3b. Techniques for plant transformation

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3b. Techniques for plant transformation

• Dr. Md. Anowar Hossain
• Associate Professor
• Dept. of Biochemistry and Molecular Biology

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Techniques for plant genetic transformation

• Indirect method- Agrobacterium mediated gene
  transfer
• Direct methods-
• Particle bombardment (biolistics)
• Microprojectile gun method
• Electroporation
• Silicon carbide fibres
• Polyethylene glycol (PEG)/protoplast fusion
• Liposome mediated gene transfer

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Agrobacterium mediated gene transfer

• Agrobacterium-
• Soil borne, gram negative, rod shaped, motile
  found in rhizosphere
• Causative agents of Crown gall disease of
  dicoltyledones
• Have ability transfer bacterial genes to plant
  genome
• Attracted to wound site via chemotaxis in
  response to chemicals (sugar and Phenolic
  molecules acetosyringone) released from damaged
  plant cells
• Contains Ti plasmid which can transfer its T-DNA
  region into genome of host plants

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Nopaline
Octopine
Acetosyringone
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Ti-plasmid features

• Two strains of Ti-plasmid
• -Octopine strains- contains two T-DNA region
  TL (14 kb) and TR ( 7 kb)
• -Nopaline strains- contain one T-DNA region(20
  kb)
• Size is about 200 kb
• Has a central role in Crown-gall formation
• Contains one or more T-DNA region that is
  integrated into the genome of host plants
• Contain a vir region 40 kb at least 811 vir
  genes
• Has origin of replication
• Contains a region enabling conjugative transfer
• Has genes for the catabolism of opines

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T-DNA

• Size 12 to 24 kb
• Left and right border sequence(24-bp) which will
  be
• transferred into genome of host plant
• Oncogenes e.g Auxin, cytokinin, opines
• tm1 gene for determining the tumour size
• the T-DNA contains eight potential genes - these
  are
• eukaryotic in nature (eukaryotic promoters,
  monocistronic,
• eukaryotic polyadenylation signals, eukaryotic
  translation
• mechanisms)
• crown gall tumorigenesis is due to the
  "activation" of
• unregulated phytohormone synthesis in the
  transformed
• cells

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Forms of T-DNA that are found in Agrobacterium

• ds circles - found only in induced bacteria, not
  (apparently) in plant cells
• ds linear T-DNA - found only in induced bacteria,
  not (apparently) in plant cells
• ss linear T-DNA - found in bacteria and plant
  cells
• what is not found - Ti plasmids with evidence
  that T-DNA has been precisely deleted

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Process of T-DNA transfer and integration

• 1. Signal recognition by Agrobacterium
• -Agrobacterium perceive signals such as sugar
  and phenolic compounds which are released from
  plants
• 2. Attachment to plants cells
• Two step processes i) initial attachment via
  polysaccharide ii) mesh of cellulose fiber is
  produced by bacteria.
• Virulence genes (chv genes) are involved in the
  attachment of bacterial cells to the plants
  cells.
• 3. Vir gene induction
• VirA senses phenolics ans subsequently
  phosphorylating and thereby activating VirG. VirG
  then induces expression of all the vir genes.
• 4. T-strand productionVirD1/virD2 complex
  recognises the LB and RB. virD2 produces
  single-stranded nicks in DNA. Then virD2 attached
  to ssDNA. virC may assist this process.

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Process of T-DNA transfer and integration
(continu)

• 5. Transfer of T-DNA out of bacterial cells
  T-DNA/VirD2 is exported from the bacterial cell
  by T-pilus composed of proteins encoded by virB
  operon and VirD2. VirE2 and VirF are also
  exported from bacterial cells.
• 6. Transfer of the T-DNA and Vir proteins into
  the plant nuclear localization T-DNA/VirD2
  complex and other Vir proteins cross the plasma
  membrane through channels formed from VirE2.
  VirE2 protect T-DNA from nucleases, facilitate
  nuclear localization and confer the correct
  conformation to the T-DNA/virD2 complex for
  passage through the nuclear pore complex (NPC).
  The T-DNA/VirD2/VirF2 /plant protein complex the
  nucleus through nuclear pore complex. And
  integrated into host chromosome.

