The Ti Plasmid and Plant Genetic Engineering
http://www.plantphys.net/article.php?ch=21&id=216
The Ti plasmid (tumor-inducing plasmid)
of Agrobacterium tumefaciens has been developed as a vehicle
for introducing foreign genes into plants (Web Figure 21.5.A).
When Agrobacterium infects plants, a region of the Ti
plasmid called the T-DNA is taken up by the plant cell and incorporated
into one of its chromosomes.
The genes in the T-DNA are referred to as phyto-oncogenes because
they induce neoplastic, or tumor-producing, growth (Matzke and
Chilton 1981). To use the Ti plasmid as a vector for introducing
new genes into plants, it is necessary to disarm the plasmid
so that it does not cause tumors. Researchers accomplished this
task by deleting the genes in the T-DNA that encode the enzymes
controlling auxin and cytokinin synthesis. In addition, it is
necessary to introduce a gene into the T-DNA that will enable
the investigator to select the transformed cells. Genes for antibiotic
resistance are normally used for this purpose.
A cloned gene can then be inserted into
the T-DNA of the engineered Ti plasmid, and the plasmid can be
used to infect cultured cells, leaf discs, or root slices. The
infected cells are placed on a culture medium that contains auxin
and cytokinin (to induce growth) and the antibiotic. Only the
transformed cells can grow in the presence of the antibiotic,
because they have received the T-DNA containing not only the
foreign gene but also the gene for antibiotic resistance.
To obtain a plant containing the foreign
gene, it is necessary to regenerate whole plants from the cultured,
transformed cells. Fortunately, methods for accomplishing this
regeneration have been developed for many plants, although not
for all important crop species yet. The regeneration involves
adjusting the ratio of cytokinin to auxin to stimulate both shoot
and root formation.
Although the transformation of cereal
crops with Agrobacterium and their regeneration from cultured
cells has been difficult, some remarkable successes have resulted
from this approach. Investigators have introduced numerous foreign
genes into plants such as tobacco, including soybean storage
protein genes. Genes for such desirable characteristics as disease
resistance, herbicide resistance, and salt tolerance have been
transferred to crop plants by these techniques, and commercial
crops now are being grown with these genetically engineered strains.
Arabidopsis
can also be transformed directly by exposing whole plants (Bechtold
et al. 1993) to a solution containing Agrobacterium that
is carrying engineered or wild-type Ti plasmids. The plants must
be treated in such a way to allow the Agrobacterium to
enter tissue, either by applying a vacuum or by treating with
detergents. The Agrobacterium penetrates the floral tissue
and transforms the developing ovules. Isolation of seeds from
these Agrobacterium-exposed plants yields up to 2% of
the seeds that are transformed with the T-DNA. This approach
is very useful for molecular genetic studies, such as for characterizing
DNA sequences involved in the control of gene expression, or
constructing large libraries of insertional mutants.