High-efficiency gene transfer to recalcitrant plants by Agrobacterium tumefaciens

Agrobacterium tumefaciens efficiently transforms most plants. A few dicotyl edonous plants and most monocotyledonous plants are, however, recalcitrant to A. tumefaciens infection. We investigated whether the constitutive synth esis of a high level of the T-strand DNA intermediate can improve the trans formation efficiency of plants. We previously described a mutation in the v ir gene regulator virG, virGN54D, that allows constitutive expression of th e vir genes. We also described the isolation of a mutant plasmid that is pr esent at a significantly high level in A. tumefaciens. The two mutations we re combined to produce an A. tumefaciens strain that synthesizes a high lev el of T-strand DNA in an inducer-independent manner. DNA transfer efficienc y of the mutant was measured by monitoring beta -glucuronidase (GUS) expres sion in a transient transfer assay. A significant increase in the efficienc y of DNA transfer to both rice and soybean was observed with the double mut ant. The presence of virGN54D has a major positive effect on transformation efficiency.