Agoforestri Jarak Pagar

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Plant Tissue Culture Application
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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.
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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 embryo-like structures
from somatic cells (Somatic embryogenesis).
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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
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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.
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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.
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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

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Tissue Culture Applications

• Micropropagation
• Germplasm preservation
• Somaclonal variation
• Haploid dihaploid production
• In vitro hybridization protoplast fusion

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Micropropagation
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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).
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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.
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Steps of Micropropagation Stage III - Rooting
explant transferred to root media. Stage IV -
Transfer to soil explant returned to soil
hardened off.
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STEPS
0. Selection preparation of the mother plant
1. Initiation of culture
2. Multiplication
3. Rooting
4. Transfer to soil
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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/environmenta
l influences.
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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.
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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
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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.

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Elimination of viruses
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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

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In vitro Collection

• Use
• Recalcitrant seeds
• Vegetatively propagated
• Large seeds

• Concern
• Security
• Availability
• cost

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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

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Cryopreservation
Storage of living tissues at ultra-low
temperatures (-196C)

• 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)

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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

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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

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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

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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)

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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)

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Somatic Hybridization
Development of hybrid plants through the fusion
of somatic protoplasts of two different plant
species/varieties
Somatic hybridization technique
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Mechanical Method
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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

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Enzymatic Method
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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

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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

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Spontaneous Fusion

• Protoplast fuse spontaneously during isolation
  process mainly due to physical contact
• Intraspecific produce homokaryones
• Intergeneric have no importance

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Induced Fusion

• Types of fusogens
• PEG
• NaNo3
• Ca 2 ions
• Polyvinyl alcohol

Chemofusion- fusion induced by chemicals

• 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

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Possible Result of Fusion of Two Genetically
Different Protoplasts
chloroplast
mitochondria
Fusion
nucleus
heterokaryon
cybrid
hybrid
cybrid
hybrid
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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

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Problem and Limitation of Somatic Hybridization

1. Application of protoplast technology requires
   efficient plant regeneration system.
2. The lack of an efficient selection method for
   fused product is sometimes a major problem.
3. The end-product after somatic hybridization is
   often unbalanced.
4. Development of chimaeric calluses in place of
   hybrids.
5. Somatic hybridization of two diploids leads to
   the formation of an amphiploids which is
   generally unfavorable.
6. Regeneration products after somatic hybridization
   are often variable.
7. It is never certain that a particular
   characteristic will be expressed.
8. Genetic stability.
9. Sexual reproduction of somatic hybrids.
10. Inter generic recombination.

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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.
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