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					      ‘Green’ biotechnology
•   Introduction
•   DNA, Chromosomes, Genomes
•   Plant Transformation
•   Modern Plant Breeding
•   Plant tissue culture
•   Molecular Marker
 What is Plant Tissue Culture?

“… the aseptic culture of plant
  protoplasts, cells, tissues or
  organs under conditions
  which lead to cell
  multiplication or
  regeneration of organs or
  whole plants “
Totipotency ….
… each living cell has a complete genetic
   complete genetic blue print
blueprint and therefore has the potential to
develop into an entire plant.
 … cells differentiate
… cells lines differentiate to form specialised
tissues and organs

   unlike animal cells, living plant cells
… living plant cells can re-differentiate can
de-differentiate and then re-differentiate to
form different cell types
Therefore,

 tissue can be regenerated from
   explants such as cotyledons,
hypocotyls, leaf, ovary, protoplast,
     petiole, root, anthers, etc.
Tobacco plants from single cells




                                   [Steeves & Sussex 1972]
Regeneration at
the microscopical
level
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).
  What for?

• Tissue culture produces clones, in
  which all product cells have the same
  genotype (unless affected by mutation
  during culture)
   What for?

• A more recent advance is the use of
  plant and animal tissue culture along
  with genetic modification using viral and
  bacterial vectors and gene guns to
  create genetically engineered
  organisms
Early Cell Culture ….

…Haberlandt .. early 1900’s
… proposed concept of totipotency
  … cells cultured under right conditions

Callus cultured from tree cambium
 (Gautheret, Nobecourt, Whire in the 1930s.

   … cells kept alive but did not develop
     First commercial use
• The first commercial use of
  plant clonal propagation on
  artificial media was in the
  germination and growth of
  orchid plants, in the 1920’s

• In the 1950’s and 60’s there
  was a great deal of research,
  but it was only after the
  development of a reliable
  artificial medium (Murashige
  & Skoog, 1962) that plant
                                     Young cymbidium orchids
  tissue culture really ‘took off’
  commercially
            What is needed?
Tissue culture has several critical requirements:

  • Appropriate tissue (some tissues culture better than
    others)

  • A suitable growth medium containing energy sources
    and inorganic salts to supply cell growth needs. This can
    be liquid or semisolid

  • Aseptic (sterile) conditions, as microorganisms grow
    much more quickly than plant and animal tissue and can
    over run a culture
         What is Needed
• Growth regulators - in plants, both auxins
  & cytokinins.

• Frequent subculturing to ensure adequate
  nutrition and to avoid the build up of waste
  metabolites
Culturing Plant Tissue - the steps

                 •    Selection of the plant
                     tissue (explant) from a
                     healthy vigorous
                     ‘mother plant’ - this is
                     often the apical bud,
                     but can be other tissue

                 • This tissue must be
                   sterilized to remove
                   microbial contaminants
Callus – an undifferentiated tissue
Elimination of microbial contaminants

Surface contaminants - principally microbial saprophytes
      that are eliminated by surface disinfestation

Internal contaminants - principally pathogens that are
       eliminated by thermotherapy (35-40 C) and culture of
       explants free of organisms or by antibiotics


Maintenance of asepsis (free from microorganisms) - during
      excision and culture - procedures are carried out in
      sterile laminar flow positive pressure hoods (0.3 µm
      filters)
Common Plant Tissue Disinfestants

                   Concentration of                       Time
Agent              Active Ingredient    Phytotoxicity    (min)
Na hypochlorite
(Laundry Bleach)        0.25-1%           Moderate       5-20
Ca hypochlorite          9-10%            Moderate       5-20
H2O2                     3-10%             High          5-20
Alcohol
(ethanol or
 isopropanol)             70%               High        <30 sec


These disinfestants can be used in combination and the
effectiveness of these solutions is enhanced by using a wetting agent
such as a detergent.
Culturing Plant Tissue - the steps
• Establishment of the
  explant in a culture
  medium. The medium
  sustains the plant cells and
  encourages cell division. It
  can be solid or liquid

