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  1. 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. 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. 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 wont use them, but they are in a growing minority of nations
  4. 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. 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. 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 plants 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. 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. 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. 9. Agrobacterium-mediated transformation• Other features of the transformation process – how T-DNA incorporates isnt known, but covalent linkage with the plants 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 plants cell growth and division
  10. 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. 11. Agrobacterium-mediated transformation• Detection of the "trait" gene – verification of the incorporation of the trait gene into the plants chromosome • by Southern hybridization • by demonstrating transfer of the trait to the original transformants progeny• Agrobacterium has a limited host range – papaya, soybean (both dicots) are susceptible – most monocots are not susceptible