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.

With the extensive adoption of transgenic crops, an understanding of transgene flow isessential to manage gene flow to non-GM crops. Thus, a flexible and accurate numericalmodel is required to assess gene flow through pollen dispersal. A three-dimensionalatmospheric model combined with a diffusion transport model would be a useful tool forpredicting pollen dispersal since it would be flexible enough to incorporate the effectsof factors such as the spatial arrangement of crop combinations, land use, topography,windbreaks, and buildings. We applied such a model to field measurements of gene flowbetween two adjacent maize (Zea mays) cultivars, with suppression effectsdue to windbreaks, in an experimental cornfield in Japan. This combined model reproducedthe measured cross-pollination distribution quite well in the case of maize plots withplant windbreaks slightly taller than the maize and without windbreaks, but the modelunderestimated the effect of a 6-m-tall windbreak net beyond 25 m from the donor pollensource on cross-pollination. The underestimation was most probably due to the problem ofassimilated wind data. The model showed that the 6-m-tall windbreak and the plant windbreak suppressed average cross-pollination rate by about 60% and 30%, respectively.Half-tall and coarser mesh windbreak net suppressed cross-pollination rates by 40% byreducing the swirl of donor pollen by reduced wind speed.

The seemingly inexorable expansion of global human population size, significant increases in the use of biofuel crops and the growing pressures of multifunctional land-use have intensified the need to improve crop productivity. The widespread cultivation of high-yielding genetically modified (GM) crops could help to address these problems, although in doing so, steps must also be taken to ensure that any gene flow from these crops to wild or weedy recipients does not cause significant ecological harm. It is partly for this reason that new GM cultivars are invariably subjected to strict regulatory evaluation in order to assess the risks that each may pose to the environment. Regulatory bodies vary in their approach to decision-making, although all require access to large quantities of detailed information. Such an exhaustive case-by-case approach has been made tractable by the comparative simplicity of the portfolio of GM crops currently on the market, with four crops and two classes of traits accounting for almost all of the area under cultivation of GM crops. This simplified situation will change shortly, and will seriously complicate and potentially slow the evaluation process. Nowhere will the increased diversity of GM crops cause more difficulty to regulators than in those cases where there is a need to assess whether the transgene(s) will enhance fitness in a non-transgenic relative and thereafter cause ecological harm. Current practice to test this risk hypothesis focuses on attempting to detect increased fitness in the recipient. In this paper we explore the merits and shortcomings of this strategy, and investigate the scope for developing new approaches to streamline decision-making processes for transgenes that could cause unwanted ecological change.

Pollen-mediated gene flow in space is well documented and isolation distances are recommended to ensure genetic purity of Brassica napus seed crops. Isolation in time could also contribute to gene flow management but has been little investigated. We assessed the effects of asynchronous and synchronous flowering on intraspecific B. napus gene flow by seeding adjacent plots of transgenic spring canola cultivars, either resistant to glyphosate or glufosinate, over a 0–4 week interval and measuring outcrossing rates and seed-set. Outcrossing rates, evaluated in the center of the first adjacent row, were reduced to the lowest level in plots flowering first when the seeding interval > 2 weeks. Increasing the time gap increased outcrossing rates in plots flowering second up to a seeding interval of two weeks. Flowers that opened during the last week of the flowering period produced fewer seed (< 10% of total seed production) and a smaller fraction of outcrossed seed (–25%). Observed time gap effects were likely caused by extraneous pollen load during the receptivity of productive seed-setting early flowers. Clearly, manipulation of B. napus flowering development through staggered planting dates can contribute to gene flow management. The approach will need to be validated by additional site-years and increased isolation distances.

It is emerging that benefits of new innovations in agricultural biotechnology may not be realised without appropriate biosafety regulatory mechanisms. The Kenyan regulatory experiences related to regulation of genetically engineered (GE) agricultural activities are explored to provide some basis for defining the challenges involved in biosafety regulation. The various shortcomings in the way the Kenyan biosafety process has been implemented are consistent with the way regulatory systems have been evolving in Africa. The proposed adoption of a “smart regulation” provides the basis for a learning process through which subsequent biotechnology policy initiatives can be improved.

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.

