TARGETED DISRUPTION OF THE TGA3 LOCUS IN ARABIDOPSIS-THALIANA

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant g enes isolated and the accelerating pace by which this collection is gr owing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the fir st successful disruption of a non-selectable gene in Arabidopsis thali ana by creating a mutant of the TGA3 locus via targeted insertion of t he bacterial neo gene conferring kanamycin (Km) resistance. A beta-glu curonidase (GUS) expression unit outside the region of homology was us ed as a screenable marker to distinguish homologous recombination even ts from those of ectopic insertions. PCR amplification coupled with So uthern blot screening identified two putative homologous recombination events among 2580 Km(r) calli. One callus line was subsequently isola ted and the structure of the targeted TGA3 allele confirmed by Souther n blot analyses. This study demonstrates the feasibility of targeting a non-selectable locus in Arabidopsis. Combined with future improvemen ts in negative selection strategies and efficient transformation metho dologies, gene replacement studies in plants could become a routine te chnique.