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Isolation of a non-tumor-inducing mutant of the Ti plasmid of Agrobacterium tumefaciens strain B6.

A nonpathogenic mutant of Agrobacterium tumefaciens strain B6 was isolated and its properties compared with the parental strain in an effort to localize the mutation. Both B6 and its mutant (B6-95) had similar colony color and morphology, were ketolactose positive, utilized octopine, and contained plasmid DNA. Kinetic analysis of DNA reannealing showed that total DNA homology and plasmid DNA homology between B6 and B6-95 was at least 90%. The length of both plasmids was found to be 58 micrometer. Plasmid DNA from both B6 and the mutant was digested with endonucleases and the fragments separated by agarose gel electrophoresis. In all cases the pattern for B6 was identical with that of B6(-95). The Ti plasmid from B6 and the mutant was transferred to an avirulent, plasmidless strain of A. tumefaciens by in vitro conjugation and transformation. All of the B6 transconjugants and transformants were virulent, whereas all of the mutant transconjugants and transformants were avirulent. Electrophoretic patterns of endonuclease-digested plasmid DNA from transformants were identical to those of plasmid DNA from B6. Therefore, we conclude that the virulence mutation lies on the Ti plasmid.