Plant to plant gene flow is a route of environmental exposure for GM plants specifically since crosses with wild relatives could lead to the formation of more vigorous hybrids, which could increase the rate of introgression and the environmental impact. Here, we test the first step in the process of potential transgene introgression: whether hybrid vigor can be inherited to the next generation, which could lead to fixation of altered, i.e., elevated, quantitative traits.

The potential for a permanent elevated fitness was tested using individual autogamous progeny lineages of hybrids between the crop Lactuca sativa (Lettuce) and the wild species Lactuca serriola (Prickly Lettuce). We compared progeny from motherplants grown under either greenhouse or field conditions. The survival of young plants depended strongly on maternal environment. Furthermore, we observed that offspring reproductive fitness components were correlated with maternal fitness.

Our study demonstrates that post-zygotic genotypic sorting at the young plants stage reduces the number of genotypes non-randomly, leading to inheritance of high levels of reproductive traits in the surviving hybrid lineages, compared to the pure wild relatives.

Consequently, directional selection could lead to displacement of the pure wild relative and fixation of more vigorous genome segments originating from crops, stabilizing plant traits at elevated levels. Such information can be used to indentify segments which are less likely to introgress into wild relative populations as a target for transgene insertion.

What happens when one nation cultivates a transgenic crop variety but neighboring nations do not? Using alfalfa as a case study, we argue that the potential for international transgene flow is substantial, and therefore, the need for international cooperation in regulatory decisions concerning transgenic crops is imperative. Alfalfa (Medicago sativa, L.) is the major forage crop in North America. Recently, genetically modified (GM) alfalfa received a moratorium on further cultivation in the US on the grounds that the approvals were based on inadequate environmental impact assessments. With their deep root system, symbiotic nitrogen fixation, prolific seed production and prolonged dormancy, alfalfa plants are capable of establishing self-perpetuating (feral) populations in unmanaged environments. Given what is known about alfalfa pollen dispersal, such feral populations could facilitate gene flow between GM and non-GM fields. The border between the US and Canada, particularly in farming areas, is very narrow (< 10 m wide). We surveyed along the US-Canada border and found both alfalfa fields and potentially feral alfalfa plants in the ditches along the border. Our survey results provide evidence of the possibility of cross-border transgene flow, suggesting a need for international co-operative risk assessment initiatives between the US and Canada. Such situations could occur for other crops, in other international border regions as well.

Genetically modified (GM) maize expressing the insecticidal protein Cry1Ab from Bacillus thuringiensis (Bt maize) is the only GM crop planted commercially in the European Union (EU). Cultivation in accordance with Directive 2001/18/EC demands post-market environmental monitoring (PMEM) to ensure the detection and prevention of adverse effects on the environment possibly deriving from commercial cultivation. Based on a seminar organized in Berlin, Germany, in April 2008 by the EU-funded Biosafenet project, the present paper reflects on experiences and hurdles faced during the implementation of PMEM for Bt maize. It reviews and reconsiders PMEM programs of Bt maize in view of existing experiences from cultivation, current monitoring activities initiated by Member States and applicants, proposed monitoring strategies and methods as well as potential environmental impacts of cultivation. Future challenges will arise from large-scale and cumulative cultivation of various events. This will demand optimized organization structures for data collation and integration to support further decision-making and management.

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.

Bagavathiannan and Van Acker propose greater international cooperation and information sharing in risk assessment for biotechnology-derived crops because pollen- and seed-mediated gene flow across political boundaries may lead to the adventitious presence of unapproved transgenes at sites along the borders of neighboring countries. However, they fail to convince us that something is wrong with the current situation and provide no details of how it could be improved.

Field experiments were conducted in Chile and western Canada to measure short-distance (0 to 100 m) outcrossing from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming to non-transgenic commodity safflower of the same variety. The transgenic safflower used as the pollen source was transformed with a construct for seed-specific expression of a high-value protein and constitutive expression of a gene conferring resistance to the broad-spectrum herbicide glufosinate. Progeny of non-transgenic plants grown in plots adjacent to the transgenic pollen source were screened for glufosinate resistance to measure outcrossing frequency. Outcrossing frequency differed among locations: values closest to the transgenic pollen source (0 to 3 m) ranged from 0.48 to 1.67% and rapidly declined to between 0.0024 to 0.03% at distances of 50 to 100 m. At each location, outcrossing frequency was spatially heterogeneous, indicating insects or wind moved pollen asymmetrically. A power analysis assuming a binomial distribution and a range of alpha values (type 1 error) was conducted to estimate an upper and lower confidence interval for the probable transgenic seed frequency in each sample. This facilitated interpretation when large numbers of seeds were screened from the outcrossing experiments and no transgenic seeds were found. This study should aid regulators and the plant molecular farming industry in developing confinement strategies to mitigate pollen mediated gene flow from transgenic to non-transgenic safflower.

