Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection.

Freeze-thaw transformation provides a simple and rapid method to transform Agrobacterium tumefaciens directly with plasmid DNA. Competent A. tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transformed with four to nine plasmids differing in size, size of insert and in some cases sensitivity to antibiotics. A threefold to fourfold increase in transformed colonies per microgram of DNA was obtained by freezing cells with liquid nitrogen vs. dry ice/ethanol. Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low concentration of appropriate antibiotics prior to plating resulted in a ninefold increase in colonies obtained compared with the procedure of freezing cells in dry ice/ethanol without the incubation period in the low concentration of antibiotics prior to plating. Restriction fragments of the expected sizes from the plasmids indicated that the procedural modifications did not cause apparent recombinations in the region of the inserts.