MICROPROPAGATION. Introduction In nature plants propagate either Sexually (seeds generation)...
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Transcript of MICROPROPAGATION. Introduction In nature plants propagate either Sexually (seeds generation)...
MICROPROPAGATION
IntroductionIntroduction In nature plants propagate eitherIn nature plants propagate either SexuallySexually (seeds generation) results heterogeneity (seeds generation) results heterogeneity
OrOr AsexuallyAsexually (vegetative multiplication) produce (vegetative multiplication) produce
genetically identical plants. genetically identical plants.
Multiplication of genetically identical copies of a Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal cultivar by asexual reproduction is called clonal propagation.propagation.
Via tissue culture called micropropagation,Via tissue culture called micropropagation,
Basic in vitro propagation ...
What is What is Micropropagation?Micropropagation?
“… the asexual or vegetative propagation (multiplication) of plants in vitro “
Implies - regeneration - multiplication - uniformity ??
MicropropagationMicropropagation
• The art and science of plant The art and science of plant multiplication multiplication in vitroin vitro
• Usually derived from meristems (or Usually derived from meristems (or vegetative buds) without a callus stagevegetative buds) without a callus stage– Tends to reduce or eliminate somaclonal Tends to reduce or eliminate somaclonal
variation, resulting in true clonesvariation, resulting in true clones
• Can be derived from other explant or Can be derived from other explant or callus (but these are often problematic)callus (but these are often problematic)
Methods of micropropagationAxillary branching
Adventitious shoot formation (organogenesis)
Somatic embryogenesis
• >95% of all micropropagation
• Genetically stable• Simple and
straightforward
• Efficient but prone to genetic instability
• Little used. Potentially phenomenally efficient
Axillary shoot proliferationGrowth of axillary buds stimulated by cytokinin treatment; shoots arise mostly from pre-existing meristems
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
Features of Features of MicropropagationMicropropagation• Clonal reproductionClonal reproduction
– Way of maintaining heterozygozityWay of maintaining heterozygozity
• Multiplication Stage can be recycled many Multiplication Stage can be recycled many times to produce an unlimited number of clonestimes to produce an unlimited number of clones– Routinely used commercially for many ornamental Routinely used commercially for many ornamental
species, some vegetatively propagated cropsspecies, some vegetatively propagated crops
• Easy to manipulate production cyclesEasy to manipulate production cycles– Not limited by field seasons/environmental Not limited by field seasons/environmental
influencesinfluences
• Disease-free plants can be producedDisease-free plants can be produced– Has been used to eliminate viruses from donor plantsHas been used to eliminate viruses from donor plants
Micropropagation advantages From one to many propagules rapidly
Multiplication in controlled laboratorium conditions
Continuous propagation year round
Potential for disease-free propagules
Inexpensive per plant once established
Precise crop production scheduling
Reduce stock plant space
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)
Propagules have enhanced growth features (multibranched character)
Micropropagation Micropropagation (contin)(contin)
Positives and negatives of Positives and negatives of micropropagationmicropropagation positivespositives
rapid multiplication ratesrapid multiplication rates low space requirementlow space requirement
negativesnegatives labor costslabor costs expensive (equipment, facilities, supplies)expensive (equipment, facilities, supplies) loss by contaminationloss by contamination danger of variationdanger of variation
Steps of Steps of MicropropagationMicropropagation• Stage 0 – Selection & preparation of the mother Stage 0 – Selection & preparation of the mother
plantplant– sterilization of the plant tissue takes placesterilization of the plant tissue takes place
• Stage I - Initiation of cultureStage I - Initiation of culture– explant placed into growth mediaexplant placed into growth media
• Stage II - MultiplicationStage II - Multiplication– explant transferred to shoot media; shoots can be explant transferred to shoot media; shoots can be
constantly dividedconstantly divided
• Stage III - RootingStage III - Rooting– explant transferred to root mediaexplant transferred to root media
• Stage IV - Transfer to soilStage IV - Transfer to soil– explant returned to soil; hardened offexplant returned to soil; hardened off
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization
Starting material formicropropagation
Leaf
Tip bud
Internode
Axillary bud
Root
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
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization
Disinfestation
Stock plant preparation
Washing in water
Disinfecting solution
Internal contaminants
Screening
The medium
Minerals
Sugar
Organic ‘growth factors’
Growth regulators
Gelling agent
Other additives
Physical Environment
Temperature
Moisture
Light
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization
Origins of new shoots ...
Terminal extension
Lateral / Axillary buds
Adventitious (de novo, re-differentiation)
Callus differentiation
Role of growth regulators ...
Cell division
Differentiation
Cell expansion
Apical dominance
auxins
abscisic acid
cytokinins
gibberelic acid
ethylene
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization
Shoot elongation ...
Basal ‘hormone free’ medium
Gibberellins
Carry-over of hormones
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Elongation
5. Root induction / formation
6. Acclimatization
Root initiation ...
Auxins
Co-factors
C : N ratio
Light / darkness
Initiation vs growth
Juvenility / rejuvenation
Genotype
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Elongation
5. Root induction / formation
6. Acclimatization
- survival of the new plant when removed
from the in vitro environment
Acclimatization (hardening)
Micropropagation of Micropropagation of almost all the fruit crops almost all the fruit crops and vegetables is possibleand vegetables is possible Some examples: dwarfing sweet cherry, Some examples: dwarfing sweet cherry,
Shade trees, Ornamental shrubs, Shade trees, Ornamental shrubs, Roses, Clematis, Lilacs, Saskatoon Roses, Clematis, Lilacs, Saskatoon berries, Nutraceutical Plants, berries, Nutraceutical Plants, Rhododendron, Azalea, mustard, corn, Rhododendron, Azalea, mustard, corn, soybeans, wheat, rice, cotton, tomato, soybeans, wheat, rice, cotton, tomato, potato, citrus, turf, legumespotato, citrus, turf, legumes
Advantages of Advantages of MicropropagationMicropropagation economical in time and space economical in time and space greater output -can produce millions of greater output -can produce millions of
uniformly flowering and yielding plantsuniformly flowering and yielding plants African Biotechnologies - fruit crops African Biotechnologies - fruit crops
banana and indoor pot flowers- 6 million banana and indoor pot flowers- 6 million pieces per yearpieces per year
disease free disease free elite plants with exceptional elite plants with exceptional
characteristicscharacteristics
Advantages Cont’dAdvantages Cont’d
facilitates safer movements of facilitates safer movements of germplasmgermplasm across nations - across nations - In vitroIn vitro germplasm assures the exchange of germplasm assures the exchange of pest and disease free material pest and disease free material
great for great for vegetatively reproduced crops vegetatively reproduced crops crops which produce few seeds or highly crops which produce few seeds or highly
heterozygous seeds.heterozygous seeds.
Uses of Uses of MicropropagationMicropropagation
Used to create transgenic, first Used to create transgenic, first generation plantsgeneration plants
Used in horticulture to produce orchids, Used in horticulture to produce orchids, African Violets, lilies, and fernsAfrican Violets, lilies, and ferns
Used in nurseries to grow fruit trees, Used in nurseries to grow fruit trees, evergreens, roses, and shade treesevergreens, roses, and shade trees
Benefits of Benefits of MicropropagationMicropropagation
Many genetically identical plants can be Many genetically identical plants can be created from one parent plantcreated from one parent plant
Because plants are clones, the uniformity Because plants are clones, the uniformity assures qualityassures quality
Allows many plants to grow in a small Allows many plants to grow in a small place in a short timeplace in a short time
In some species this method will produce In some species this method will produce healthier plantshealthier plants