Transcript Agoforestri Jarak Pagar

Plant Tissue Culture
Application
Definitions
• Plant cell and tissue culture: cultural techniques
for regeneration of functional plants from
embryonic tissues, tissue fragments, calli, isolated
cells, or protoplasts.
• Totipotency: the ability of undifferentiated plant
tissues to differentiate into functional plants
when cultured in vitro.
• Competency: the endogenous potential of a given
cell or tissue to develop in a particular way.
Definitions
• Organogenesis: The process of initiation
and development of a structure that shows
natural organ form and/or function.
• Embryogenesis: The process of initiation
and development of embryos or embryolike structures from somatic cells (Somatic
embryogenesis).
Basic for Plant Tissue Culture
• Two Hormones Affect Plant Differentiation:
– Auxin
: Stimulates Root Development
– Cytokinin : Stimulates Shoot Development
• Generally, the ratio of these two hormones can
determine plant development:
– ↑ Auxin ↓Cytokinin = Root Development
– ↑ Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development
Factors Affecting Plant Tissue Culture
• Growth Media
– Minerals, growth factors, carbon source,
hormones.
• Environmental Factors
– Light, temperature, photoperiod, sterility, media.
• Explant Source
– Usually, the younger, less differentiated the
explant, the better for tissue culture.
Factors Affecting Plant Tissue Culture
• Genetics
– Different species show differences in
amenability to tissue culture.
– In many cases, different genotypes within a
species will have variable responses to tissue
culture; response to somatic embryogenesis
has been transferred between melon cultivars
through sexual hybridization.
Development of
superior cultivars
Germplasm storage
Somaclonal variation
Embryo rescue
Ovule and ovary cultures
Anther and pollen cultures
 Callus and protoplast culture
Protoplasmic fusion
In vitro screening
 Multiplication
Tissue Culture Applications
Micropropagation
Germplasm preservation
Somaclonal variation
Haploid & dihaploid production
In vitro hybridization – protoplast fusion
Micropropagation
Micropropagation
• The art and science of plant multiplication in vitro.
• Usually derived from meristems (or vegetative
buds) without a callus stage.
– Tends to reduce or eliminate somaclonal
variation, resulting in true clones.
• Can be derived from other explant or callus (but
these are often problematic).
Steps of Micropropagation
• Stage 0 - Selection & preparation of the mother plant
– sterilization of the plant tissue takes place.
• Stage I - Initiation of culture
– explant placed into growth media.
• Stage II - Multiplication
– explant transferred to shoot media; shoots can be
constantly divided.
Steps of Micropropagation
• Stage III - Rooting
– explant transferred to root media.
• Stage IV - Transfer to soil
– explant returned to soil; hardened off.
0. Selection & preparation
of the mother plant
1. Initiation of culture
2. Multiplication
3. Rooting
4. Transfer to soil
Features of Micropropagation
• Clonal reproduction
– Way of maintaining heterozygozity.
• Multiplication stage can be recycled many times to
produce an unlimited number of clones
– Routinely used commercially for many ornamental
species, some vegetatively propagated crops.
• Easy to manipulate production cycles
– Not limited by field seasons/environmental
influences.
Potential Uses for Micropropagation in Plant
Breeding
• Eliminate virus from infected plant selection
– Either via meristem culture or sometimes via heat
treatment of cultured tissue (or combination).
• Maintain a heterozygous plant population for
marker development
– By having multiple clones, each genotype of an F2
can be submitted for multiple evaluations.
Potential Uses for Micropropagation in Plant
Breeding
• Produce inbred plants for hybrid seed production
where seed production of the inbred is limited
– Maintenance or production of male sterile lines
– Poor seed yielding inbred lines
– Potential for seedless watermelon production
Ways to eliminate viruses
Heat treatment.
Plants grow faster than viruses at high temperatures.
Meristemming.
Viruses are transported from cell to cell through
plasmodesmata and through the vascular tissue.
Apical meristem often free of viruses. Trade off
between infection and survival.
Not all cells in the plant are infected.
Adventitious shoots formed from single cells can
give virus-free shoots.
