Oilseed rape (Brassica napus) is sexually compatible with its wild and weedy relative B. rapa, and introgression of genes from B. napus has been found to occur over a few generations. We simulated the early stages of transgene escape by producing F1 hybrids and the first backcross generation between two lines of transgenic B. napus and two populations of weedy B. rapa. Transgene expression and the fitness of the hybrids were examined under different environmental conditions. Expression of the transgenes was analyzed at the mRNA level by quantitative PCR and found to be stable in the hybrids, regardless of the genetic background and the environment, and equal to the level of transcription in the parental B. napus lines. Vigor of the hybrids was measured as the photosynthetic capability; pollen viability and seed set per silique. Photosynthetic capability of first generation hybrids was found to be at the same level, or higher, than that of the parental species, whereas the reproductive fitness was significantly lower. The first backcross generation had a significantly lower photosynthetic capability and reproductive fitness compared to the parental species. This is the first study that examines transgene expression at the mRNA level in transgenic hybrids of B. napus of different genetic background exposed to different environmental conditions. The data presented clarify important details of the overall risk assessment of growing transgenic oilseed rape.

Over the last 20 years, there has been much research aimed at improving environmental risk assessment of transgenic crops. Despite large amounts of data, decisions to allow or prohibit the release of transgenic crops remain confused and controversial. We argue that part of the reason for confusion is the lack of clear definitions of components of the environment that should be protected, and, as a consequence, there is no way to judge the relevance of data collected under the auspices of ‘environmental risk assessment’. Although this criticism applies to most aspects of environmental risk assessment of transgenic crops, it is most pertinent to effects that might result from an increase in plant fitness, often referred to as increased weediness. Environmental risk assessment of weediness is regarded as complicated: an increase in the fitness of a transgenic plant compared with non-transgenic counterparts will be the result of an interaction between the altered plant phenotype and an enormous number of environmental variables. This has led to the idea that risk assessment of weediness needs to “understand” these interactions, with the implication that exhaustive data are required. Here we argue that environmental risk assessment of the weediness of transgenic plants need not be complicated. Analysis of the conditions that must be met for increased weediness to occur suggests a series of studies that starts with simple tests in the laboratory under “worst case” assumptions, and becomes increasingly complex and realistic should the simpler studies not indicate negligible risk with sufficient certainty. We illustrate how the approach might work for assessing the risks of increased weediness using the example of possible introgression of a gene for Turnip mosaic virus (TuMV) resistance from oilseed rape to certain wild Brassica species.

The ongoing discussion on the co-existence between genetically modified (GM) and non-GM crops becomes more important in the European Union (EU). With the recent inscription of 17 GM maize varieties in the common EU catalogue of varieties of agricultural plant species, the acreage of transgenic maize for market purposes is expected to increase in some European countries. In the EU, specific tolerance thresholds have been established for the adventitious and technically unavoidable presence of GM material in non-GM produce, and member states are elaborating legal frames to cope with co-existence. As maize is a cross-pollinated crop relying on wind for the dispersal of its pollen, technical management measures will be imposed to reduce cross-fertilization between transgenic and non-transgenic maize. Various biological, physical and analytical parameters have been identified to play a role in the study of cross-fertilization in maize. This variability may hamper the comparison between research results and may complicate the definition of appropriate isolation distances and/or pollen barriers in order to limit out-crossing. The present review addresses these parameters and proposes containment measures in order to not exceed the legal labeling thresholds in maize.