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Practical application of Agrobacterium-mediated
plant transformation

• 1. Agrobacterium mediated transformation methods
  are thought to induce less rearrangement of the
  transgene.
• Lower transgene copy number that direct DNA
  delivery methods.
• Successful production of transgenic plants
  depends on the suitable transformation protocols.
  

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Agrobacterium-mediated transformation of Tobacco

• Several factors have to be considered in the
  design and implementation of any plant
  transformation study
• -1. plant tissue to be transformed. The explant
  should be capable of producing whole plant and
  should contain high number of cells that are
  competent for transformation.
• 2. The vector used to deliver the transgene into
  the genome of plant. Vector should Ti-plasmid
  binary vector which have LB and RB of Ti-plasmid,
  bacterial selectable marker gene, selectable
  marker gene for selection of transformed plant.
  and Multiple cloning sites.
• 3. Strain of Agrobacterium used The choice of
  strain for crop plants is not critical to the
  success of transformation but for recalcitrant
  plants, choice of strains is a major factor to
  successful transformation. For dicotyledons
  plants LBA4404, GV3001 etc., For cereals (
  which are not infected by naturally infected)
  Supervirulent strain such as EHA101, EHA105 are
  used

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The basic protocol used for any Agrobacteruim
mediated transformation experiments

• 1. Identify a suitable explants Suitable plant
  tissue is removed and sterilized. Leaf is used
  for Tobacco.
• 2. Co-cultivate with the Agrobacterium Leaf
  tissue is cut into small pieces and placed into a
  culture of Agrobacterium for about 30 mins. The
  explants are subsequently removed from the
  bacterial culture and placed on to the MS medium
  that contain no selective agent. The incubation
  of explants with Agrobacterium is allowed to
  continue for 2 days to allow transfer of the
  T-DNA transfer to the plant cells.
• 3. Kill the Agrobacterium with a suitable
  antibiotic The explants are removed from the
  medium and washed in antibiotic (cefotaxime)
  solution that kill Agrobacterium cells.

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The basic protocol used for any Agrobacteruim
mediated transformation experiments (Conti-)

• 4. Select for transformed plant cells The
  explant are transferred to fresh solid medium
  supplemented with a selective agent (kanamycin).
  It also contains cefotaxime. Auxin, Cytokinin are
  used to encourage the regeneration of by
  organogenesis. High cytokinin to auxin ratio
  promotes shoot formation from the explants.
• 5. Regeneration of whole plant The shoot can be
  rooted by placing them on solid medium containing
  a high auxin to cytokinin ratio.

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Direct gene transfer methods

• The trem Direct gene transfer is used to
  discriminate between the methods of plant
  transformation that rely on Agrobacterium
  (indirect method) and those that do not (direct
  methods). Direct gene transfer methods all rely
  on the delivery of large amount of naked DNA
  whilst plant is transiently permeabilised.

Direct methods- Particle bombardment
(biolistics) Microprojectile gun
method Electroporation Silicon carbide
fibres Polyethylene glycol (PEG)/protoplast
fusion Liposome mediated gene transfer
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Advantages and disadvantages of direct gene
transfer

• Adv- Widespread use of transformation of cereal
  crops that initially proved difficult to
  transformation with Agrobacterium.
• Disadv- They tend to lead higher frequency of
  transgene rearrangement and higher copy number.
  This can lead to high frequency of gene silencing.

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Particle bombardment

• Why Biolistics or Biolistic bombardment?
• Is the most powerful method for introducing
  nucleic acids into plants, because the helium
  pressure can drive microcarriers through cell
  walls
• Is much easier and less time consuming than
  microinjecting nucleic acids into plant cells or
  embryos
• Allows transformation of animal cells that have
  unique growth requirements and that are not
  amenable to gene transfer using any other method
• Requires less DNA and fewer cells than other
  methods, and can be used for either transient or
  stable transformation

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Principle

• The gold or tungsten particles are coated with
  the DNA that is used to be transform the plant
  tissue.
• The particles are propelled at high speed into
  the target plant material where the DNA is
  released within then cell and can integrate into
  the genome.
• Two types of plant tissues are used for particle
  bombardment
• a) Primary explants that are bombarded and then
  induced to become embryogenic
• b) Proliferating embryonic cultures that are
  bombarded and then allowed to proliferate further
  and subsequently regenerate.

