Vectors Based on Ti and Ri Plasmids - The following properties of Ti plasmids did not allow their direct use as vectors: (i) large size,

(ii) absence of uniqe restriction enzyme sites and

(iii) tumour induction property.

Therefore, only the useful attributes of Ti plasmid have been used in designing several plant transformation vectors. Since tumour cells can not develop into normal shoots, disarming of Ti plasmids was an essential step in designing useful vectors. This was achieved by replacement of tumour inducting genes in T-DNA (T-DNA is found in both Ti and Ri plasmids) by selectable markers (like npt II), providing resistance against antibiotics like kanamycin.

Promoters and polyadenylation signal isolated from octopine or nopaline synthase genes were used for expression of selectable markers. Other powerful promoters included CaMV35S and CaMV19S isolated from cauliflower mosaic virus.

One of the first cointegrative vectors, pGV3850 was developed from a nopaline type Ti plasmid (C58), where almost all T-DNA has been deleted and replaced by pBR322, a common small E. coli cloning vector. The intermediate vector (e.g. pGVllO3) based on pBR322 is conjugated into pGV3850 at the region of pBR322 homology.

After cointegration, pGV3850 : : 1103 T-DNA contains the whole of intermediate vector including the unwanted DNA, which is also transferred to host plant along with the gene intended to be transferred. To overcome this difficulty the right border repeat or both borders are introduced to flank a cloning site in the intermediate vector, so that only a part of intermediate vector cointegrates. The utility of this vector system is however limited.

(ii) Binary vectors are based on the principle that vir genes may be located on a 'helper' Ti plasmid having the whole of T-DNA deleted (e.g. pAL4404), because vir genes can function even in trans configuration.

Bacterial colonies containing pBin19 are recognized by loss of blue colour on IPTG/X-GAL plates. Unlike the cointegrative vectors, binary vectors need not have ai1Y homology with resident Ti plasmid and are capable of autonomous replication.

Binary vectors based on Ri plasmid of A. rhizogenes have also been prepared. One such binary vector (pARS 8) in a virulent strain (A4) of A. rhizogenes was used for the production of transgenic plants in tomato. Other examples of vectors based on Ri plasmids are also available in literature.