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Agrobacterium-mediated transformation

• Transformation – the process of obtaining
  transgenic plants
• Transgenic plant – a plant with a foreign gene
  (or genes) from another plant/animal that is
  incorporated into its chromosome
• Most common genes (and traits) in transgenic or
  biotech crops
  – herbicide resistance (e.g., Round-up Ready
    soybeans)
Agrobacterium-mediated transformation

• Most common genes (and traits) in transgenic or
  biotech crops
  – Bt genes (European corn borer toxin) in field corn
    (maize)
  – virus-resistance (coat-protein) genes (papaya)
• Viewpoints of these biotech crops have stirred
  debate pro and con
  – growers are generally in the "pro" camp
Agrobacterium-mediated transformation

• Viewpoints (contin)
  – environmentalists are suspicious
  – organic farmers are in the "con" camp
  – some consumers wonder where the benefit is to
    them, others worry about potential allergens
  – Europeans won't use them, but they are in a growing
    minority of nations
Agrobacterium-mediated transformation

• Regardless of the debate over biotech crops,
  they seem certain to be with us for the
  foreseeable future
• We will concentrate on the way in which in vitro
  methods are currently used for producing
  transgenics
• Two major methods for making transgenics
  – Agrobacterium-mediated transformation
  – particle bombardment
Agrobacterium-mediated transformation

• Basic tissue-culture process for Agrobacterium-
  mediated transformation
  – disinfestation of an explant
  – isolation and growth of the Agrobacterium vector (with
    a DNA sequence that is to be transferred)
  – co-culture of the explant with the Agrobacterium
  – growth of the explant on a selective medium
  – regeneration/rooting of the transformed shoots
  – testing for the inserted DNA sequence and
    propagation of the tranformed plant
Agrobacterium-mediated transformation

• The infection process
  – wild-type Agrobacterium attaches to a plant cell, then
    transfers a discrete portion of its tumor-inducing (Ti)
    plasmid to the plant's chromosome
  – The Ti plasmid contains 2 critical regions:
      • the oncogene-containing T-DNA (the DNA that is
        transferred)
      • the virulence (vir) genes that encode proteins
        required for T-DNA transfer
Agrobacterium-mediated transformation

• Binary vectors (an Agrobacterium w/2 plasmids)
  – 1st plasmid contains a T-DNA region with the "trait"
    gene and an antibiotic resistance gene inserted into
    the T-DNA in place of the oncogenes
      • removal of oncogenes, which cause crown gall
        disease, make these "disarmed" vectors
      • the antibiotic resistance gene can be used as a
        selectable marker
  – 2nd plasmid (aka "helper") contains the vir genes
    necessary for infection, but w/o T-DNA
Agrobacterium-mediated transformation

• Binary vectors (an Agrobacterium w/2 plasmids)
  – an advantage of binary vectors is that it is easier to do
    the actual genetic engineering in E. coli, then transfer
    that smaller, engineered plasmid into Agrobacterium
• Other features of the transformation process
  – left (LB) and right (RB) border regions are ca. 23 bp
    repeats and are important in the transfer process
  – DNA strand synthesis starts at RB, so those genes
    closest to RB are most efficiently transferred
Agrobacterium-mediated transformation

• Other features of the transformation process
  – how T-DNA incorporates isn't known, but covalent
    linkage with the plant's DNA has been proven
• Regeneration and selection
  – for shoot organogenesis, cytokinin (and usu. lower
    amounts of auxin) are required
  – two antibiotics are required
      • an antibiotic to kill the Agrobacterium, while not
        affecting the plant's cell growth and division
Agrobacterium-mediated transformation

• Regeneration and selection
   – two antibiotics are required
      • a second antibiotic allows growth of transformed
        shoots (w/selectable marker) but inhibits growth of
        untranformed plant cells
• Detection of the "trait" gene
   – PCR methods can detect the presence of the "trait"
     DNA
   – protein detection methods are used where a gene
     product is produced that defines the trait
Agrobacterium-mediated transformation

