A study of crop-to-crop gene flow using farm scale sites of fodder maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) in the UK

From 2000 to 2003 a range of Farm Scale Evaluation (FSE) trials were established in the UK to assess the effect of the release and management of herbicide tolerant (HT) crops on arable weeds and invertebrates. The FSE trials for maize were also used to investigate crop-to-crop gene flow and to develop a statistical model for the prediction of gene flow frequency that can be used to evaluate current separation distance guidelines for GM crops. Seed samples were collected from the non-GM half of 55 trial sites and 1,055 were tested for evidence of gene flow from the GM HT halves using a quantitative PCR assay specific to the HT (pat) gene. Rates of gene flow were found to decrease rapidly with increasing distance from the GM source. Gene flow was detected in 30% of the samples (40 out of 135) at 150 m from the GM source and events of GM to non-GM gene flow were detected at distances up to and including 200 m from the GM source. The quantitative data were subjected to statistical analysis and a two-step model was found to provide the best fit for the data. A dynamic whole field model predicted that a square field (150 m x 150 m in size) of grain maize would require a separation distance of 3 m for the adjacent crop to be below a 0.9% threshold (with <2% probability of exceeding the threshold). The data and models presented here are discussed in the context of necessary separation distances to achieve various possible thresholds for adventitious presence of GM in maize.     

Effect of volunteers on maize gene flow

Regulatory approvals for deliberate release of GM maize events into the environment have lead to real situations of coexistence between GM and non-GM, with some fields being cultivated with GM and conventional varieties in successive seasons. Given the common presence of volunteer plants in maize fields in temperate areas, we investigated the real impact of GM volunteers on the yield of 12 non-GM agricultural fields. Volunteer density varied from residual to around 10% of plants in the field and was largely reduced using certain cultural practices. Plant vigour was low, they rarely had cobs and produced pollen that cross-fertilized neighbour plants only at low—but variable—levels. In the worst-case scenario, the estimated content of GMO was 0.16%. The influence of GM volunteers was not enough to reach the 0.9% adventitious GM threshold but it could potentially contribute to adventitious GM levels, especially at high initial densities (i.e. above 1,000 volunteers/ha).     

Assessment of Real-time PCR Based Methods for Quantification of Pollen-mediated Gene Flow from GM to Conventional Maize in a Field Study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.     

Design of a decision support tool for managing coexistence between genetically modified and conventional maize at farm and regional levels

Over the last 15 years, several studies on coexistence have used simulation results of spatially explicit gene flow models. These models predict the adventitious presence (AP) of GM grains in non-GM fields at the landscape scale. However, result uncertainty is not quantified. Moreover, most of the models require an important amount of input data on climate, land use, and crop management practices which might not always be available. A comprehensive Bayesian statistical approach has been implemented in the case of gene flow. This approach makes it possible to inform the decision-maker on AP, whatever the amount of information available in a given situation, to provide information on the uncertainty of the predictions and to model the variability of AP within a field, which helps set up sampling strategies.The resulting decision-support tool (DST) can compute the expected AP and its probability distribution in non-GM maize fields at different times of the growing season and under different management scenarios. Integrated through a web interface, the DST is designed to be operationally helpful for managing coexistence between GM and non-GM maize crops for a wide range of stakeholders from farmers to policy makers.     

Low frequency of transgene flow from Bt/CpTI rice to its non-transgenic counterparts planted at close spacing

Crop-to-crop transgene flow will affect seed purity of non-GM rice varieties, leading to unwanted consequences. To assess the maximum probability of transgene outflow in rice (Oryza sativa), gene flow experiments were conducted with three cultivation patterns with different mixed-planting proportions of adjacent GM and non-GM rice at two sites in Fujian and Hainan Provinces of China. Three GM rice lines containing two insect-resistance genes (Bt/CpTI) and their non-GM counterparts were used in the experiments to allow natural hybridization to occur. A hygromycin resistance gene was used as a selective marker for identifying hybrids. Based on the examination of > 645 700 geminated seeds, the result showed low frequencies (0.05-0.79%) of transgene flow from GM to non-GM rice at close spacing, although with significant variation among mixed-planting proportions. It is concluded that rice transgene flow will occur at a very low frequency (< 1.0%), even if the GM rice is planted at close spacing with non-GM rice, and high densities of GM rice cultivated in the neighborhood of non-GM rice will increase the probability of outcrossing with the non-GM rice.     

