SUMMARY

Plants are genetically engineered to alter a variety of their traits. Goals of these efforts are to optimize plant properties of economic interest including plant performance, food and feed values, biomolecule production, and bioremediation. Concerns on the use of transgenic plants in the environment have been expressed, mainly related to unintended changes of the engineered plants themselves but also of other plants or possibly microorganisms accidentally incorporating the engineered genes into their genomes (Dietz, 1993).

In previous studies we found that plant DNA may persist in decomposing plant tissue in soilmicrocosms (Widmer et al., 1996a) and in the field (Widmer et al., 1996b) for several months, indicating that in such locations microorganisms may be exposed to relatively high concentrations of plant DNA. The goal of this study is to evaluate the potential of gene transfer from transgenic plants to microorganisms. For this purpose we designed a model system for gene transfer, based on marker rescue transformation of Bacillus subtilis (Weinrauch and Dubnau 1983) with a transgenic plant encoded recombinant neomycin phophotransferase II (rNPT-II) marker which confers kanamycin resistance (Flavell et al., 1992).

The approach is to restore a truncated RNPT-II gene, encoded on a plasmid in B. subtilis BD170, by transformation and homologous recombination with DNA types encoding the functional RNPTII gene. For efficient expression in B. subtilis, we fused the NPT-IL coding region to the neomycin promoter from plasmid pUB110 (McKenzie et al., 1986). The resulting NEO/NPT-Il construct was inserted into a plasmid (modification of pBD8, Gryczan and Dubnau 1978) and conferred high kanamycin resistance in B. subtilis. The NEO/NPT-11 gene was truncated by excising an internal 885 bp NcoI/BfrI fragment. The deletion was flanked by 549 bp and 558 bp residual sequences, offering the target for homologous recombination. This construct (NEO/NPT-II ) was inserted into the same plasmid and did not confer kanamycin resistance to B. subtilis.

Marker rescue transformation of competent B. subtilis was performed with plasmid DNA (pFW1, Widmer et al., 1996a) and purified genomic DNA from a transgenic tobacco (Johnson et al. 1989). Both DNA types encoded the same rNPT-II gene, sharing the approximately 550 bp homologous sequences on either site of the deletion in NEO/NPT-II . Control marker rescue transformation was performed with B. subtilis 168 genomic DNA to restore the chromosomal tryptophan-auxotrophy marker in B subtilis BD 170.

Even though marker rescue in the control experiment (tryptophan-auxotrophy marker) occurred at a frequency of 3.6 x 10^-5, we were not able to detect marker rescue of the rNPT-II gene for either plasmid or transgenic tobacco chromosomal DNA. These preliminary results indicate a low transfer frequency of the rNPT-II gene in our system, possibly due to the relatively small homologous regions on the plasmid in B. subtilis as compared to the extensive homology of the chromosomal control marker. The size of the homologous region in our model system, however, closely reflects the composite nature of transgenes present in engineered plants. Future experiments will be focused on further defining the sensitivity of RNPT-II gene transfer detection with our system and determining the potential of gene transfer from plant to microorganisms.

REFERENCES

Dietz A (1993) Risk assessment of genetically modified plants introduced into the environment. In: Transgenic Organisms, Risk Assessment of Deliberate Release., (ed. Wöhrmann K, Tomiuk J), pp. 209-227. Birkhäuser Verlag, Basel, Switzerland.

Flavell RB, Dart E, Fuchs RL, Fraley RT (1992) Selectable marker genes: safe for plants? BiolTechnology 10, 141-144.

Gryczan TJ, Dubnau D (1978) Construction and properties of chimeric plasmids in B. subtilis. Proceedings of the National Academy of Sciences of the United States of America, 75, 1428-1432.

Johnson R, Narvdez J, Gynheung A, Ryan CA (1989) Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manduca sexta larvae. Proceeding of the National Academy of Sciences of the USA, 86, 9871-9875.

McKenzie T, Hoshino T, Tanaka T, Sueoka N (1986) The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15, 93-103.

Weinrauch Y, Dubnau D (1983) Plasmid marker rescue transformation in Bacillus subtilis. Journal of Bacteriology, 154, 1077-1087.

Widmer F, Seidler RJ, Watrud LS (1996a) Sensitive detection of transgenic plant marker gene persistence in soil microcosms. Molecular Ecology, in press.

Widmer F, Seidler RJ, Donegan KK, Reed GL (1996b) Quantification of Transgenic Plant Marker Gene Persistence in the Field. Molecular Ecology, in press.