An easy method for generating deletion mutants in Agrobacterium tumefaciens using a simple replacement vector

Abstract

We have developed a method to make precise mutations in the A.tumefaciens genome at frequencies high enough to allow direct identification of mutants by PCR or other screening methods rather than by selection. This method utilized a novel pUC18-based gene replacement vector that was used as a donor plasmid carrying the desired mutation in the target cell. Two sites of single-crossover occurred resulting in efficient replacement of the wild type allele on the chromosome with the modified sequence. The usefulness of this method was demonstrated by making deletion mutants in citrate synthase genes of A. tumefaciens.