There is much discussion of the probability of transgene flow from transgenic crop varieties to landraces and wild relatives in centers of origin or diversity, and its genetic, ecological, and social consequences. Without costly research on the variables determining gene flow, research on transgene frequencies in landrace (or wild relative) populations can be valuable for understanding transgene flow and its effects. Minimal research requirements include (1) understanding how farmer practices and seed systems affect landrace populations, (2) sampling to optimize Ne/n (effective /census population size), (3) minimizing variance at all levels sampled, and (4) using Ne to calculate binomial probabilities for transgene frequencies. A key case is maize in Mexico. Two peer-reviewed papers, based on landrace samples from the Sierra Juárez region of Oaxaca, Mexico, reached seemingly conflicting conclusions: transgenes are present (Quist and Chapela, 2001, Nature414: 541–543; 2002, Nature416: 602) or “detectable transgenes” are absent (Ortiz-García et al., 2005, Proc. Natl. Acad. Sci. USA102: 12338–12343 and 18242). We analyzed these papers using information on Oaxacan maize seed systems and estimates of Ne. We conclude that if Quist and Chapela’s results showing presence are accepted, Ortiz-García et al.’s conclusions of no evidence of transgenes at detectable levels or for their introgression into maize landraces in the Sierra de Juárez of Oaxaca are not scientifically justified. This is because their samples are not representative, and their statistical analysis is inconclusive due to using n instead of Ne. Using estimates of Ne based on Ortiz-García et al.’s n, we estimate that transgenes could be present in maize landraces in the Sierra Juárez region at frequencies of ~1–4%, and are more likely to be present in the 90% of Oaxacan landrace area that is not mountainous. Thus, we have no scientific evidence of maize transgene presence or absence in recent years in Mexico, Oaxaca State, or the Sierra Juárez region.

Genetically modified plants (GMPs) may soon be cultivated commercially in several member countries of the European Union (EU). According to EU Directive 2001/18/EC, post-market monitoring (PMM) for commercial GMP cultivation must be implemented, in order to detect and prevent adverse effects on human health and the environment. However, no general PMM strategies for GMP cultivation have been established so far. We present a conceptual framework for the design of environmental PMM for GMP cultivation based on current EU legislation and common risk analysis procedures. We have established a comprehensive structure of the GMP approval process, consisting of pre-market risk assessment (PMRA) as well as PMM. Both programs can be distinguished conceptually due to principles inherent to risk analysis procedures. The design of PMM programs should take into account the knowledge gained during approval for commercialization of a specific GMP and the decisions made in the environmental risk assessments (ERAs). PMM is composed of case-specific monitoring (CSM) and general surveillance. CSM focuses on anticipated effects of a specific GMP. Selection of case-specific indicators for detection of ecological exposure and effects, as well as definition of effect sizes, are important for CSM. General surveillance is designed to detect unanticipated effects on general safeguard subjects, such as natural resources, which must not be adversely affected by human activities like GMP cultivation. We have identified clear conceptual differences between CSM and general surveillance, and propose to adopt separate frameworks when developing either of the two programs. Common to both programs is the need to put a value on possible ecological effects of GMP cultivation. The structure of PMM presented here will be of assistance to industry, researchers, and regulators, when assessing GMPs during commercialization.

Guidelines to ensure the efficient coexistence of genetically modified (GM) and conventional crops are currently being considered across the European Union. The purpose of this strategy is to describe the measures a farmer must adopt to minimize the admixture of GM and non-GM crops. Minimizing pollen / seed-mediated gene flow between GM and non-GM crops is central to successful coexistence. However no system is currently available to permit the numeric quantification of a crop’s propensity for pollen/seed-mediated gene flow. The provision of such a system could permit a background level of gene flow, specific for a particular conventional crop, to be calculated. Here we present a gene flow index model implemented using the principal arable crops in Ireland as a model dataset. The objective of this research was to establish a baseline gene flow data set for Ireland’s primary conventional crops through the provision of a simple numerical index. This Gene Flow Index (GFI) incorporates four strands of crop-mediated gene flow (crop pollen-to-crop, crop pollen-to-wild, crop seed-to-volunteer and crop seed-to-feral) into a format that permits the calculation of a crop’s gene flow potential. Responsive to regional parameters, we have applied the model to sugar beet, oilseed rape, potato, ryegrass, maize, wheat and barley. We propose that the attained indices will highlight those crops that require additional measures in order to minimize gene flow in accordance with anticipated coexistence guidelines.

Cassava is an important subsistence crop grown only in the tropics, and represents a major source of calories for many people in developing countries. Improvements in the areas of resistance to insects and viral diseases, enhanced nutritional qualities, reduced cyanogenic content and modified starch characteristics are urgently needed. Traditional breeding is hampered by the nature of the crop, which has a high degree of heterozygosity, irregular flowering, and poor seed set. Biotechnology has the potential to enhance crop improvement efforts, and genetic engineering techniques for cassava have thus been developed over the past decade. Selectable and scorable markers are critical to efficient transformation technology, and must be evaluated for biosafety, as well as efficiency and cost-effectiveness. In order to facilitate research planning and regulatory submission, the literature on biosafety aspects of the selectable and scorable markers currently used in cassava biotechnology is surveyed. The source, mode of action and current use of each marker gene is described. The potential for toxicity, allergenicity, pleiotropic effects, horizontal gene transfer, and the impact of these on food or feed safety and environmental safety is evaluated. Based on extensive information, the selectable marker genes nptII, hpt, bar/pat, and manA, and the scorable marker gene uidA, all have little risk in terms of biosafety. These appear to represent the safest options for use in cassava biotechnology available at this time.

