Drawbacks
Several drawbacks are associated with disarmed Ti- vector systems as discussed below;

• Necessity to carry out enzymatic assays on all potential transformants.
• Not convenient as experimental gene vectors due to large size.
• Difficulty in in vitro manipulation and
• Absence of unique restriction sites in the T-DNA.

Several approaches are employed to overcome the limitations associated with disarmed Ti vectors. The transformed plant cells can be identified on the basis of drug or herbicide resistance by inserting selectable markers (Table 5-5.7.1. of Lecture 5 of Module 5) into the T-DNA.

5-4.4.2. Co- integrate vectors

Co-integrate vectors are the deletion derivatives of Ti-plasmids. The DNA to be introduced into the plant transformation vector is sub cloned in a conventional Escherichia coli plasmid vector for easy manipulation, producing a so-called intermediate vector. These vectors are incapable of replication in A. tumefaciens and also lack conjugation functions. Transfer is achieved using a ‘triparental mating’ in which three bacterial strains are mixed together:

(i) An E. coli strain carrying a helper plasmid able to mobilize the intermediate vector in trans;
(ii) The E. coli strain carrying the recombinant intermediate vector;
(iii) A. tumefaciens carrying the Ti plasmid.

Conjugation between the two E. coli strains transfers the helper plasmid to the carrier of the intermediate vector, which in turn is mobilized and transferred to the recipient Agrobacterium. Homologous recombination between the T-DNA sequences of the Ti plasmid and intermediate vector forms a large co- integrate plasmid resulting in the transfer of recombinant T-DNA to the plant genome.