specialized equipment/facilities required
• More technical expertise required
• Protocols not optimized for all species
• Plants produced may not fit industry standards
• Relatively expensive to set up?

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Micropropagation Applications

• Rapid increase of stock of new varieties
• Elimination of diseases
• Cloning of plant types not easily propagated by
  conventional methods (few offshoots/ sprouts/
  seeds date palms, ferns, nandinas)
• Propagules have enhanced growth features
  (multibranched character Ficus, Syngonium)

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• Explant
• Cell, tissue or organ of a plant that is used to
  start in vitro cultures
• Many different explants can be used for
  micropropagation, but axillary buds and meristems
  are most commonly used

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Choice of explant

• Desirable properties of an explant
• Easily sterilizable
• Juvenile
• Responsive to culture

• Shoot tips
• Axillary buds
• Seeds
• Hypocotyl (from germinated seed)
• Leaves

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Methods of micropropagation

• gt95 of all micropropagation
• Genetically stable
• Simple and straightforward
• Efficient but prone to genetic instability
• Little used. Potentially phenomenally efficient

• Axillary branching
• Adventitious shoot formation
• Somatic embryogenesis

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Axillary shoot proliferation Growth of axillary
buds stimulated by cytokinin treatment shoots
arise mostly from pre-existing meristems
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• Shoot Culture Method Overview
• Clonal in vitro propagation by repeated enhanced
• formation of axillary shoots from shoot-tips or
• lateral meristems cultured on media
• supplemented with plant growth regulators,
• usually cytokinins.
• Shoots produced are either rooted first in vitro
• or rooted and acclimatized ex vitro

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• ADVANTAGES
• Reliable rates and consistency of shoot
  multiplication
• 3 -8 fold multiplication rate per month
• Pre-existing meristems are least susceptible to
• genetic changes

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• mericloning  A propagation method using shoot
  tips in culture to proliferate multiple buds,
  which can then be separated, rooted and planted
  out

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• First commercially used with orchids -
  conventional propagation rate of 1 per year.
• Through protocorms, 1,000,000 per year.

Corm (Swollen stem)
Chop into pieces
Maturation
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Axillary shoot production

• Selection of plant material
• Establish aseptic culture
• Multiplication
• Shoot elongation
• Root induction / formation
• Acclimatization

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Mother Block A slowly multiplying indexed and
stabilized set of cultures Serve as source of
cultures (explants) for Stage II multiplication
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Stage I - Sterilisation

• Bacteria and fungi will overgrow the explant on
  the medium unless they are removed
• Pre-treatments to clean up the explant
• Detergents
• Sterilants and Antibiotics

• Pre-treatments
• Transfer plants to a greenhouse to reduce endemic
  contaminants
• Force outgrowth of axillary buds
• Washing removes endemic surface contaminants

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• STAGE II Shoot Production
• Stage II selection of cytokinin type and
• concentration determined by
• Shoot multiplication rate
• Length of shoot produced
• Frequency of genetic variability
• Cytokinin effects on rooting and survival

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• STAGE II Shoot Production
• Subculture shoot clusters at 4 -5 week intervals
• 3 -8 fold increase in shoot numbers
• Number of subcultures possible is
• species/cultivar dependent

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• STAGE III Pretransplant (rooting)
• Goals
• Preparation of Stage II shoots/shoot clusters
• for transfer to soil (prehardening)
• Elongation of shoots prior to ex vitro rooting
• Fulfilling dormancy requirements

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STAGE IV Transfer to Natural Environment
Ultimate success of shoot culture depends on
ability to acclimatize vigorously growing
quality plants from in vitro to ex vitro
conditions
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• STAGE IV Transfer to Natural Environment
• Acclimatization
• Process whereby plants physiologically and
• anatomically adjust from in vitro to ex vitro
• cultural and environmental conditions
• Two reasons micropropagated plants may be
  difficult to acclimatize ex vitro Low
  photosynthetic competence
• (heterotrophic nutrition)
• Poor control of water loss

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