• Each plant species has
  particular medium
  requirements that must be
  established by trial and
  error
    Culture Medium constituents

•   Inorganic salt formulations
•   Source of carbohydrate
•   Vitamins
•   Water
•   Plant hormones - auxins, cytokinins, GA’s
•   Solidifying agents
Composition
of tissue
culture
medium is
complex
• 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
2iP/IAA 0.5/2.5

                  Effect of
Kin/IAA 0.5/2.5   different
                  auxine and
Kin/IBA 0.5/2.5   cytokinine
                  concentration
Kin/IBA 0.5/0.5
                  on tissue
                  developemt

Kin/NAA 0.5/0.5
Culturing Plant Tissue - the steps

                 • The rooted shoots are
                   potted up (deflasked)
                   and ‘hardened off’ by
                   gradually decreasing
                   the humidity


                 • This is necessary as
                   many young tissue
                   culture plants have no
                   waxy cuticle to prevent
                   water loss
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
• 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
Why do Plant Tissue Culture?
• A single explant can be multiplied into several
  thousand plants in less than a year - this
  allows fast commercial propagation of new
  cultivars

• Taking an explant does not usually destroy
  the mother plant, so rare and endangered
  plants can be cloned safely

• Once established, a plant tissue culture line
  can give a continuous supply of young plants
  throughout the year
  Why do Plant Tissue Culture? II
• In plants prone to virus diseases, virus free
  explants (new meristem tissue is usually virus
  free) can be cultivated to provide virus free
  plants

• Plant ‘tissue banks’ can be frozen, then
  regenerated through tissue culture

• Plant cultures in approved media are easier to
  export than are soil-grown plants, as they are
  pathogen free and take up little space (most
  current plant export is now done in this
  manner)
Tissue Culture Applications
- Micropropagation
- Germplasm preservation
- Somaclonal variation & mutation selection
- Embryo Culture
- Haploid & Dihaploid Production
- In vitro hybridization – Protoplast Fusion
- Industrial Products from Cell Cultures
Micropropagation
A single explant can be
multiplied into several
thousand plants in less than
a year - this allows fast
commercial propagation of
new cultivars
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
Micropropagation

– generation of genetical
     identical plants
 Micropropagation of almost all
 the fruit crops and vegetables
         is possible now

• Some examples: dwarfing sweet cherry,
  Shade trees, Ornamental shrubs, Roses,
  Clematis, Lilacs, Saskatoon berries,
  Nutraceutical Plants, Rhododendron,
  Azalea, mustard, corn, soybeans, wheat,
  rice, cotton, tomato, potato, citrus, turf,
  legumes
Micropropagation
- allows rapid
  propagation of new varieties
- economical in time and space
- disease free
- elite propagules




 example: violets
 Germplasm Preservation
Slow growth techniques

  o e.g.: ↓ Temp., ↓ Light, media supplements
    (osmotic inhibitors, growth retardants), tissue
    dehydration, etc…
  o Medium-term storage (1 to 4 years)
Germplasm Preservation

Example:
Titan Arum
(Amorphophallus titanum)
    Germplasm Preservation

Cryopreservation
  o Ultra low temperatures (-196 °C)
  o Stops cell division &
    metabolic processes
  o Very long-term (indefinite?)
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 cryoprotectants
  and nursed back to normal growth
   – Avoid callus production to maintain genetic stability
Cryopreservation Requirements
• Preculturing
   – Usually a rapid growth rate to create cells with small
     vacuoles and low water content
• Cryoprotection
   – Glycerol, DMSO, PEG, etc…, 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
        Embryo Culture
Embryo culture developed from the need
 to rescue embryos (embryo rescue)
 from wide crosses where fertilization
 occurred, but embryo development did
 not occur
     Embryo Culture Uses
• Rescue F1 hybrid from a wide cross
• Overcome seed dormancy, usually with
  addition of hormone to media (GA)
• To overcome immaturity in seed
  – To speed generations in a breeding program
  – To rescue a cross or self (valuable genotype) from
    dead or dying plant
 Embryo Culture Uses

