The principle behind Agrobacterium mediated gene transfer is any DNA fragment can replace T-DNA and that will be transferred and integrated into the plant genome under specific conditions.
Why wild type Ti plasmid cannot be used for gene transfer?
1)Large size of Ti plasmid; difficult to handle
2) presence of oncogenes or tumor causing genes (auxin and cytokinin)
3) lack of unique restriction sites and marker site within T-DNA
Disarmed Ti plasmid
Ti plasmid where oncogenes (genes for auxin and cytokinin) are removed and replaced by our gene of interest.
Prerequisites for T-DNA transfer
Any DNA fragment can be transferred into the plant genome by replacing T DNA with our gene of interest provided that:
1) LB and RB repeats should be there
2) Vir regions are required to mediate transfer
3) Chromosomal genes (chv genes) are required that facilitates intimate contact between bacterial cell and plant cell.
Two Ti plasmid based vectors are co-integrate vector strategy and binary vector strategy .