Understanding the process of crown gall formation through Agrobacterium tumefaciens has shown that virulent strains of this bacteria are able to introduce a part of their genome material into the plant cell. This results in not only successful integration and transformation of the plant cells, but also prompt replication of bacterial genes, along with the plant genome. This is however possible only in dicots having a wound response mechanism. This natural ability of the bacterium to transform plant cells led to the development of effective vectors for introducing foreign genes into higher plants.

These are of 2 types—co-integrating vectors and binary vectors. They are based on a small circular non-chromosomal DNA of the virulent strains of Agrobacterium tumefaciens called Ti-plasmid. Only a portion of this plasmid is introduced into the host cell and they carry genes for the synthesis of opines and phytohormones. This portion, the T-DNA carries prokaryotic promoters that enable it to be expressed in eukaryotic organisms. T-DNA is identified by specific repeat border sequences on either side and any piece of DNA inserted between these two borders gets introduced into the plants’ genome. A certain length of base pair regions lying just outside the right border of T-DNA is important for transferring it into plant cells. Thus being responsible for virulence of the bacteria, it is called the “vir” region. When the entire wild type Ti-plasmid with a small fragment between the border sequences replaced by the desired gene is used as vector, it is called a co-integrating vector. The replaced fragment represents oncogenes that code for synthesis of phytohormones—cytokinin and auxin in the transformed cells, making them proliferate uncontrollably. This may interfere with recognition of morphologically normal transformed plants. Hence, when they are replaced, the Ti-plasmid is disarmed and so used in plant genetic transformation.

While the “vir” regions are important for transferring genes into the plant cells, they need not be physically linked to the T-DNA. Hence, these are carried separately on a plasmid disarmed with respect to the oncogenes. Another plasmid without the “vir”-regions, carries the desired foreign genes between the border regions. These two vectors or binary vectors are used for transformation of the plant cell genome, thus overcoming problems of manipulating the large-sized native Ti-plasmid.

The desired gene (DNA) to be inserted into the T-DNA is first inserted into an E-coli plasmid with DNA sequences homologous to those between the border regions of the T-DNA. Once cloned, such a carrier vector with the desired DNA is transferred from E-coli to Agrobacterium (having the disarmed Ti-plasmid) by conjugation using a helper plasmid. In the bacterium, this carrier vector is first unable to replicate. However, recombination happens between it and the disarmed Ti-plasmid and the so combined plasmid, having both the inserted gene as well as the native “vir” gene continues to remain in the bacterium. This is then used for transfecting plant cells.

In the case of binary vector, the “vir” gene of T-DNA (with the desired gene inserted) is replaced with a plasmid replication region so that it can replicate both in E. coli and Agrobacterium. It is cloned in E. coli and transferred to Agrobacterium having the disarmed Ti-plasmid with “vir” genes. Now the Agrobacterium has both vectors and it is used to transfect plant cells.