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.