Sensitive detection of transgenic plant marker gene persistence in soil microcosms

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.

The ecology of environmental DNA and implications for conservation genetics

“…As was the case with eDNA transport, much of study of eDNA persistence has been motivated by concern over the release of transgenes from genetically modified organisms. Such studies have examined persistence of genes from transgenic crops in terrestrial soils (Widmer et al 1996(Widmer et al , 1997Hay et al 2002;Gebhard and Smalla 2006) and nearby groundwater and riverine environments (Matsui et al 2001;Zhu 2006). Across these studies, transgenic material remained detectable for long periods of time (days to years) in terrestrial soils, but much shorter periods of detection (hours to days) occurred in aquatic environments (Table 1).…”

Horizontal gene transfer between bacteria

“…Mainly two aspects of natural transformation in the environment have been studied: the persistence of free DNA, and the ability of different bacterial species to become competent and take up free DNA under environmental conditions. Different studies have shown that in spite of the ubiquitous occurrence of DNases, highmolecular free DNA could be detected and its persistence in different environments has been demonstrated (Gebhard and Smalla, 1999;Nielsen et al, 1997;Paget and Simonet, 1997;Widmer et al, 1996;. Natural transformation is considered to also be the mechanism by which competent bacteria could capture DNA from transgenic plants (de Vries and Wackernagel, 1998;2004;Gebhard and Smalla, 1998).…”

“…A more rapid breakdown of DNA was observed at higher soil H. Heuer and K. Smalla humidity and temperature. Both factors are thought to contribute to a higher microbial activity in soil (Blum et al, 1997;Widmer et al, 1996). In the study of Demanèche et al (2001a) plasmid DNA adsorbed on clay particles was found to be incompletely degradable even at high nuclease concentrations.…”

Impact environnemental des cultures transgéniques

“…L'absence de transgènes détectables dans le tractus digestif (Chambers et al 2001), les fèces (Harrison et al 1996;Netherwood et al 2004) ou les tissus (Chowdhury et al 2003a, b;Jennings et al 2003) d'animaux ou de sujets humains nourris de végétaux transgéniques suggère d'ailleurs une hydrolyse rapide de ces molécules, bien qu'incomplète sous certaines conditions (Chowdhury et al 2003a, b;MartinOrue et al 2002;Mercer et al 1999), au cours du processus de digestion alimentaire. En revanche, les molécules d'ADN pourraient montrer une certaine stabilité dans l'eau (Paul et al 1989) et une fraction non négligeable des molécules d'ADN relâchées des tissus végétaux dans le sol ne serait pas hydrolysée rapidement par les nucléases microbiennes et persisterait, adsorbée aux particules argileuses, sur des périodes suffisamment longues pour permettre l'inté-gration de ces molécules au génome d'éventuelles bactéries compétentes (Gebhard et Smalla 1999;Lorenz et Wackernagel 1992;Widmer et al 1996Widmer et al , 1997.…”