Rescue F1 hybrid from a wide cross


 Example: Anthurium
    Embryo Rescue Process
• Make cross between two species
• Dissect embryo (usually immature)
   – The younger the embryo, the more difficult to culture
• Grow on culture medium using basic tissue culture
  techniques, use for breeding if fertile
• Many times, resulting plants will be haploid because
  of lack of pairing between the chromosomes of the
  different species
   – This can be overcome by doubling the chromosomes,
     creating allotetraploids
      Embryo rescue process



15 days




30 days

                   50 days    80 days
Regeneration of grape plants via
somatic embryosgenesis
Potential uses for tissue culture
       in plant breeding
• Eliminate virus from infected plant
  selection



  – Either via meristem culture or sometimes via
    heat treatment of cultured tissue (or
    combination)
                    Phytosanitation

Bacteria or Virus
infected plant
                       Infection of shoot       Regeneration of infected
                       meristem                 plants




                    In a number of plants the
                    shoot meristem doe‘s not                 Regeneration of healthy
                    get infected                             pathogen-free plants
Eliminate virus from infected plant selection.


often used for potato, strawberry, banana, citrus




                 Isolation of the shoot meristem
             Somaclonal Variation

• There are two general types of Somaclonal Variation:
   – Heritable, genetic changes (alter the DNA)
   – Stable, but non-heritable changes (alter gene
     expression, epigenetic)

   – used in mutation breeding
Somaclonal variability




                  Kohleria „Orange Glow“, eine durch mutagene
                  Behandlung von Gewebekulturen erhaltene Mutante
                  (Oben) im Vergleich zur Ausgangsform (links).
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 possible)
• 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
Somaclonal/Mutation Breeding
• Advantages
  – Screen very high populations (cell based)
  – Can apply selection to single cells
• Disadvantages
  – Many mutations are non-heritable
  – Requires dominant mutation (or double recessive
    mutation); most mutations are recessive
Industrial Products from Cell Cultures

• Secondary metabolites produced by plants
  – Alkaloids, Terpenoids, Steroids, Anthocyanins,
    Polyphenols
• Often unclear function in the plant
• Often restricted production (specific species,
  tissue or organ)
• Many are commercially valuable
• Cell culture techniques allow large-scale
  production of specific secondary metabolites
Secondary
metabolites
Example:
Production of Shikonin via cell
culture of Lithospermum                  Shikonin crystals
erythrorhizon




       ムラサキ科                      Shikonin-products
                                  „bio-soap, bio-lipstick“
Tissue Culture Applications
- Micropropagation
- Germplasm preservation
- Somaclonal variation & mutation selection
- Embryo Culture
- Haploid & Dihaploid Production
- In vitro hybridization – Protoplast Fusion
- Industrial Products from Cell Cultures
Early tissue culture ….
- dependent on discovery of
               “growth regulators”

] Cell enlargement … role of auxins
] Cell division ... role of cytokinins

] Regeneration from tobacco pith ..
  (Skoog and Miller) … interaction of auxin
  and cytokinin gives differentiation.
           Essential Nutrients



Macronutrients (required content in the plant - 0.1% or
% per dry weight) - C, H, O, P, K, N, S, Ca, Mg


Micronutrients (requirement - ppm/dry weight) - Fe,
Mn, Zn, Cu, B, Cl, Mo


Na, Se and Si are essential for some plants
              Hormone Balance
Auxin                                Cytokinin
High                                     Low
          Root formation on cuttings
                Embryogenesis
      Adventitious root formation in callus
                Callus initiation
         Adventitious shoot formation
             Axillary shoot growth

Low                                       High
 Further development …

] GA for growth of shoots

] Aux + Cyt + sucrose
       > vascular development

] Culture of ‘thin layers’
 … many interacting factors eg pH
 Carrot plants from root cells – Stewart in 1964




[Steeves & Sussex 1972]
Plant Organ Culture ….

Murashige and Skoog 1962 - mineral media

→ micropropagation
  2 Types of Cell & Tissues

Many different types of cells
Varying degrees of specialisation

   - Meristematic
   - Embryonic
   - Reproductive
     Meristematic tissues ...

