Genetically modified organisms (GMOs), e.g. viral vectors, could threaten the environment if by their release they spread hazardous gene products. Even in contained use, to prevent adverse consequences, viral vectors carrying genes from mammals or humans should be especially scrutinized as to whether gene products that they synthesize could be hazardous in their new context. Examples of such potentially hazardous gene products (PHGPs) are: protein toxins, products of dominant alleles that have a role in hereditary diseases, gene products and sequences involved in genome rearrangements, gene products involved in immunomodulation or with an endocrine function, gene products involved in apoptosis, activated proto-oncogenes. For contained use of a GMO that carries a construct encoding a PHGP, the precautionary principle dictates that safety measures should be applied on a “worst case” basis, until the risks of the specific case have been assessed. The potential hazard of cloned genes can be estimated before empirical data on the actual GMO become available. Preliminary data may be used to focus hazard identification and risk assessment. Both predictive and empirical data may also help to identify what further information is needed to assess the risk of the GMO. A two-step approach, whereby a PHGP is evaluated for its conceptual dangers, then checked by data bank searches, is delineated here.

Hybridization and introgression between crops and wild relatives may have important evolutionary and ecological consequences such as gene swamping or increased invasiveness. In the present study, we investigated hybridization under field conditions between crop lettuce (Lactuca sativa) and its wild relative prickly lettuce (L. serriola), two cross-compatible, predominantly autogamous and insect pollinated species. In 2003 and 2004, we estimated the rates of hybridization between L. sativa and L. serriola in close-to-reality field experiments carried out in two locations of Northern Switzerland. Seeds set by the experimental wild plants were collected and sown (44 352 in 2003 and 252 345 in 2004). Progeny was screened morphologically for detecting natural hybrids. Prior to the experiment, specific RAPD markers were used to confirm that morphological characters were reliable for hybrid identification. Hybridization occurred up to the maximal distance tested (40 m), and hybridization rates varied between 0 to 26%, decreasing with distance. More than 80% of the wild plants produced at least one hybrid (incidence of hybridization, IH) at 0 m and 1 m. It equaled 4 to 5% at 40 m. In sympatric crop-wild populations, cross-pollination between cultivated lettuce and its wild relative has to be seen as the rule rather than the exception for short distances.

The effect of root-released compounds of transplastomic tobacco (Nicotiana tabacum) on the soil bacterial community structure, and their potential to support horizontal gene transfer (HGT) to bacteria have been studied. Soil microcosms were exposed to root-released compounds collected from transplastomic and non-transgenic tobacco cultivars. Cluster analysis of automated ribosomal intergenic spacer analysis (ARISA) profiles of the soil bacterial community after 48 h incubation grouped the transgenic cultivar apart from the non-transgenic, indicating that it had a rhizodeposition pattern different from the parental plants. However, these differences were less than between the two non-transgenic tobacco cultivars studied. NMR characterization of the root-released compounds showed some differences in chemical fingerprinting pattern between the transplastomic and the parental cultivar. However, the effect on bacterial community structure was transient, and tended to disappear after 96 h of incubation. The potential of root-released compounds as a source of transforming DNA for bacteria was investigated by using four potential recipient species. No transformants were obtained following exposure of all the recipients to the root-released compounds. Root-released compounds amended to transgene donor DNA decreased the transformation frequency of Acinetobacter baylyistrain ADP1200, while Azospirillum, Agrobacterium, and Sinorhizobium strains failed to develop competence also in the presence of an external added transgene source. Detection of plastid sequences by PCR suggested that a very low amount of fragmented plastid donor DNA was present in the root-released compounds.