Elimination of viruses
Plant from the field
Pre-growth in the greenhouse
Active
growth
Heat treatment
35oC / months
‘Virus-free’ Plants
Adventitious
Shoot
formation
Meristem culture
Virus testing
Micropropagation cycle
Plant germplasm preservation
 In situ : Conservation in ‘normal’ habitat
–rain forests, gardens, farms
 Ex Situ :
–Field collection, Botanical gardens
–Seed collections
–In vitro collection: Extension of micropropagation
techniques
•Normal growth (short term storage)
•Slow growth (medium term storage)
•Cryopreservation (long term storage
 DNA Banks
In vitro Collection
Use :
 Recalcitrant seeds
 Vegetatively propagated
 Large seeds
Concern:
 Security
Availability
cost
Ways to achieve slow growth
 Use of immature zygotic embryos
(not for vegetatively propagated species)
 Addition of inhibitors or retardants
 Manipulating storage temperature and light
 Mineral oil overlay
 Reduced oxygen tension
 Defoliation of shoots
Cryopreservation
Storage of living tissues at ultra-low temperatures (-196°C)
Conservation of plant germplasm
• Vegetatively propagated species (root and tubers, ornamental, fruit trees)
• Recalcitrant seed species (Howea, coconut, coffee)
Conservation of tissue with specific characteristics
• Medicinal and alcohol producing cell lines
• Genetically transformed tissues
• Transformation/Mutagenesis competent tissues (ECSs)
Eradication of viruses (Banana, Plum)
Conservation of plant pathogens (fungi, nematodes)
Cryopreservation Steps
 Selection
 Excision of plant tissues or organs
 Culture of source material
 Select healthy cultures
 Apply cryo-protectants
 Pre-growth treatments
 Cooling/freezing
 Storage
 Warming & thawing
 Recovery growth
 Viability testing
 Post-thawing
Cryopreservation Requirements
 Preculturing
 Usually a rapid growth rate to create cells with small vacuoles
and low water content
 Cryoprotection
 Cryoprotectant (Glycerol, DMSO/dimetil sulfoksida, PEG) to
protect against ice damage and alter the form of ice crystals
 Freezing
 The most critical phase; one of two methods:
 Slow freezing allows for cytoplasmic dehydration
 Quick freezing results in fast intercellular freezing with little dehydration
Cryopreservation Requirements
 Storage
 Usually in liquid nitrogen (-196oC) to avoid changes in ice
crystals that occur above -100oC
 Thawing
 Usually rapid thawing to avoid damage from ice crystal growth
 Recovery
 Thawed cells must be washed of cryo-protectants and nursed
back to normal growth
 Avoid callus production to maintain genetic stability
Somaclonal Variation
 Variation found in somatic cells dividing mitotically in culture
 A general phenomenon of all plant regeneration systems that
involve a callus phase
Some mechanisms:
 Karyotipic alteration
 Sequence variation
 Variation in DNA Methylation
Two general types of Somaclonal Variation:
 Heritable, genetic changes (alter the DNA)
 Stable, but non-heritable changes (alter gene expression,
epigenetic)
Haploid Plant Production
 Embryo rescue of interspecific
crosses
 Creation of alloploids
 Anther culture/Microspore
culture
 Culturing of Anthers or Pollen
grains (microspores)
 Derive a mature plant from a
single microspore
 Ovule culture
 Culturing of unfertilized
ovules (macrospores)
Somatic Hybridization
Development of hybrid plants through the fusion of somatic protoplasts of
two different plant species/varieties
Somatic
hybridization
technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
Isolation of Protoplast
(Separartion of
protoplasts from plant tissue)
1. Mechanical Method
2. Enzymatic Method
Mechanical Method
Cells Plasmolysis
Plant Tissue
Microscope Observation of cells
Cutting cell wall with knife
Release of protoplasm
Collection of protoplasm
Mechanical Method
Used for vacuolated cells like onion bulb scale, radish
and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability
Enzymatic Method
Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase
Pectinase
Protoplasm
released
Release of
isolated cells
Protoplasm released
cellulase
Isolated
Protoplasm
Enzymatic Method
Used for variety of tissues and organs including
leaves, petioles, fruits, roots, coleoptiles, hypocotyls,
stem, shoot apices, embryo microspores
 Mesophyll tissue - most suitable source
 High yield of protoplast
 Easy to perform
 More protoplast viability
Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion
Intraspecific
Intergeneric
2. Induced Fusion
Chemofusion
Mechanical
Fusion
Electrofusion
Uses for Protoplast Fusion
Combine two complete genomes
 Another way to create allopolyploids
In vitro fertilization
Partial genome transfer
 Exchange single or few traits between species
 May or may not require ionizing radiation
Genetic engineering
 Micro-injection, electroporation, Agrobacterium
Transfer of organelles
 Unique to protoplast fusion
 The transfer of mitochondria and/or chloroplasts between
species
Spontaneous Fusion
 Protoplast fuse spontaneously during isolation
process mainly due to physical contact


Intraspecific produce homokaryones
Intergeneric have no importance
Induced Fusion
Chemofusion- fusion induced by chemicals
•
Types of fusogens
 PEG
 NaNo3
 Ca 2+ ions
 Polyvinyl alcohol
 Mechanical Fusion- Physical fusion of protoplasts under
microscope by using micromanipulator and perfusion
micropipette
 Electrofusion- Fusion induced by electrical stimulation
 Fusion of protoplasts is induced by the application of
high strength electric field (100kv m-1) for few
microsecond
Possible Result of Fusion of Two
Genetically Different Protoplasts
= chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon
cybrid
hybrid
hybrid
cybrid
Advantages of somatic
hybridization
 Production of novel interspecific and intergenic hybrid
 Pomato (Hybrid of potato and tomato)
 Production of fertile diploids and polypoids from sexually
sterile haploids, triploids and aneuploids
 Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other quality
characters
 Production of heterozygous lines in the single species
which cannot be propagated by vegetative means
 Studies on the fate of plasma genes
 Production of unique hybrids of nucleus and cytoplasm
Problem and Limitation of Somatic
Hybridization
1. Application of protoplast technology requires efficient plant
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3.
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regeneration system.
The lack of an efficient selection method for fused product is
sometimes a major problem.
The end-product after somatic hybridization is often unbalanced.
Development of chimaeric calluses in place of hybrids.
Somatic hybridization of two diploids leads to the formation of an
amphiploids which is generally unfavorable.
Regeneration products after somatic hybridization are often variable.
It is never certain that a particular characteristic will be expressed.
Genetic stability.
Sexual reproduction of somatic hybrids.
Inter generic recombination.
One Last Role of Plant Tissue Culture
• Genetic engineering would not be possible without
the development of plant tissue
– Genetic engineering requires the regeneration of
whole plants from single cells.
– Efficient regeneration systems are required for
commercial success of genetically engineered
products.