Genetically engineered transgenic crop varieties (TGVs) have spread rapidly in the last 10 years, increasingly to traditionally-based agricultural systems (TBAS) of the Third World both as seed and food. Proponents claim they are key to reducing hunger and negative environmental impacts of agriculture. Opponents claim they will have the opposite effect. The risk management process (RMP) is the primary way in which TGVs are regulated in the US (and many other industrial countries), and proponents claim that the findings of that process in the US and its regulatory consequences should be extended to TBAS. However, TBAS differ in important ways from industrial agriculture, so TGVs could have different effects in TBAS, and farmers there may evaluate risks and benefits differently. To evaluate some potential impacts of TGVs in TBAS we used the RMP as a framework for the case of Bt maize in Mesoamerica and Cuba. We interviewed 334 farmers in Cuba, Guatemala and Mexico about farming practices, evaluations of potential harm via hypothetical scenarios, and ranking of maize types. Results suggest high potential for transgene flow via seed, grain and pollen; differences in effects of this exposure in TBAS compared with industrial agriculture; farmers see some potential consequences as harmful. Perceptions of harm differ among farmers in ways determined by their farming systems, and are different from those commonly assumed in industrial systems. An RMP including participation of farmers and characteristics of TBAS critical for their functioning is necessary to ensure that investments in agricultural technologies will improve, not compromise these agricultural systems.

Cleveland et al. (2005, Environ. Biosafety Res.4: 197–208) offer useful suggestions for monitoring transgenes in landraces of maize, but we disagree with their statement that the scientific conclusions of our paper (Ortiz-García et al., 2005, Proc. Natl. Acad. Sci. USA102: 12338–12343) are not justified. First, contrary to their perception, our survey was not designed to evaluate transgenes in the whole State of Oaxaca, but rather to monitor a specific portion of the District of Ixtlán de Juárez where the presence of transgenes had been reported previously by Quist and Chapela (2001, Nature414: 541–543). Second, our paper described two methods for estimating frequencies of undetected transgenic seeds, while Cleveland et al. recommend a third approach that explicitly estimates effective population size. They argue that the effective population size of our seed samples is smaller than we assumed, leading to false claims about our detection accuracy. However, we employed a robust statistical approach to compensate for possible bias by using numbers of maternal plants, in addition to numbers of seeds, to provide a conservative estimate of the minimum number of independent samples. When we re-analyzed our 2004 data using effective population sizes, our conclusion that transgenic seeds were “absent or extremely rare” did not change, nor did the general range of possible frequencies of undetected transgenic seeds. Unlike Cleveland et al., we advocate using combined probability tests to analyze data across localities. Third, our critics argue that we accepted the null hypothesis that transgenes were absent. Actually, we assumed that transgenes were present in local landraces, and we used parameter estimation methods to calculate the probability of failing to detect transgenic individuals at a range of frequencies. In agreement with Cleveland et al., we reiterate that there is a clear need for additional surveys with rigorous sampling methods to provide estimates of transgene frequencies over broad geographic areas in Mexico.

An investigation was carried out to monitor the escape and spread of oilseed rape (Brassica napus) transgenic plants and the introgression of transgenes to its closely related feral species in Japan. We screened a total of about 7500 feral B. napus, 300 B. rapa, and 5800 B. juncea seedlings from maternal plants in 143 locations at several ports, roadsides, and riverbanks. The presence of glufosinate-resistance or glyphosate-resistance transgenes in these seedlings was confirmed by means of herbicide treatments and also immunochemical and DNA analyses. B. napus plants with herbicide-resistant transgenic seeds were found at five of six major ports and along two of four sampled roadsides in the Kanto District. Transgenic oilseed rape plants have not been commercially cultivated in Japan, suggesting that the transgenes would probably have come from imported transgenic seeds that were spilled during transportation to oilseed processing facilities. No transgenes were detected in seeds collected from B. napus plants growing along riverbanks in the Kanto District or in seeds from closely related species (B. rapa and B. juncea). To our knowledge, this is the first published example of feral, transgenic populations occurring in a nation where the transgenic crop has not been cultivated commercially.