Impact assessments of virus resistance transgene introgression into wild, free-living populations are important for determining whether these transgenes present a risk to agriculture or the environment. Transgenic virus-resistant Cucurbita pepo ssp. ovifera var. ovifera L. (squash) cultivars have been commercialized, and may be cultivated in close proximity to cross-compatible wild, free-living relatives (C. pepo subsp. pepo vars. ozarkana and texana). Therefore, the potential impact of these virus resistance transgenes was studied by surveying the incidence and fluctuations of virus infection (as assayed by ELISA), virus symptoms (which may not be seen in an infected plant) and population size in forty-three free-living C. pepo populations in Illinois, Missouri, Arkansas, Mississippi, Louisiana, and Texas. Ten of these populations were studied over three consecutive seasons. Depending on the year, 61% to 78% percent of the populations had at least one individual infected by at CMV, ZYMV or WMV2, but the median incidence of infection within populations was 13%. The observed infection level in free-living populations was consistent with levels defined as “low” in field plot experiments conducted by others, leading to the conclusion that transgenic virus resistance should not provide a significant fitness advantage to the free-living populations examined. Viral symptoms were detected in only 2% of plants observed, indicating that severity of viral infection was low. CMV, ZYMV, and WMV2 were not the only viruses infecting these populations, further reducing the likelihood that resistance to these viruses would release populations from constraints imposed by virus diseases.

Horizontal gene transfer (HGT) is the stable transfer of genetic material from one organism to another without reproduction or human intervention. Transfer occurs by the passage of donor genetic material across cellular boundaries, followed by heritable incorporation to the genome of the recipient organism. In addition to conjugation, transformation and transduction, other diverse mechanisms of DNA and RNA uptake occur in nature. The genome of almost every organism reveals the footprint of many ancient HGT events. Most commonly, HGT involves the transmission of genes on viruses or mobile genetic elements. HGT first became an issue of public concern in the 1970s through the natural spread of antibiotic resistance genes amongst pathogenic bacteria, and more recently with commercial production of genetically modified (GM) crops. However, the frequency of HGT from plants to other eukaryotes or prokaryotes is extremely low. The frequency of HGT to viruses is potentially greater, but is restricted by stringent selection pressures. In most cases the occurrence of HGT from GM crops to other organisms is expected to be lower than background rates. Therefore, HGT from GM plants poses negligible risks to human health or the environment.

The combination or stacking of different traits or genes in plants is rapidly gaining popularity in biotech crop production. Here we review the existing terminology regarding gene stacking in plants, and its implications in relation to genetics, biosafety, detectability and European regulations. Different methods of production of stacked gene traits, as well as the status of their cultivation and approval, are reviewed. Related to the different techniques of transformation and production, including classical breeding, and to differences in global authorization and commercialization practices, there are many types, definitions, and perceptions of stacking. These include: (1) stacking of traits and (2) stacking of events, which are the most widely accepted perceptions of stacking, and (3) stacking of genes, which from the analytical and traceability point of view may be a more appropriate perception. These differences in perceptions and definitions are discussed, as are their implications for analytical detection and regulatory compliance according to (in particular) European Union (EU) regulations. A comprehensive terminology regarding gene stacking with regulatory relevance is proposed. The haploid genome equivalent is proposed as the prevailing unit of measurement at all stages throughout the chain, in order to ensure that terminology and definitions of gene stacks are adapted to analytical detection, traceability, and compliance with EU regulations.

Different techniques to assess bacterial community structure and diversity were evaluated in silages prepared with four different maize cultivars, three conventional and one transgenic (cv. Tundra, event Bt-176). Plants were cultivated in the greenhouse and harvested after 30 days of growth. Silage samples were collected at successive times during fermentation and analyzed for bacterial counts and by various DNA-based fingerprinting techniques. Bacterial counts were similar between cultivars for the total culturable bacteria, sporeforming, and mesophilic and thermophilic lactic acid bacteria (LAB). Further analysis of the species composition of 388 LAB strains by intergenic transcribed spacer (ITS) PCR followed by sequencing of 16S rRNA gene did not reveal differences between cultivars. In contrast, molecular fingerprinting methods targeting whole bacterial communities, such as automated ribosomal intergenic spacers analysis (ARISA) and 16S rRNA gene length heterogeneity-PCR (LH-PCR), indicated that different maize silage batches or cultivars hosted different bacterial communities. Thus, ARISA and LH-PCR fingerprinting techniques offer a fast and sensitive method to compare bacterial communities, and to detect differences in silage bacterial communities.

