Positive-Negative selection for homologous recombination in Arabidopsis

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct con tained two flanking regions of the target alcohol dehydrogenase gene as hom ologous sequences, and neomycin phosphotransferase and cytosine deaminase a s positive and negative markers, respectively. A root explant transformatio n procedure was used to obtain transgenic calli. Among 6250 transformants i solated by positive selection, 39 were found to be resistant to negative se lection as well. Of these 39, at least one had undergone homologous recombi nation correlated with a unidirectional transfer of information. Although t he ADH locus was not changed, our data demonstrate that a homologous recomb ination event can be selected by positive negative selection in plants. (C) 2001 Elsevier Science B.V. All rights reserved.