Over the last decade, plant virus-based vectors have been developed and successfully exploited for high-yield production of heterologous proteins in plants. However, widespread application of recombinant viruses raises concerns about possible risks to the environment. One of the primary safety issues that must be considered is the uncontrolled spread of the genetically engineered virus from experimental plants to susceptible weeds or crops. Using a movement-deficient Potato virus X (PVX)-based transient gene expression vector which harbors the β-glucuronidase (gus) gene, we established a plant viral expression system that provides containment of the recombinant virus and allows for safe and efficient protein production. By deletion of the viral 25k movement protein gene, systemic spread of the modified virus in non-transgenic Nicotiana benthamiana plants was successfully inhibited. In transgenic N. benthamiana plants expressing the 25K viral movement protein, this deficiency was complemented, thus resulting in systemic infection with the movement-deficient virus. While no differences in virus spread and accumulation were observed compared to infection caused by wild-type PVX in non-transgenic plants, the movement protein transgenic plants exhibited none of the normal symptoms of viral infection. Several biosafety aspects were investigated including the potential for recombination between the defective virus and the movement protein transgene, as well as complementation effects in non-transgenic plants doubly infected with the defective and the wild-type virus. Furthermore, the applicability of the safety system for the production of heterologous proteins was evaluated with gus as a model gene. With respect to the stability of the gus insert and the expression level of the GUS protein, there were no differences between the novel system developed and the conventional PVX-based expression system.

Despite all the achieved benefits and potential promises from recombinant DNA technology of plants, the potential of transgene spread to wild relatives and to non-transgenic crops is still of a wide-spread concern. We continue to develop recoverable block of function (RBF) technology for gene flow control in transgenic plants. RBF consists of two elements: blocking construct (BC) and recovering construct (RC). Natural expression of the BC (barnase) in embryos and sprouts blocks a physiological function essential for survival or reproduction of the transgenic plant (mRNA synthesis and germination). Artificially induced (heat shock treatment) RC (barstar) recovers the blocked function enabling transgenic plant to reproduce. In natural conditions without artificial induction of RC the transgenic plant can not reproduce itself. However, a single RBF may still fail because of the potential for mutations and gene silencing of the inserted constructs. To minimize the frequency of such an inactivation, we developed a double RBF, in which a single insert comprising two BC, flanking a transgene of interest, was constructed and transferred into tobacco (Nicotiana tabacum (L.)). We used a barstar gene driven by a heat shock or 35S promoter as a RC, and two different promoters were used for barnase genes in the BC. One BC contained a seed germination specific cysteine endopeptidase promoter (BC1) and the other contained the cruciferin promoter (BC2), which is active during fruit development and embryogenesis. Three alternative constructs of double RBF are described, and a segregating two-insert as well as a one-insert cassettes, were compared. One-insert system comprising two BC with different nucleotide sequences but degenerate codons that expressed the same Barnase protein appeared to be the most reliable choice. The biological and molecular data obtained suggest that double RBF is a potent transgene containment technique that can safely be applied in agriculture.

Previous studies have shown that the widely used plant transformation vector Agrobacterium tumefaciens can persist in genetically engineered plants in vitro and in transgenic greenhouse-grown plants, despite the use of counter-selective antibiotics. However, little is known regarding Agrobacterium persistence in tree species. To understand the kinetics of A. tumefaciens decline and persistence in transformation experiments, we assayed for the presence of A. tumefaciens in spruce and pine embryogenic tissue for up to 10 weeks post-transformation. The A. tumefaciens populations declined rapidly in the first five days post-cocultivation but generally declined more slowly in pine, relative to spruce. No bacteria were detected in spruce embryogenic tissue beyond four weeks after cocultivation, however in pine there were ~100 colony forming units per g tissue at 10 weeks post-cocultivation. We present evidence that the detection limit for PCR using virD2 primers to detect A. tumefaciens in a background of pine needle DNA was approximately 109–1010 A. tumefaciens cells per g of tissue. We also assayed for A. tumefaciens in transgenic pine and spruce embryogenic tissue and from needles, branches, stems and roots of transformed plants, up to four years post-inoculation. Occasionally A. tumefaciens was detected in embryogenic tissue up to 12 months post-inoculation. A. tumefaciens was never detected in cultured embryogenic tissue more than twelve months after inoculation, nor in developing somatic embryos or germinating plantlets, nor any of the parts of greenhouse-grown plants. From these data we conclude that if A. tumefaciens persists in transgenic conifers, it does so beneath our ability to detect it.