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.

Regulations 1829/2003/CE and 1830/2003/CE have allowed the placing on the European market of GM products in food and feed chains, and have defined their rules of traceability and labeling. For some supply chains, like for soy and its derived products that are used in the production of feed, manufacturers have to face both non-GM and GM production, although there are no labeling requirements for animal products derived from animals fed with GMOs. This study presents the strategies of stakeholders involved in the feed production chain to maintain concurrent production of compound feed with GM and non-GM soy products, by dealing with the coexistence between those two crops. The stakeholders include importers, traders, soy processors, feed processors and retailers. The study shows that many tools are in place to ensure and maintain the current coexistence. However, a profound harmonization of procedures and methods at a European level should be encouraged.

Monitoring for escape of genetically modified (GM) oilseed rape (Brassica napus) during transport can be performed by means of roadside evaluations in areas where cultivation of this GM crop is not conducted, such as in Japan. We performed a survey of oilseed rape plants growing along a 20-km section of Japan's Route 51, one of the main land transportation routes in central Japan for imports of GM oilseed rape from the Port of Kashima into Keiyo District. Oilseed rape plants were found each year, but the number of plants varied substantially during the three years of our study: 2162 plants in 2005, 4066 in 2006, and only 278 in 2007. The low number in 2007 was probably caused by roadwork. Herbicide-resistant individuals were detected in the three consecutive years (26, 8, and 5 individuals with glyphosate resistance), but glufosinate-resistant plants (9 individuals) were detected only in 2005. The roadside plants occurred mainly along the inbound lane from Kashima to Narita. These plants are likely to have their origin in seeds spilled during transportation of cargo from the port, since there are no potential natural seed source plants for B. napus near Route 51. This is the first detailed report on the transition and distribution of herbicide-resistant oilseed rape plants following loss and spillage along Japanese roads.

Risk assessments of new insect-resistant crops will need to estimate the potential for increased weediness of wild crop relatives as a consequence of gene flow. When field experiments are precluded by containment concerns, simulation experiments can identify hazards or measure expected differences between GMOs and parental plants. To measure plant fitness consequences of wild plant protection from Bt-susceptible herbivores, we used topical sprays of bacterial Bacillus thuringiensis larvacide (Bt) on Brassica rapa. Spontaneous crosses between B. rapa and Bt cole crops cannot be precluded, especially if adoption of Bt varieties leads to high exposure. We compared survivorship and seed output of B. rapa that were either protected from or exposed to Bt-susceptible Lepidoptera in the various conditions where hybrids are likely to occur: cultivated (disked) soil, uncultivated agricultural field margins, and nearby non-crop habitats (meadows and ruderal areas). The relative effect of herbivore protection varied among years, habitats, and populations of seedlings. In 2003–2004, Bt sprays did not result in lower herbivory on B. rapa, and plant fitness was not increased. However, in 2004–2006 B. rapa seedlings protected from Bt-susceptible herbivores lived 25% longer, on average, than those that were exposed to these herbivores. In addition, an average B. rapa seedling sprayed with Bt throughout its lifetime was twice as likely to produce siliques and had 251% of the seed output of a seedling exposed to herbivores. The fitness advantage of Bt-based plant protection was apparent in 2004–2005 in half the plants that experienced higher herbivory, and for 2005–2006, was more pronounced in agricultural habitats than in meadows with established, perennial vegetation and less disturbance. Positive effects of Bt-based plant protection and greater fitness in disturbed habitats suggest that crop-wild gene flow may benefit weed populations, and that field tests with herbivore exclusion/addition experiments are feasible alternatives when molecular containment of transgenes restricts field experiments with insect resistant crop-wild hybrids.

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.