Many varieties of transgenic rice are under development in countries where wild and weedy relatives co-occur with the crop. To evaluate possible risks associated with pollen-mediated transgene dispersal, we conducted a two-year survey in Vietnam to examine overlapping flowering periods of rice (Oryza sativa L.), weedy rice (O. sativa), and wild rice (O. rufipogon Griff.), all of which are inter-fertile. We surveyed populations in two regions of the Mekong Delta, northern and southern, and at three sites in each of three habitats per region: fresh water, saline water, and acid sulfate soil. Weedy rice frequently flowered simultaneously with neighboring cultivated rice plants. Flowering was more seasonal in wild rice and often peaked in November and December. Peak flowering times of wild rice overlapped with adjacent rice fields at all of the saline sites and half of the acid sulfate sites. The longer flowering season of wild rice ensured that crop-to-wild gene flow was possible in fresh water habitats as well. Our second objective was to determine whether wild and weedy rice populations are exposed to pests that could be targeted by future transgenes, which may then provide fitness benefits. These populations shared many pathogen and insect herbivore species with cultivated rice (leaffolder, locust, cricket, planthoppers, rice bug, stem borer, sheath blight, blast, bacterial leaf blight, and brown spot). Damage by leaffolders and locusts was the most frequently observed insect feeding damage on all three rice types. Indicator species analysis revealed that most of the insect herbivores were associated with particular habitats, demonstrating the importance of broad geographic sampling for transgenic rice risk assessment. These survey data and the strong likelihood of gene flow from cultivated rice suggest that further studies are needed to examine the effects of transgenic traits such as resistance to pests on the abundance of wild and weedy rice.

This study evaluates a survey about Swedish farmers' attitude towards genetically modified (GM) crops, and their perception concerning potential benefits and drawbacks that cropping of an insect resistant (IR) GM variety would involve. The questions were “tick a box” choices, included in a yearly omnibus survey sent to 1000 Swedish farmers (68% response rate). The results showed that a majority of the farmers were negative, although almost one third claimed to be neutral to GM crops. The farmers recognized several benefits both in terms of agricultural production and for the environment, but they were also highly concerned about the consumers' unwillingness to buy GM products. Farmers perceived an increase in yield, but nearly as many farmers thought that there would be no benefits with growing an IR GM crop. Several differences in hopes and concerns of the farmers surveyed were revealed when they were divided in positive, neutral and negative groups. Farmers negative to GM were more concerned than positive farmers about IR GM crops being dangerous for humans, livestock or other organisms to consume. GM-positive farmers seemed to be most concerned about potential problems with growing a marketable crop and expensive seeds, but saw a reduced health risk to the grower, due to less use of pesticides, as a possible benefit. The results among the GM-neutral farmers were in most cases closely related to the positive farmers' choices, implying that they believe that there are advantages with growing an IR GM crop, but also fear potential drawbacks. This general uncertainty about GM IR crops may prevent them from accepting the new technology.

Outcrossing of non-Bt cotton (Gossypium hirsutum (L.)) in refuges by transgenic Bt cultivars could reduce the efficacy of refuges for delaying resistance in seed-feeding pests. Based on reports that outcrossing decreased as distance from Bt cotton increased in small-scale studies, we hypothesized that increasing refuge width or distance from Bt fields would reduce outcrossing. In a large-scale study in Arizona, we quantified Bt seed in refuges of experimental and commercial fields, comparing outcrossing between in-field (narrow) and external (wide) refuges and among rows of refuges at various distances from Bt fields. Some refuges, including those in tightly controlled experimental plots, contained up to 8% adventitious Bt plants. Some, but not all, Bt plants likely resulted from Bt seed in the non-Bt seed bags. We did not detect a difference in outcrossing between in-field and external refuges. However, statistical power was low because outcrossing was low (< 0.4% of seeds) in both treatments. Higher outcrossing levels (≤ 4.6% of seeds) were observed in the studies measuring outcrossing at various distances from Bt fields, yet outcrossing did not decrease as the distance from Bt fields increased. We hypothesize that Bt plants in refuges cross-pollinated surrounding non-Bt plants, overshadowing the expected association between distance from Bt fields and outcrossing.

