T-DNA Transformation

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

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).

E-mail inquiries to U. Melcher------------Last Updated: 12 August, 2008