• Detection of the "trait" gene
   – verification of the incorporation of the trait gene into
     the plant's chromosome
       • by Southern hybridization
       • by demonstrating transfer of the trait to the original
         transformant's progeny
• Agrobacterium has a limited host range
   – papaya, soybean (both dicots) are susceptible
   – most monocots are not susceptible

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Agro

  • 1. Agrobacterium-mediated transformation • Transformation – the process of obtaining transgenic plants • Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome • Most common genes (and traits) in transgenic or biotech crops – herbicide resistance (e.g., Round-up Ready soybeans)
  • 2. Agrobacterium-mediated transformation • Most common genes (and traits) in transgenic or biotech crops – Bt genes (European corn borer toxin) in field corn (maize) – virus-resistance (coat-protein) genes (papaya) • Viewpoints of these biotech crops have stirred debate pro and con – growers are generally in the "pro" camp
  • 3. Agrobacterium-mediated transformation • Viewpoints (contin) – environmentalists are suspicious – organic farmers are in the "con" camp – some consumers wonder where the benefit is to them, others worry about potential allergens – Europeans won't use them, but they are in a growing minority of nations
  • 4. Agrobacterium-mediated transformation • Regardless of the debate over biotech crops, they seem certain to be with us for the foreseeable future • We will concentrate on the way in which in vitro methods are currently used for producing transgenics • Two major methods for making transgenics – Agrobacterium-mediated transformation – particle bombardment
  • 5. Agrobacterium-mediated transformation • Basic tissue-culture process for Agrobacterium- mediated transformation – disinfestation of an explant – isolation and growth of the Agrobacterium vector (with a DNA sequence that is to be transferred) – co-culture of the explant with the Agrobacterium – growth of the explant on a selective medium – regeneration/rooting of the transformed shoots – testing for the inserted DNA sequence and propagation of the tranformed plant
  • 6. Agrobacterium-mediated transformation • The infection process – wild-type Agrobacterium attaches to a plant cell, then transfers a discrete portion of its tumor-inducing (Ti) plasmid to the plant's chromosome – The Ti plasmid contains 2 critical regions: • the oncogene-containing T-DNA (the DNA that is transferred) • the virulence (vir) genes that encode proteins required for T-DNA transfer
  • 7. Agrobacterium-mediated transformation • Binary vectors (an Agrobacterium w/2 plasmids) – 1st plasmid contains a T-DNA region with the "trait" gene and an antibiotic resistance gene inserted into the T-DNA in place of the oncogenes • removal of oncogenes, which cause crown gall disease, make these "disarmed" vectors • the antibiotic resistance gene can be used as a selectable marker – 2nd plasmid (aka "helper") contains the vir genes necessary for infection, but w/o T-DNA
  • 8. Agrobacterium-mediated transformation • Binary vectors (an Agrobacterium w/2 plasmids) – an advantage of binary vectors is that it is easier to do the actual genetic engineering in E. coli, then transfer that smaller, engineered plasmid into Agrobacterium • Other features of the transformation process – left (LB) and right (RB) border regions are ca. 23 bp repeats and are important in the transfer process – DNA strand synthesis starts at RB, so those genes closest to RB are most efficiently transferred
  • 9. Agrobacterium-mediated transformation • Other features of the transformation process – how T-DNA incorporates isn't known, but covalent linkage with the plant's DNA has been proven • Regeneration and selection – for shoot organogenesis, cytokinin (and usu. lower amounts of auxin) are required – two antibiotics are required • an antibiotic to kill the Agrobacterium, while not affecting the plant's cell growth and division
  • 10. Agrobacterium-mediated transformation • Regeneration and selection – two antibiotics are required • a second antibiotic allows growth of transformed shoots (w/selectable marker) but inhibits growth of untranformed plant cells • Detection of the "trait" gene – PCR methods can detect the presence of the "trait" DNA – protein detection methods are used where a gene product is produced that defines the trait
  • 11. Agrobacterium-mediated transformation • Detection of the "trait" gene – verification of the incorporation of the trait gene into the plant's chromosome • by Southern hybridization • by demonstrating transfer of the trait to the original transformant's progeny • Agrobacterium has a limited host range – papaya, soybean (both dicots) are susceptible – most monocots are not susceptible