We have developed a screening method that can be used during the problem formulation phase of risk assessment to identify and prioritize non-target invertebrates for risk analysis with any transgenic plant. In previously published protocols for this task, five criteria predominated. These criteria have been combined by our method in a simple model which assesses: (1) the possible level of risk presented by the plant to each invertebrate species (through measurements of potential hazard and exposure, the two principal criteria); (2) the hypothetical environmental impact of this risk (determined by the currently known status of the species' population in the ecosystem and its potential resilience to environmental perturbations); (3) the estimated economic, social and cultural value of each species; and (4) the assessed ability to conduct tests with the species. The screening method uses information on each of these criteria entered into a specially designed database that was developed using Microsoft $^{\circledR}$ Access 2003. The database holds biological and ecological information for each non-target species, as well as information about the transgenic plant that is the subject of the risk assessment procedure. Each piece of information is then ranked on the basis of the value of the information to each criterion being measured. This ranking system is flexible, allowing the method to be easily adapted for use in any agro-ecosystem and with any plant modification. A model is then used to produce a Priority Ranking of Non-Target Invertebrates (PRONTI) score for each species, which in turn allows the species to be prioritized for risk assessment. As an example, the method was used to prioritize non-target invertebrates for risk assessment of a hypothetical introduction of Bacillus thuringiensis (Bt) Cry1Ac-expressing Pinus radiata trees into New Zealand.

It is widely acknowledged that plant-made pharmaceuticals (PMPs) offer numerous benefits, including inexpensive production, biological safety and the facility for production at agricultural scale. At the same time, it is important to minimize any potential risk associated with this new technology, including the potential release of bioactive proteins into the environment. To address this issue, we studied transgenic Nicotiana benthamiana and Nicotiana tabacum plants expressing two recombinant single-chain variable fragment (scFv) antibodies, respectively scFvB9 and scFvH10. ScFvB9 was raised against glycoprotein G1 of Tomato spotted wilt virus (TSWV), and scFvH10 was raised against human tumor-associated antigen tenascin-C. Both antibodies were targeted to the secretory pathway using the N-terminal signal peptide from Phaseolus vulgaris polygalacturonase-inhibiting protein (PGIP), and scFvH10 carried in addition a C-terminal KDEL tetrapeptide for retention in the endoplasmic reticulum (ER). Sterile hydroponic cultures were established, allowing us to investigate whether scFvB9 and scFvH10 were present in root exudates. Intercellular fluids extracted from different plant tissues were analyzed by western blotting revealing the presence of scFvB9. Successful secretion of scFvB9 in hydroponic medium was also demonstrated, whereas no scFvH10 could be detected in the leaf, stem or root apoplast, nor secreted into the hydroponic medium. Our results show that scFvH10 release or diffusion from the roots of transgenic plants was not occurring, suggesting that the KDEL signal might contribute to the environmental biosafety of crops producing PMPs.

Spatially isolating genetically modified (GM) maize fields from non-GM maize fields is a robust on-farm measure to keep the adventitious presence of GM material in the harvest of neighboring fields due to cross-fertilizations below the European labeling threshold of 0.9%. However, the implementation of mandatory and rigid isolation perimeters can affect the farmers' freedom of choice to grow GM maize on their fields if neighboring farmers do not concur with their respective cropping intentions and crop plans. To minimize the presence of non-GM maize within isolation perimeters implemented around GM maize fields, a method was developed for optimally allocating GM maize to a particular set of fields. Using a Geographic Information System dataset and Monte Carlo analyses, three scenarios were tested in a maize cultivation area with a low maize share in Flanders (Belgium). It was assumed that some farmers would act in collaboration by sharing the allocation of all their arable land for the cultivation of GM maize. From the large number of possible allocations of GM maize to any field of the shared pool of arable land, the best field combinations were selected. Compared to a random allocation of GM maize, the best field combinations made it possible to reduce spatial co-existence problems, since at least two times less non-GM maize fields and their corresponding farmers occurred within the implemented isolation perimeters. In the selected field sets, the mean field size was always larger than the mean field size of the common pool of arable land. These preliminary data confirm that the optimal allocation of GM maize over the landscape might theoretically be a valuable option to facilitate the implementation of rigid isolation perimeters imposed by law.


