Transcript Micropropagation 2009 lec16 - An

Micropropagation
“… the art and
science of
multiplying plants
in vitro.”
Plant Propagation by Tissue
Culture
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GJ DeKlerk, EF George, MA Hall (eds)
3rd Edition, 2008
Springer
http://www.springerlink.com/content/n5tm3
0/?p=2d1b86aedb5a437f987c7bd63962bb
c8&pi=0
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
Toward Commercial Micropropagation 1950s
Morel & Martin 1952
Meristem-tip culture for disease elimination
Morel
1960
Wimber
1963
Disease
eradication
& in vitro
production of
orchids
Commercialization of Micropropagation 1970s & 1980s
Murashige 1974
Broad commercial application
Clone
Genetically identical assemblage of
individuals propagated entirely by
vegetative means from a single plant.
Conventional Propagation
• Cuttings
• Budding, grafting
• Layering
Conventional Propagation
Advantages
• Equipment costs minimal
• Little experience or technical expertise needed
• Inexpensive
• Specialized techniques for growth control (e.g.
grafting onto dwarfing rootstocks)
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
Micropropagation
Advantages
• Precise crop production scheduling
• Reduce stock plant space
• Long-term germplasm storage
• Production of difficult-to-propagate species
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?
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)
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
Choice of explant
Desirable properties of an
explant:
• Easily sterilizable
• Juvenile
• Responsive to culture
• Importance of stock
plants
• Shoot tips
• Axillary buds
• Seeds
• Hypocotyl (from
germinated seed)
• Leaves
Methods of micropropagation
• Axillary branching
>95% of all micropropagation
Genetically stable
Simple and straightforward
• Adventitious shoot
formation
Efficient but prone to genetic
instability
• Somatic
embryogenesis
Little used, but potentially
phenomenally efficient
Axillary shoot proliferation
Growth of axillary buds stimulated by cytokinin treatment;
shoots arise mostly from pre-existing meristems
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
ADVANTAGES
•Reliable rates and consistency of shoot multiplication
•3 -8 fold multiplication rate per month
•Pre-existing meristems are least susceptible to
genetic changes
• mericloning A propagation method using shoot
tips in culture to proliferate multiple buds, which
can then be separated, rooted and planted out
• 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
Axillary shoot production
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Selection of plant material
Establish aseptic culture
Multiplication
Shoot elongation
Root induction / formation
Acclimatization
Selection of plant material
• Part of plant
• Genotype
• Physiological condition
• Season
• Position on plant
• Size of explant
Physiological state - of stock plant
• Vegetative / Floral
• Juvenile / Mature
• Dormant / Active
• Carbohydrates
• Nutrients
• Hormones
Stage 1
Disinfestation
• Stock plant preparation
• Washing in water
• Disinfecting solution
• Internal contaminants
• Screening
Mother Block:
A slowly multiplying indexed and
stabilized set of cultures
Serve as source of cultures (explants)
for Stage II multiplication
Stage I - Sterilization
• 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
• Antibiotics, fungicides,
Admire, others
The medium
• Minerals
• Sugar
• Organic ‘growth factors’
• Growth regulators
• Gelling agent
• Other additives
Physical Environment
• Temperature
• Moisture
• Light
Problems
• Contamination
• Browning of the explant (phenolics)
• Dormant buds (woody plants)
STAGE II: Shoot Production
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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
Origins of new shoots
• Terminal extension
• Lateral / Axillary buds
• Adventitious (de novo, re-differentiation)
• Callus differentiation
Problems
• Vitrification – a glassy appearance to tissue
• Long acclimation time
• Callus formation leading to mutations
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
STAGE II: Shoot Production
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
Shoot elongation ...
• Basal ‘hormone free’ medium
• Gibberellins
• Carry-over of hormones
Root initiation
• Auxins
• Charcoal
• C : N ratio
• Light / darkness
• Initiation vs growth
• Juvenility / rejuvenation
• Genotype
STAGE III: Pretransplant (rooting)
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
Stage IV
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