Sensitive detection of transgenic plant marker gene persistence in soil microcosms

Authors

  • F. WIDMER,

    1. US Environmental Protection Agency, Western Ecology Division, National Health and Environmental Effects Research Laboratory, Terrestrial Branch/Biotechnology Program, 200 SW 35th Street, Corvallis, OR 97333, USA
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  • R. J. SEIDLER,

    Corresponding author
    1. US Environmental Protection Agency, Western Ecology Division, National Health and Environmental Effects Research Laboratory, Terrestrial Branch/Biotechnology Program, 200 SW 35th Street, Corvallis, OR 97333, USA
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  • L. S. WATRUD

    1. US Environmental Protection Agency, Western Ecology Division, National Health and Environmental Effects Research Laboratory, Terrestrial Branch/Biotechnology Program, 200 SW 35th Street, Corvallis, OR 97333, USA
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  • This study was performed as part of the Biotechnology Program's Transgenic Plant Risk Assessment Project at the US EPA National Health and Environmental Effects Research Laboratory in Corvallis, Oregon. F. Widmer is a molecular biologist and a Swiss National Foundation Research Fellow at the laboratory. R. J. Seidler is a research microbiologist and L. S. Watrud is a research ecologist with the US EPA. This research team is interested in developing molecular tools to evaluate effects of natural and anthropogenic perturbations on ecosystem components.

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Abstract

Genetic engineering offers the opportunity to generate plants with useful new traits conferred by genes originating from a variety of organisms. The objectives of this study were to establish methods for investigating 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 recombinant neomycin phosphotransferase II (rNPT-II), a neomycin/kanamycin resistance marker, used in plant genetic engineering. The recombinant nature of the target (i.e. fusion of nopaline synthase promoter and NPT-II coding region) allowed us to design a rNPT-II-specific PCR primer pair. DNA preparation and quantitative PCR protocols were established. Effects of temperature and moisture, on DNA persistence in soil were determined in two laboratory test systems. In the first system, purified plasmid DNA was added to soil and incubated under controlled conditions. 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 sequences were detectable. Under most experimental conditions, leaf tissue-derived and plasmid DNA were initially degraded at a high rate. A small proportion of the added DNA resisted degradation and was detectable for several months. We hypothesize that this DNA may have been adsorbed to soil particles and was protected from complete degradation.

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