This paper analyzes some important issues surrounding possible deployment of genetically engineered (GE) insect-resistant potato in Peru, based on a large farmer survey held in Peru in 2003. We found that the formal seed system plays a limited role compared with the informal seed system, especially for smallholder farmers. Although 97% of smallholder farmers would buy seed of an insect-resistant variety, a majority would buy it only once every 2 to 4 years. Survey data show that farmers would be willing to pay a premium of 50% on seed cost for insect resistant varieties. Paying price premiums of 25% to 50%, farmers would still increase their net income, assuming insect resistance is high and pesticide use will be strongly reduced. Of all farmers, 55% indicated preference for insect-resistant potato in varieties other than their current varieties. The survey indicates that smallholder farmers are interested to experiment with new varieties and have a positive perception of improved varieties. Based on these findings, and considering the difficulties implementing existing biosafety regulatory systems such as those in place in the U.S. and E.U., we propose to develop a variety-based segregation system to separate GE from conventionally bred potatoes. In such a system, which would embrace the spread of GE potatoes through informal seed systems, only a limited number of sterile varieties would be introduced that are easily distinguishable from conventional varieties.

Non-target organisms associated with the soil might be adversely affected by exposure to the Cry1Ab protein from Bacillus thuringiensis (Bt) in transgenic corn (Zea mays L.). To check for such exposure, we used ELISA to test for Cry1Ab in ground beetles collected live from fields with Bt corn residues and Bt corn (Bt/Bt), Bt corn residues and non-Bt crops (Bt/non-Bt), or non-Bt corn residues and non-Bt crops (non-Bt/non-Bt). In fields with Bt corn residues (Bt/Bt and Bt/non-Bt), Cry1Ab was present in all seven species of ground beetles examined (Agonum placidum, Bembidion rupicola, Clivina impressefrons, Cyclotrachelus iowensis, Harpalus pensylvanicus, Poecilus chalcites, and Poecilus lucublandus). For the two most abundant species, P. chalcites and P. lucublandus, the proportion of beetles with Cry1Ab was significantly higher in Bt/Bt fields (0.50–1.0) and Bt/non-Bt fields (0.41–0.50) than in non-Bt/non-Bt fields (0.0). This is the first field evidence that some ground beetle species are exposed to Cry1Ab. The implications of exposure on the performance of these non-target organisms are unclear.

Horizontal gene transfer (HGT) is an important mechanism by which bacteria recombine and acquire novel genes and functions. Risk scenarios where novel plant transgenes transfer horizontally into bacteria have been addressed in numerical theoretical assessments and experimental studies. A key outcome of these studies has been that the frequencies of such inter-domain transfer are very low, if occurring at all, suggesting that such transfers would not occur at a level that is biologically significant. The relationship between transfer frequencies and the subsequent selection or genetic drift of transgene carrying bacteria often remains unresolved in these studies and assessments. Here we present a stochastic model to better understand the initial establishment and population dynamics of rare bacterial transformants carrying horizontally acquired (trans)genes. The following key parameters are considered: initial transformant numbers, strength of selection, bacterial population size and bacterial generations (time). We find that the initial number of transformants is important for the subsequent persistence of transformants only in the range of 1 to ~50 independent HGT events. Our simulations show that transformant populations under a wide range of HGT rates and selection coefficients undergo stochastic developments where they persist at low frequencies for up to several years (at frequencies that are below detection using available field sampling methodology), after which they eventually may go to fixation. Stochastic variability may thus play a crucial but disregarded role in the design of field monitoring strategies e.g. in biosafety assessments. We also estimate the time required for transformants to reach 0.0002% prevalence in a bacterial population, a threshold that allows experimental detection of transgene carrying bacteria through sampling of the larger bacterial populations.