Agrobacterium Mediated Gene Transfer -

The appropriate gene construct is inserted within the T-region of a disarmed Ti plasmid; either a cointegrate or a binary vector is used. The recombinant DNA is placed into Agrobacterium, which is co-cultured with the plant cells or tissues to be transformed for about 2 days. In case of many plant species, small leaf discs are excised from surface sterilized leaves and used for co cultivation.

In general, the transgene construct includes a selectable reporter gene, e.g., the bacterial neo gene. The neo gene is linked with suitable regulatory sequences that are functional in plant cells.

The use of leaf disc for co-culture is better than that of protoplasts or cultured cells since they are likely to show somaclonal variation.
Efficient transformation of monocot cells can be obtained by providing acetosyringone during the co-culture of plant cells with Agrobacterium. Some plant species may secrete compounds, which inhibit the induction of vir operons by acetosyringone.

This problem can be overcome by addition of an excess acetosyringone. Using this approach, successful transformation has been obtained in barley, wheat, maize and rice.