THE ANTI-NPTII GENE - A POTENTIAL NEGATIVE SELECTABLE MARKER FOR PLANTS

An efficient negative selection procedure is crucial to the isolation of rare homologous recombinants in gene targeting. Although gene targe ting is a common practice in lower eukaryotes and is becoming routine in mammals, its application to plants has not been achieved. In this r eport, we have evaluated an antisense construct against the neomycin p hosphotransferase gene (nptII) as a negative selectable marker. The an ti-nptII gene construct was able to suppress nptII expression both tra nsiently and in transformed tobacco (Nicotiana tabacum) calli. A const ruct was made which includes both a hygromycin-resistance gene and the sense plus antisense genes for neomycin phosphotransferase. Hygromyci n-resistant calli were obtained after Agrobacterium-mediated transform ation. Subsequently, hygromycin-resistant calli were tested for kanamy cin sensitivity. The growth on kanamycin medium of calli harboring bot h the sense and antisense gene constructs was retarded, whereas that o f control calli transformed with only the sense nptII gene was not inh ibited. Southern blot analysis confirmed the presence of both nptII an d anti-nptII genes. Northern blot analyses revealed that antisense tra nscripts of the nptII gene were made and that the level of sense trans cripts was greatly reduced in transgenic calli. These results suggest that the anti-nptII gene could potentially be used as a negative selec table marker for gene targeting in plants.