DETECTION OF NPTII (KANAMYCIN RESISTANCE) GENES IN GENOMES OF TRANSGENIC PLANTS BY MARKER-RESCUE TRANSFORMATION

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in t ransgenic plant genomes. The assay is based on the recombinational rep air of an nptII gene with an internal 10-bp deletion located on a plas mid downstream of a bacterial promoter. Uptake of an nptII gene by tra nsformation restores kanamycin resistance. In Escherichia coli, promot erless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapt ed to natural transformation: which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cel ls containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (S olanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Ly copersicon esculentum), kanamycin-resistant transformants were obtaine d roughly in proportion to the concentration of nptII genes in the pla nt DNA. The rescue of nptII genes occurred in the presence of a more t han 6 x 10(6)-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 x 10(3) genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).