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					Plant Tissue Culture
  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

        Germplasm preservation
          Somaclonal variation
   Haploid & dihaploid production
In vitro hybridization – protoplast fusion
 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
• Disease-free plants can be produced
   – Has been used to eliminate viruses from donor plants
 Microcutting propagation
• It involves the production of shoots from pre-existing
                      meristems only.
         • Requires breaking apical dominance
      • This is a specialized form of organogenesis
  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
• Stage III - Rooting
   – explant transferred to root media
• Stage IV - Transfer to soil
   – explant returned to soil; hardened off
                Conventional                       Micropropagation

Duration:       6 years                            2 years

Labor:          Dig & replant every 2 years;       Subculture every 4 weeks;
                unskilled (Inexpensive)            skilled (more expensive)

Space:          More, but less expensive (field)   Less, but more expensive
Required to
prevent viral   Screening, fumigation, spraying    None
 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           Heat treatment   Adventitious
growth           35oC / months    Shoot
 ‘Virus-free’ Plants              formation

 Meristem culture                  Virus testing

                                   Micropropagation cycle
   Indirect Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination

           1.Callus induction
    2. Embryogenic callus development
• Auxins required for induction
  – Proembryogenic masses form
  – 2,4-D most used
  – NAA, dicamba also used
 Auxin must be removed for embryo development
 Continued use of auxin inhibits embryogenesis
 Stages are similar to those of zygotic embryogenesis
   –   Globular
   –   Heart
   –   Torpedo
   –   Cotyledonary
   –   Germination (conversion)
• Require complete maturation with apical
  meristem, radicle, and cotyledons
• Often obtain repetitive embryony
• Storage protein production necessary
• Often require ABA for complete maturation
• ABA often required for normal embryo
  – Fasciation
  – Precocious germination
• May only obtain 3-5% germination
• Sucrose (10%), mannitol (4%) may be required
• Drying (desiccation)
  – ABA levels decrease
  – Woody plants
  – Final moisture content 10-40%
• Chilling
  – Decreases ABA levels
  – Woody plants
   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

       Security
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
        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
  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
• 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,
  the study of gene regulation that does not involve making changes
  to the SEQUENCE of the DNA, but rather to the actual BASES
            within the nucleotides and to the HISTONES

The three main mechanisms for regulation are:
   CpG island methylation
   acetylation and methylation of histone H3
   the production of antisense RNA
 Somaclonal Breeding Procedures

• Use plant cultures as starting material
   – Idea is to target single cells in multi-cellular culture
   – Usually suspension culture, but callus culture can
     work (want as much contact with selective agent as
   – Optional: apply physical or chemical mutagen
• Apply selection pressure to culture
   – Target: very high kill rate, you want very few cells to
     survive, so long as selection is effective
• Regenerate whole plants from surviving cells
 Requirements for Somaclonal Breeding
• Effective screening procedure
   – Most mutations are deleterious
      • With fruit fly, the ratio is ~800:1 deleterious to beneficial
   – Most mutations are recessive
      • Must screen M2 or later generations
      • Consider using heterozygous plants?
          – But some say you should use homozygous plants to be sure effect is mutation
            and not natural variation
      • Haploid plants seem a reasonable alternative if possible
   – Very large populations are required to identify desired mutation:
      • Can you afford to identify marginal traits with replicates & statistics?
        Estimate: ~10,000 plants for single gene mutant
• Clear Objective
   – Can’t expect to just plant things out and see what happens; relates
     to having an effective screen
   – This may be why so many early experiments failed
       Embryo Culture Uses

• Rescuing interspecific and intergeneric hybrids
– wide hybrids often suffer from early spontaneous abortion
– cause is embryo-endosperm failure
– Gossypium, Brassica, Linum, Lilium
• Production of monoploids
– useful for obtaining "haploids" of barley, wheat, other cereals
– the barley system uses Hordeum bulbosum as a pollen parent
              Bulbosum Method
                 Hordeum                         Hordeum
                                    X           Wild relative
                                                2n = 2X = 14
                2n = 2X = 14
                               Embryo Rescue

                               Haploid Barley
                                 2n = X = 7
                                H. Bulbosum

• This was once more efficient than microspore culture in creating
                          haploid barley
  • Now, with an improved culture media (sucrose replaced by
    maltose), microspore culture is much more efficient (~2000
                     plants per 100 anthers)
       Bulbosum technique
H. vulgare is the seed parent
zygote develops into an embryo with elimination of HB
eventually, only HV chromosomes are left
embryo is "rescued“ to avoid abortion