Monitoring is a statutory requirement for the cultivation of genetically modified (GM) crops in the European Community. Questionnaires for farmers to report on observations of effects linked with the cultivation of GM crops can form a useful part of a monitoring regime. A questionnaire for GM maize (Zea mays L.) was designed, with questions focusing on potential effects related to the GM maize grown, as well as on background information about cultivation methods and on individual field situations. In this paper we present the methodological approach of the monitoring regime, the structuring of the data, and the contents and structure of the questionnaire. The statistical requirements and background for an appropriate evaluation and interpretation of the data are described. Results of interviews made from 2001 to 2005 are also presented. It is envisaged that this approach will be developed for monitoring other cultivated GM plants and traits, and may be applicable in monitoring certain non-farmed environments.

Chitinases are hydrolytic enzymes that have been employed in biotechnology in attempts to increase plants' resistance against fungal pathogens. Genetically modified plants have given rise to concerns of the spreading of transgenes into the environment through vertical or horizontal gene transfer (HGT). In this study, chitinase-like sequences from silver birch (Betula pendula) EST-libraries were identified and their phylogenetic relationships to other chitinases were studied. Phylogenetic analyses were used to estimate the frequency of historical gene transfer events of chitinase genes between plants and other organisms, and the usefulness of phylogenetic analyses as a source of information for the risk assessment of transgenic silver birch carrying a sugar beet chitinase IV gene was evaluated. Thirteen partial chitinase-like sequences, with an approximate length of 600 bp, were obtained from the EST-libraries. The sequences belonged to five chitinase classes. Some bacterial chitinases from Streptomyces and Burkholderia, as well as a chitinase from an oomycete, Phytophthora infestans, grouped together with the class IV chitinases of plants, supporting the hypothesis that some class IV chitinases in bacteria have evolved from eukaryotic chitinases via horizontal gene transfer. According to our analyses, HGT of a chitinase IV gene from eukaryotes to bacteria has presumably occurred only once. Based on this, the likelihood for the HGT of chitinase IV gene from transgenic birch to other organisms is extremely low. However, as risk is a function of both the likelihood and consequences of an event, the effects of rare HGT event(s) will finally determine the level of the risk.

To address some concerns about the expansion of genetically engineered pharmaceutical and industrial crops to outdoor plantings and potential impacts on the human food supply, we determined whether commercial agriculture seeds of maize or corn Zea mays L., barley Hordeum vulgare L., safflower Carthamus tinctorius L. and rice Oryza sativa L. are digested or pass viably through the digestive tract, or are transported externally, by captive mallard ducks Anas platyrhynchos L., ring-necked pheasants Phasianus colchicus L., red-winged blackbirds Agelaius phoeniceus (L.) and rock pigeons Columba livia Gmelin (with the exception of whole maize seeds which were too large to feed to the blackbirds). These crop seeds, whether free-fed or force-fed, did not pass through the digestive tract of these bird species. The birds nonetheless did retain viable seeds in the esophagus/crop and gizzard for several hours. For example, after foraging for 6 h, mallards had retained an average of 228 ± 112 barley seeds and pheasants 192 ± 78 in the esophagus/crop, and their germination rates were 93 and 50%, respectively. Birds externally transported seeds away from the feeding location, but in only four instances were seeds found attached to their muddy feet or legs and in no case to feathers. Risk of such crop seeds germinating, establishing and reproducing off site after transport by a bird (externally or internally) or movement of a carcass by a predator, will depend greatly on the crop and bird species, location, environmental conditions (including soil characteristics), timing, and seed condition.