The T-DNA of the Ti plasmid of Agrobacterium tumefaciens can be used to introduce modified genes into plants.
- The Ti plasmid is too large to manipulate easily in vitro.
- Two strategies evolved to overcome this manipulation difficulty.
- Both strategies make use of the fact that hormone and opine synthesis genes in T-DNA are not needed for transfer. The vir genes, essential for transfer of T-DNA, are located outside of T-DNA on the Ti plasmid.
- One of the introduced genes is usually an antibiotic resistance gene allowing selection of transformed plant cells on antibiotic-containing media.
Integrating Vector Strategy
- In one strategy, the integrating vector strategy, manipulations are done in vitro on a plasmid, such as pMON200, containing a small segment from within the T-DNA.
- The plasmid, engineered to contain the gene to be transferred as well as a selectable marker, is mobilized from E. coli to A. tumefaciens containing a "disarmed" (no hormone genes) Ti plasmid.
- Homologous recombination at the region of homology between the pMON200 derivative and the resident Ti plasmid results in integration of the entire engineered plasmid into the T-DNA.
- On infection of plants, the T-DNA with the insert is transferred to a plant nuclear chromosome.
Binary Vector Strategy
- In a second strategy, a plasmid containing the left and right borders of T-DNA is modified in vitro to carry the gene to be transferred and a selectable marker. The modified plasmid DNA is transformed into E. coli.
- The modified plasmid is transferred by conjugation to an A. tumefaciens strain containing a helper plasmid that provides the vir functions.
- On infection of a plant, the activated vir functions recognize the left border sequence of the modified plasmid and transfer all DNA between left and right borders to a plant chromosome.
- This strategy is called the binary vector strategy
Implications and Further Information:
- The use of a natural genetic engineer results in a high efficiency of transformation.
- T-DNA mediated transformation is the predominant route used to create transgenic dicotyledonous plants.
- T-DNA transfer to monocotyledonous plants has only recently become possible.
- The site of integration of the T-DNA appears random.
- Agrobacterium rhizogenes, the causative organism of hairy root disease, contains a plasmid (Ri plasmid, for "Root inducing") analogous to the Ti plasmid. It also has been used in genetic engineering of plants.
- Many T-DNA vectors are available (ref).
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E-mail inquiries to U. Melcher------------Last Updated: 12 August, 2008