In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

Previous European guidance for environmental risk assessment of genetically modified plants emphasized the concepts of statistical power but provided no explicit requirements for the provision of statistical power analyses. Similarly, whilst the need for good experimental designs was stressed, no minimum guidelines were set for replication or sample sizes. Furthermore, although substantial equivalence was stressed as central to risk assessment, no means of quantification of this concept was given. This paper suggests several ways in which existing guidance might be revised to address these problems. One approach explored is the `bioequivalence' test, which has the advantage that the error of most concern to the consumer may be set relatively easily. Also, since the burden of proof is placed on the experimenter, the test promotes high-quality, well-replicated experiments with sufficient statistical power. Other recommendations cover the specification of effect sizes, the choice of appropriate comparators, the use of positive controls, meta-analyses, multivariate analysis and diversity indices. Specific guidance is suggested for experimental designs of field trials and their statistical analyses. A checklist for experimental design is proposed to accompany all environmental risk assessments.

The presence of recombinant DNA in soil cultivated with white poplars (Populus alba L.) expressing either the bar transgene for herbicide tolerance or the StSy transgene for resveratrol production, respectively, was investigated in a greenhouse over a 20-month period. The bar trial included the transgenic lines 5P56 and 6EA22P56 and the untransformed line, while the StSy trial was established with the transgenic lines 5EAC1 and 12EAC1 and with the untransformed line. All the transgenic poplars harbored the nptII marker gene. Plantlets were cultivated in pots, and soil samples were mixed in order to obtain composite pools which were used for molecular analyses. The 35SCaMV-bar (1504 bp), 35SCaMV-StSy (1403 bp) and NosP-nptII (1188 bp) sequences were detected in total DNA extracted from soil samples taken at different times after planting, using PCR/Southern blot hybridization. Microcosm experiments, carried out to assess the effects of temperature and DNA purity on transgene persistence, revealed only a partial correlation between the intensity of hybridization signals and the parameters tested.

Public debate about the possible risk of genetically modified plants often concerns putative effects of pollen dispersal and out-crossing into conventional fields in the neighborhood of transgenic plants. Though Vitis vinifera (grapevine) is generally considered to be self-pollinating, it cannot be excluded that vertical gene transfer might occur. For monitoring pollen flow and out-crossing events, transgenic plants of Vitis vinifera cv. `Dornfelder' harboring the gus-int gene were planted in the center of a field experiment in Southwest Germany in 1999. The rate of pollen dispersal was determined by pollen traps placed at radial distances of 5–150 m from the pollen-donor plants, at 1.00 and 1.80 m above ground. Transgenic pollen was evaluated by GUS staining, and could clearly be distinguished from pollen originating from non-transgenic grapevine plants. Transgenic pollen was observed up to 150 m from the pollen donors. The rate of out-crossing was determined by sampling seeds of selected grapevines at a distance of 10 m to the pollen source, and of a sector at 20 m distance, respectively, followed by GUS analysis of seedlings. The average cross-pollination rate during the experiment (2002–2004) was 2.7% at a distance of 20 m. The results of this first pilot study present a good base for further assessment under the conditions of normal viticulture practice.

When growing different transgenic herbicide-resistant oilseed rape cultivars side by side, seeds with multiple herbicide resistance can arise, possibly causing problems for the management of volunteer plants. Large-scale field experiments were performed in the years 1999/2000 and 2000/2001 in order to investigate the frequencies and the consequences of the transfer of herbicide resistance genes from transgenic oilseed rape to cultivars grown on neighboring agricultural fields. Transgenic oilseed rape with resistance to glufosinate-ammonium (LibertyLink, LL) and with glyphosate resistance (RoundupReady, RR), respectively, was sown in adjacent 0.5 ha plots, surrounded by about 8 ha non-transgenic oilseed rape. The plots and the field were either in direct contact (0.5 m gap width) or they were separated by 10 m of fallow land. Seed samples taken during harvest in the transgenic plots at different distances were investigated for progeny with resistance to the respective other herbicide. It was found that outcrossing frequencies were reduced to different extents by a 10 m isolation distance. In addition to pollen-mediated transgene flow as a result of outcrossing, we found considerable seed-mediated gene flow by adventitious dispersal of transgenic seeds through the harvesting machine. Volunteer plants with double herbicide resistance emerging in the transgenic plots after harvest were selected by suitable applications of the complementary herbicides Basta® and Roundup Ultra®. In both years, double-resistant volunteers were largely restricted to the inner edges of the plots. Expression analysis under controlled laboratory conditions of double-resistant plants generated by manual crosses revealed stability of transgene expression even at elevated temperatures. Greenhouse tests with double-resistant oilseed rape plants gave no indication that the sensitivity to a range of different herbicides is changed as compared to non-transgenic oilseed rape.

The reliable use of purified Cry1Ab protein standards is a prerequisite for ecological studies and resistance monitoring programs of Cry1Ab-expressing transgenic corn. In this study the stability and activity of different Cry1Ab protein batches expressed in and purified from Escherichia coli were determined during two-year storage at different temperature conditions (4 °C, –20 °C, and –80 °C). SDS-Polyacrylamide gel electrophoresis showed degradation of the protein stored at 4 °C over four months, whereas no difference in the band intensity of the Cry1Ab proteins stored at –20 °C and –80 °C was observed. Bioassays with neonate larvae of Ostrinianubilalis indicated that the biological activity of Cry1Ab varied from batch to batch, depending on the production process. Cry1Ab protein stored at 4 °C for four months showed a significantly decreasing activity measured as median lethal concentration (LC50), whereas the protein activity declined less than 11-fold after two years storage at –20 °C. When stored at –80 °C the toxin activity remained relatively stable for at least 30 months, as indicated by low LC50 values of 7–10 ng Cry1Ab per cm2 diet. These experiments demonstrate that appropriate long-term storage conditions of Cry1Ab protein standards are crucial for resistance monitoring programs of Bt corn, and storage at –80 °C is recommended.