Genetic engineering techniques offer the opportunity to generate plants with a variety of new traits that may be useful in pest control, have added value for food products, be used as sources of biomolecules, or facilitate bioremediation. Genes conferring these novel activities in plants may originate from a wide variety of organisms ranging from viruses to animals. The fate of the genes encoding these novel traits in plants and the potential effects of the accumulated compounds on the ecosystem continues to be a subject of risk assessment discussions. We have focused our efforts on developing methods to track the fate and biological activities of plant transgenes following their introduction into soil.

As a model system, we have studied the stability of naked, linear plasmid DNA and that of DNA contained in transgenic plant tissue after introduction into non-sterile agricultural soil. Each type of DNA encodes recombinant neomycin phosphotransferase II (rNPT-II), an antibiotic marker gene which confers kanamycin resistance and which is routinely used in plant genetic engineering. A modified DNA extraction technique (Porteous et al., 1994) was used to recover DNA from environmental samples as small as 100 mg. Further purification of extracted soil DNA allowed for sensitive polymerase chain reaction (PCR) amplification of target sequences. Novel PCR primers specific for two engineered marker genes were designed by taking advantage of the fusion of the transposon Tn5 derived NPT-II coding sequence (Beck et al., 1982) to two different promoters. Both the nopaline synthetase promoter from the Agrobacterium Ti plasmid (Shaw et al., 1984) and the cauliflower mosaic virus 35S promoter (Sanders et al., 1987) are commonly used in plant engineering and confer expression of the marker inplant cells. A modified PCR protocol (Romanowski et al., 1993) was used to quantify the stability of rNPT-II sequences in agricultural soil under different experimental conditions.

The effects of environmental factors, i.e., temperature and soil moisture, on persistence of DNA in soil was determined in two laboratory test systems (Widmer and Seidler, 1995; Widmer et al., 1996a). In the first system, purified plasmid DNA was added to soil and incubated under defined temperature and moisture conditions, i.e., 4C, 20C, or 36C and 10 %, 40 %, or 80 % water holding capacity (WHC). Aliquots were recovered after selected time intervals and prepared for analysis. Incubation at higher temperatures decreased the persistence of added nucleic acids. DNA breakdown was optimal at 40 % WHC. Except for the 36C incubation, rNPT-II target sequences were detectable throughout the 40 days experiment. In the second test system fresh leaf tissue of transgenic tobacco (Johnson et al., 1989) was ground in liquid nitrogen, added to soil, and incubated under controlled conditions. Subsequently samples were recovered and analyzed for persistence of the rNPT-II marker gene. Leaf tissue derived genomic rNPT-II target sequence was still detectable after 120 days incubation.

To compare the laboratory observations with a field situation, dried tansgenic tobacco leaves were placed in fiber-glass screen bags (litter bags) and buried at 10 cm depth on a field plot of the Willamette Research Station in Corvallis, Oregon. After selected time intervals, litter bags were recovered and leaf litter was analyzed for rNPT-II gene presence. In this field experiment, the genomic rNPT-II marker gene sequence was found to be detectable after 77 days incubation of the leaf tissue in field soil (Widmer et al., 1996b).

In summary, the results of this study show that under the experimental conditions tested, DNA added to soil was initially degraded at a high rate. A small proportion of the added DNA however, resisted degradation and was detectable for several months in soil. We hypothesize that this DNA may have been adsorbed to soil particles and thus was protected from complete degradation.

REFERENCES

Beck, E., G. Ludwig, E.A. Auerswald, B. Reiss, and H. Schaller. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327-336.

Johnson, R., J. Narvaez, A. Gynheung, C. Ryan. 1989. Expression of proteinaseinhibitors I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA 86:9871-9875.

Porteous, L.A., J.L. Armstrong, R.J. Seidler, L.S. Watrud. 1994. An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis. Curr Microbiol 29:301-307.

Romanowski, G., M.G. Lorenz, W. Wackernagel. 1993. Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils. Appl Env. Micro. 59:3438-3446.

Sanders, P.R., J.A.Winter, A.R. Barnason, S.G. Rogers, and R.T. Fraley RT. 1987. Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucleic Acids Res 15:1543-1558.

Shaw, C.H., G.H. Carter, M.D. Watson, and C.H. Shaw. 1984. A functional map of the nopaline synthase promoter. Nucleic Acids Res 12:7831-7846.

Widmer, F. and R.J. Seidler. 1995. Fate and Microbial Transformation Potential of Plant-Derived Transgenes in Nonsterile Agricultural soil. In: Levin M, Grim C, and Angle JS (eds) Proceedings of the Biotechnology Risk Assessment Symposium, June 22-24, 1994, College Park, Maryland, pp.137-147.

Widmer, F., R.J. Seidler, and L.S. Watrud. 1996a. Sensitive Detection of Transgenic Plant Marker Gene Persistence in Soil Microcosms. Mol Ecol, in press.

Widmer, F., R.J. Seidler, K.K. Donegan, and G.L. Reed. 1996b. Quantification of Transgenic Plant Marker Gene Persistence in the Field. Mol Ecol, submitted.