ENHANCED RECOVERY OF TRANSFORMANTS OF AGROBACTERIUM-TUMEFACIENS AFTERFREEZE-THAW TRANSFORMATION AND DRUG SELECTION

Freeze-thaw transformation provides a simple and rapid method to trans form Agrobacterium tumefaciens directly with plasmid DNA. Competent A. tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transfor med with four to nine plasmids differing in size, size of insert and i n some cases sensitivity to antibiotics. A threefold to fourfold incre ase in transformed colonies pet microgram of DNA was obtained by freez ing cells with liquid nitrogen vs. dry ice/ethanol. Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low c oncentration 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 th e low concentration of antibiotics prior to plating. Restriction fragm ents of the expected sizes from the plasmids indicated that the proced ural modifications did nor cause apparent recombinations in the region of the inserts.