Shoot ... apical,
     … axillary
    Culturing Plant Tissue - the steps

                                • Multiplication- The
                                  explant gives rise to a
                                  callus (a mass of
                                  loosely arranged cells)
                                  which is manipulated by
        Dividing shoots           varying sugar
                                  concentrations and the
                                  auxin (low): cytokinin
                                  (high) ratios to form
                                  multiple shoots
                                • The callus may be
                                  subdivided a number of
                                  times

Warmth and good light are essential
Culturing Plant Tissue - the steps

• Root formation - The
  shoots are
  transferred to a
  growth medium with
  relatively higher
  auxin: cytokinin
  ratios
                         The pottles on these racks
                         are young banana plants and
                         are
                         growing roots
              new leaf
   tunica
             apical
             meristem
 corpus
             leaf trace

             axillary
             meristem
procambium



  cortex     pith
         Advantages Cont’d
• facilitates safer movements of germplasm
  across nations - In vitro germplasm assures
  the exchange of pest and disease free
  material
• great for
  – vegetatively reproduced crops
  – crops which produce few seeds or highly
    heterozygous seeds.
Vegetative Propagation in Nature
 • Layering - a drooping lower branch contacts
   the soil (pressed down by snow or
   vegetation); roots form at point of soil
   contact forming a new genetically identical
   tree
 • When trees of some species are cut down,
   new shoots emerge from the stump
 • strawberries spread through sending out
   above-ground horizontal shoots called
   runners, also called stolons.
   Successes of Somaclonal/Mutation Breeding
Herbicide Resistance and Tolerance
• Resistance: able to break-down or metabolize the herbicide –
  introduce a new enzyme to metabolize the herbicide
• Tolerance: able to grow in the presence of the herbicide –
  either ↑ the target enzyme or altered form of enzyme
   – Most successful application of somaclonal breeding have been
     herbicide tolerance
   – Glyphosate resistant tomato, tobacco, soybean (GOX enzyme)
   – Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated EPSP
                                               )
      (enolpyruvyl shikimate phosphate synthase)

        • But not as effective as altered EPSP enzyme (bacterial sources)
   – Imazaquin (Sceptor) tolerant maize
• Theoretically possible for any enzyme-targeted herbicide – it’s
  relatively easy to change a single enzyme by changing a single
  gene
  Other Targets for Somaclonal Variation
• Specific amino acid accumulators
  – Screen for specific amino acid production
  – e.g. Lysine in cereals
• Abiotic stress tolerance
  – Add or subject cultures to selection agent
  – e.g.: salt tolerance, temperature stresses, etc…
• Disease resistance
  – Add toxin or culture filtrate to growth media
  – Examples shown on next slide →
      Disease Resistant Success using Somaclonal Variation

     Crop                    Pathogen                              Toxin
     Alfalfa             Colletotrichum sp.                    Culture filtrate
    Banana                  Fusarium sp.                        Fusaric acid
     Coffee              Colletotrichum sp.           Partially purified culture filtrate
     Maize           Helminthosporium maydis                       T-toxin
      Oat*           Helminthosporium victoriae                   Victorin
 Oilseed Rape*             Phoma lingam                        Culture filtrate
     Peach                Xanthomonas sp.                      Culture filtrate
    Potato**           Phytophthora infestans                  Culture filtrate
     Rice*              Xanthomonas oryzae                     Culture filtrate
  Sugarcane**          Helminthosporium sp.                    Culture filtrate
  Sugarcane**        Helminthosporium sachari           Partially purified HS toxin
   Tobacco*              Psedomonas tabaci                Methionine-sulfoximine
   Tobacco*              Alternaria alternata             Partially purified toxin
*Shown to be heritable through sexual propagation
**Shown to be stable through vegetative propagation
  Requirements for Somaclonal/Mutation 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
Tissue Culture in Plant Breeding

• rescue crosses which would otherwise abort
• Only method of reproduction for sterile
  plants such as triploids (e.g., bananas)
• combine desirable root characteristics with
  desirable shoot characteristics (e.g., grapes)
• Haploid generation through anther/pollen
  culture is an important area in crop
  improvement

				
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