SENSITIVE DETECTION OF TRANSGENIC PLANT MARKER GENE PERSISTENCE IN SOIL MICROCOSMS

Genetic engineering offers the opportunity to generate plants with use ful new traits conferred by genes originating from a variety of organi sms. The objectives of this study were to establish methods for invest igating persistence of recombinant plant marker DNA after introduction into soil and to collect data from controlled laboratory test systems . As a model system, we studied the stability of DNA encoding recombin ant neomycin phosphotransferase II (rNPT-II), a neomycin/kanamycin res istance marker, used in plant genetic engineering. The recombinant nat ure of the target (i.e. fusion of nopaline synthase promoter and NPT-I I coding region) allowed us to design a rNPT-II-specific PCR primer pa ir. DNA preparation and quantitative PCR protocols were established. E ffects of temperature and moisture, on DNA persistence in soil were de termined in two laboratory test systems. In the first system, purified plasmid DNA was added to soil and incubated under controlled conditio ns. Up to 0.08% of the rNPT-II target sequences were detectable after 40 days. In the second system, fresh leaf tissue of transgenic tobacco was ground, added to soil, and incubated under controlled conditions. After 120 days, up to 0.14% of leaf tissue-derived genomic rNPT-II se quences were detectable. Under most experimental conditions, leaf tiss ue-derived and plasmid DNA were initially degraded at a high rate. A s mall proportion of the added DNA resisted degradation and was detectab le for several months. We hypothesize that this DNA may have been adso rbed to soil particles and was protected from complete degradation.