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PDS-1000/He bombardment System
Fig Schematic representation of the PDS-1000/He
system upon activation. The arrows indicate the
direction of helium flow
Fig The PDS-1000/He system, shown here with
magnified view of the Hepta adaptor.
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How the PDS-1000/He System Works

• The sample to be transformed is placed in the
  bombardment chamber, which is evacuated to
  subatmospheric pressure
• The instrument is fired helium flows into the
  gas acceleration tube and is held until the
  specific pressure of the rupture disk is reached
• The disk bursts, and the ensuing helium shock
  wave drives the macrocarrier disk (which carries
  the coated microparticles) a short distance
  toward the stopping screen
• The stopping screen retains the macrocarrier,
  while the microparticles pass through the screen
  into the bombardment chamber and penetrate the
  target cells
• The launch velocity of microcarriers depends on a
  number of adjustable parameters the helium
  pressure (rupture disk selection, 4502,200 psi),
  the amount of vacuum, the distance from the
  rupture disk to the macrocarrier, the distance
  from the microcarrier launch assembly to the
  stopping screen, and the distance between the
  stopping screen and target cells. Adjusting these
  parameters allows you to produce a range of
  velocities to optimally transform many different
  cell types.

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Polyethylene glycol (PEG) mediated transformation
method

• Plant protoplast can be transformed with naked
  DNA by treatment with PEG in the presence of
  divalent cations e. g., Calcium.
• PEG and divalent cations destabilize the plasma
  membrane of the plant protoplast and rendered it
  permeable to naked DNA.
• DNA enters the nucleus and integrates into the
  host genome.

• Disadvantage and advantages
• Regeneration of fertile plants from protoplasts
  is a problematic for some species.
• The DNA used for transformation is also
  susceptible to degradation and rearrangement.
• Despite the limitations, the technique have the
  advantages and protoplast can isolated and
  transformed in number of plants species.

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Electroporation

• It can be used to deliver DNA into plant cells
  and protoplasts.
• The genes of interest require plant regulatory
  sequence.
• Plant materials is incubated in a buffer solution
  containing DNA and subjected to high-voltage
  electric pulse.
• The DNA then migrates through high-voltage-induced
  pores in the plasma membrane and integrates into
  the genome.
• It can be used to transform all the major cereals
  particularly rice, wheat, maize.
• Advantages and disadvantages
• Both intact cells and tissue can be transformed.
• The efficiency of transformation depends upon the
  plant materials, electroporation and tissue
  treatment conditions used for transformation.
• 40 to 50 incubated cells receive DNA
• 50 of the transformed cells can survive

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Silicon carbide fibres-Whiskers

• Plant materials (Cells in suspension culture,
  embryos and embryo-derived callus) is introduced
  into a buffer containing DNA and the silicon
  fibers which is then vortexed.
• The fibers (0.3-0.6 µm in diameter and 10-100µm
  long) penetrate the cell wall and plasma
  membrane, allowing the DNA to gain access to the
  inside of the cells.
• Disadvantages and advantages
• The drawbacks of this technique relate to the
  availability of suitable plant material and the
  inherent dangers of the fibers, which require
  careful handing.
• Many cereals, produce embryonic callus that is
  hard and compact and not easily transformed with
  this technique.
• Despite the some disadvantages, this method is
  recently used for successful transformation of
  wheat, baerly, and maize without the need to cell
  suspension.

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Microinjection

• Microinjection techniques for plant protoplasts
  utilize a holding pipette for immobilizing the
  protoplast while an injection pipette is utilized
  to inject the macromolecule.
• In order to manipulate the protoplasts without
  damage, the protoplasts are cultured for from
  about 1 to 5 days before the injection is
  performed to allow for partial regeneration of
  the cell wall.
• It was found that injection through the partially
  regenerated cell wall could still be accomplished
  and particular compartments of the cell could be
  targeted.
• The methods are particularly useful for
  transformation of plant protoplasts with
  exogenous genes.

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