        Excision of the immature embryo:
    Hand pollination of freshly opened flowers
    Surface sterilization – EtOH on enclosing structures
    Dissection – dissecting under microscope necessary
    Plating on solid medium – slanted media are often used to
     avoid condensation
     Culture Medium
– Mineral salts – K, Ca, N most important
  – Carbohydrate and osmotic pressure
              – Amino acids
       – Plant growth regulators
              Culture Medium
–Carbohydrate and osmotic pressure
»   2% sucrose works well for mature embryos
»   8-12% for immature embryos
»   transfer to progressively lower levels as embryo grows
»   alternative to high sucrose – auxin & cyt PGRs
– amino acids
» reduced N is often helpful
» up to 10 amino acids can be added to replace N salts, incl.
  glutamine, alanine, arginine, aspartic acid, etc.
» requires filter-sterilizing a portion of the medium
             Culture Medium
– natural plant extracts
»   coconut milk (liquid endosperm of coconut)
»   enhanced growth attributed to undefined hormonal factors
    and/or organic compounds
»   others – extracts of dates, bananas, milk, tomato juice
– PGRs
»   globular embryos – require low conc. of auxin and cytokinin
»   heart-stage and later – usually none required
»   GA and ABA regulate "precocious germination“
»   GA promotes, ABA suppresses
  “Wide” crossing of wheat and rye
requires embryo rescue and chemical
 treatment to double the number of

    Haploid Plant Production
 Embryo rescue of interspecific
   – Creation of alloploids
 Anther culture/Microspore
   – Culturing of Anthers or
      Pollen grains (microspores)
   – Derive a mature plant from a
      single microspore
 Ovule culture
   – Culturing of unfertilized
      ovules (macrospores)
   Specific Examples of DH uses
• Evaluate fixed progeny from an F1
   – Can evaluate for recessive & quantitative traits
   – Requires very large dihaploid population, since no prior selection
   – May be effective if you can screen some qualitative traits early
• For creating permanent F2 family for molecular marker
• For fixing inbred lines (novel use?)
   – Create a few dihaploid plants from a new inbred prior to going to
     Foundation Seed (allows you to uncover unseen off-types)
• For eliminating inbreeding depression (theoretical)
   – If you can select against deleterious genes in culture, and screen
     very large populations, you may be able to eliminate or reduce
     inbreeding depression
   – e.g.: inbreeding depression has been reduced to manageable level
     in maize through about 50+ years of breeding; this may reduce
     that time to a few years for a crop like onion or alfalfa
      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

                                           Release of protoplasm
Cutting cell wall with knife

                                                       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

Plasmolysed                          Plasmolysed
cells                                cells

              Pectinase +cellulase                    Pectinase

                                     Release of                           Protoplasm
Protoplasm released                                                       released
                                     isolated cells

           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                                 2. Induced Fusion

                                        Chemofusion         Mechanical
Intraspecific   Intergeneric                                             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
          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
              Induced Fusion

• 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
                                         = nucleus


cybrid     hybrid                         cybrid
       Identifying Desired Fusions
• Complementation selection
   – Can be done if each parent has a different selectable marker (e.g.
     antibiotic or herbicide resistance), then the fusion product
     should have both markers
• Fluorescence-activated cell sorters
   – First label cells with different fluorescent markers; fusion
     product should have both markers
• Mechanical isolation
   – Tedious, but often works when you start with different cell types
• Mass culture
   – Basically, no selection; just regenerate everything and then screen
     for desired traits
        Advantages of somatic
• 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
• 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
    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.
   True in vitro fertilization
  A procedure that involves retrieval of eggs and
sperm from the male and female and placing them
     together in a laboratory dish to facilitate
  Using single egg and sperm cells and fusing them
  Fusion products were cultured individually in 'Millicell'
   inserts in a layer of feeder cells
  The resulting embryo was cultured to produce a fertile
  Requirements for plant genetic
• Trait that is encoded by a single gene
• A means of driving expression of the gene in
  plant cells (Promoters and terminators)
• Means of putting the gene into a cell (Vector)
• A means of selecting for transformants
• Means of getting a whole plant back from the
  single transformed cell (Regeneration)

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