Called co-integrated vectors or hybrid Ti plasmids, these vectors were among the first types of modified and engineered Ti plasmids devised for Agrobacterium -mediated transformation, but are not widely used today.
These vectors are constructed by homologous recombination of a bacterial plasmid with the T-DNA region of an endogenous Ti plasmid in Agrobacterium. Integration of the two plasmids requires a region of homology present in both.
Three vectors are necessary in this system:
- Disarmed Agrobacterium Ti plasmids
In these Ti plasmids, the oncogenes located in the T-DNA region have been replaced by exogenous DNA.
Examples of these vectors include:
- SEV series: the right border of the T-DNA together with the phytohormone genes coding for cytokinin and auxin are removed and replaced by a bacterial kanamycin resistance gene while the left border and a small part of the left segment (TL) of the original T-DNA (referred to as Left Inside Homology (LIH)) are left intact.
- pGV series: the phytohormone genes are excised and substituted by part of pBR322 vector sequence. The left and right border sequences as well as the nopaline synthase gene of the Ti plasmid are conserved.
- Intermediate vectors
These are small pBR322-based plasmids (E. coli vectors) containing a T-DNA region. They are used to overcome the problems derived from the large size of disarmed Ti plasmids and their lack of unique restriction sites. Intermediate vectors are replicated in E.coli and are transferred into Agrobacterium by conjugation. They cannot replicate in A. tumefaciens and therefore, carry DNA segments homologous to the disarmed T-DNA to permit recombination to form a co-integrated T-DNA structure.
- Helper vectors
These are small plasmids maintained in E. coli that contain transfer (tra) and mobilization (mob) genes, which allow the transfer of the conjugation-deficient intermediate vectors into Agrobacterium.
A resulting co-integrated plasmid assembled by in vitro manipulation normally contains:
- the vir genes,
- the left and right T-DNA borders,
- an exogenous DNA sequence between the two T-DNA borders, and
- plant and bacterial selectable markers.
Although co-integrated vectors have been designed to allow site-specific recombination based on the recombination system of the phage P1 (e.g., wP1loxP-Cre series), co-integrated vectors in general are less popular due to:
- long homologies required between the Ti plasmid and the E. coli plasmids making them difficult to engineer and use, and
- relatively inefficient gene transfer compared to the binary vectors.