GEN 314

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GEN 314

• Gene Manipulation Lecture Nine
• Gene transfer to plants

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Plant vectors based on Ti-plasmids

• Wild-type Ti-plasmids are not suitable as general
  gene vectors for plant transformation
• they will cause disorganized growth of the
  recipient plant cells
• gt in order to be able to regenerate plants
  efficiently, one must use a vector with modified
  (disarmed) T-DNA regions, i.e. non-oncogenic
• For example, Zambryski et al. (1983) constructed
  a disarmed vector called pGV3850
• substituted pBR322 sequences for almost all of
  the T-DNA of pTiC58 leaving only the left right
  borders and the opine biosynthetic (nos) gene
• Agrobacterium carrying this plasmid
  transferred the modified T-DNA into the plant
  cells (no tumour formation)
• positive transformants were screened for
  nopaline production
• oncogenic transformation doesnt occur gt
  phytohormone-independent callus production not
  possible to use for self-selecting recipient
  plant cells ?other selectable markers necessary

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Disarmed vector pGV3850
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Inserting DNA into T-DNA region

• Wild-type Ti-plasmids are too large ? it is not
  easy to find adequate unique restriction sites in
  the T-DNA region alone
• intermediate vectors (IV) have therefore being
  developed
• T-DNA region has been subcloned into a
  conventional pBR322-based plasmid vector of E.
  coli
• DNA transfer using IVs will involve
• an IV, containing foreign DNA in the T-region,
  being transferred into Agrobacterium by
  conjugation
• IV is conjugation-deficient, therefore
  conjugation is mediated by a helper plasmid, i.e.
  conjugation-proficient plasmid which can mobilize
  IV (helper plasmid cannot replicate in
  Agrobacterium)
• Transfers are conveniently brought about by
  triparental mating
• E. coli carrying the conjugation-proficient
  helper plasmid, E. coli carrying recombinant IV
  and the recipient Agrobacterium cell
• Following incubation, helper plasmid (tra and mob
  genes) is transferred to the E. coli strain
  carrying recombinant IV, which is then mobilized
  and transferred into Agrobacterium
• In Agrobacterium, in vivo homologous
  recombination is used to insert IV into a
  non-recombinant Ti-plasmid (produce co-integrate)

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Co-integrate disarmed Ti-plasmid
IV transferred into Agrobacterium by conjugation.
Unable to replicate autonomously in Agrobacterium
Disarmed Ti-vector pGV3850 resident in
Agrobacterium
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Homologous recombination between DNA regions
derived from pBR322

• neo marker gene to allow selection of
  recombinant T-DNA in plant cells

Co-integrate Ti-plasmid
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Binary Ti-vectors

• Produced by bringing together a disarmed T-DNA
  carrying foreign DNA and the vir genes that are
  located on a separate disarmed Ti-plasmid
• Construction involves,
• use a modified T-DNA region carrying foreign
  DNA in a small plasmid which replicates in E.
  coli (termed mini-Ti or micro-Ti)
• the mini-Ti/micro-Ti plasmid can then be
  transferred conjugatively in a triparental mating
  into A. tumefaciens containing a compatible
  plasmid-carrying virulence gene
• the vir functions are supplied in trans,
  resulting in the transfer of recombinant T-DNA
  into the plant genome

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Selectable markers

• These are gene products, produced by the plant
  transformation vector, that can be used as a
  means for selecting the transformed plant tissue
• Typically, selection is based on the inclusion of
  a substance that is toxic to plants
• selectable marker on the vector confers
  resistance to the toxic substance when expressed
  in transformed plant tissues, e.g. kanamycin,
  bleomycin,
  spectinomycin, hygromycin, etc
  (Table
  4.1, Slater et al. 2003).
• Also, other methods of selectable markers have
  been used, e.g.
  reporter genes. These include
• GUS, GFP, Luciferases and Chloramphenicol
  acetyltransferase (CAT)

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Direct gene transfer methods

• Briefly means the delivery of large amounts of
  naked DNA into plant cells whilst being
  transiently permeabilized
• Several methods have been developed but
  biolistics or gene bombardment have
  particularly received attention
• Disadvantages of direct gene transfer methods
  (vs. Agrobacterium) is the possibility to produce
  a higher frequency of transgene rearrangement and
  higher transgene copy number
• Methods
• particle bombardment
• PEG-mediated transformation
• electroporation electric pulse to
  protoplasts in a buffer gt permeable
• silicon carbide fibres (WhiskersTM)

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End Lecture Nine