Sequence-Based Analysis of the Intestinal Microbiota of Sows and Their Offspring Fed Genetically Modified Maize Expressing a Truncated Form of Bacillus thuringiensis Cry1Ab Protein (Bt Maize)

The aim was to investigate transgenerational effects of feeding genetically modified (GM) maize expressing a truncated form of Bacillus thuringiensis Cry1Ab protein (Bt maize) to sows and their offspring on maternal and offspring intestinal microbiota. Sows were assigned to either non-GM or GM maize dietary treatments during gestation and lactation. At weaning, offspring were assigned within sow treatment to non-GM or GM maize diets for 115 days, as follows: (i) non-GM maize-fed sow/non-GM maize-fed offspring (non-GM/non-GM), (ii) non-GM maize-fed sow/GM maize-fed offspring (non-GM/GM), (iii) GM maize-fed sow/non-GM maize-fed offspring (GM/non-GM), and (iv) GM maize-fed sow/GM maize-fed offspring (GM/GM). Offspring of GM maize-fed sows had higher counts of fecal total anaerobes and Enterobacteriaceae at days 70 and 100 postweaning, respectively. At day 115 postweaning, GM/non-GM offspring had lower ileal Enterobacteriaceae counts than non-GM/non-GM or GM/GM offspring and lower ileal total anaerobes than pigs on the other treatments. GM maize-fed offspring also had higher ileal total anaerobe counts than non-GM maize-fed offspring, and cecal total anaerobes were lower in non-GM/GM and GM/non-GM offspring than in those from the non-GM/non-GM treatment. The only differences observed for major bacterial phyla using 16S rRNA gene sequencing were that fecal Proteobacteria were less abundant in GM maize-fed sows prior to farrowing and in offspring at weaning, with fecal Firmicutes more abundant in offspring. While other differences occurred, they were not observed consistently in offspring, were mostly encountered for low-abundance, low-frequency bacterial taxa, and were not associated with pathology. Therefore, their biological relevance is questionable. This confirms the lack of adverse effects of GM maize on the intestinal microbiota of pigs, even following transgenerational consumption.     

Potential subchronic food safety of the stacked trait transgenic maize GH5112E-117C in Sprague-Dawley rats

The food safety of stacked trait genetically modified (GM) maize GH5112E-117C containing insect-resistance gene Cry1Ah and glyphosate-resistant gene G2-aroA was evaluated in comparison to non-GM Hi-II maize fed to Sprague-Dawley rats during a 90-day subchronic feeding study. Three different dietary concentrations (12.5, 25 and 50 %, w/w) of the GM maize were used or its corresponding non-GM maize. No biologically significant differences in the animals’ clinical signs, body weights, food consumption, hematology, clinical chemistry, organ weights and histopathology were found between the stacked trait GM maize groups, and the non-GM maize groups. The results of the 90-day subchronic feeding study demonstrated that the stacked trait GM maize GH5112E-117C is as safe as the conventional non-GM maize Hi-II.     

Definition and feasibility of isolation distances for transgenic maize cultivation

A major concern related to the adoption of genetically modified (GM) crops in agricultural systems is the possibility of unwanted GM inputs into non-GM crop production systems. Given the increasing commercial cultivation of GM crops in the European Union (EU), there is an urgent need to define measures to prevent mixing of GM with non-GM products during crop production. Cross-fertilization is one of the various mechanisms that could lead to GM-inputs into non-GM crop systems. Isolation distances between GM and non-GM fields are widely accepted to be an effective measure to reduce these inputs. However, the question of adequate isolation distances between GM and non-GM maize is still subject of controversy both amongst scientists and regulators. As several European countries have proposed largely differing isolation distances for maize ranging from 25 to 800 m, there is a need for scientific criteria when using cross-fertilization data of maize to define isolation distances between GM and non-GM maize. We have reviewed existing cross-fertilization studies in maize, established relevant criteria for the evaluation of these studies and applied these criteria to define science-based isolation distances. To keep GM-inputs in the final product well below the 0.9% threshold defined by the EU, isolation distances of 20 m for silage and 50 m for grain maize, respectively, are proposed. An evaluation using statistical data on maize acreage and an aerial photographs assessment of a typical agricultural landscape by means of Geographic Information Systems (GIS) showed that spatial resources would allow applying the defined isolation distances for the cultivation of GM maize in the majority of the cases under actual Swiss agricultural conditions. The here developed approach, using defined criteria to consider the agricultural context of maize cultivation, may be of assistance for the analysis of cross-fertilization data in other countries.     

Detection of gene flow from GM to non-GM watermelon in a field trial

Gene flow from genetically modified (GM) crops to conventional non-GM crops is a serious concern for protection of conventional and organic farming. Gene flow from GM watermelon developed for rootstock use, containing cucumber green mottle mosaic virus (CGMMV)-coat protein (CP) gene, to a non-GM isogenic control variety “Clhalteok” and grafted watermelon “Keumcheon” was investigated in a small scale field trial as a pilot study. Hybrids between GM and non-GM watermelons were screened from 1304 “Chalteok” seeds and 856 “Keumcheon” seeds using the duplex PCR method targeting theCGMMV- CP gene as a marker. Hybrids were found in all pollen recipient plots. The gene flow frequencies were greater for “Chaiteok” than for “KeumcheonD; with 75% outcrossing in the “Chaiteok” plot at the closest distance (0.8 m) to the GM plot. A much larger scale field trial is necessary to identify the isolation distance between GM and non-GM watermelon, as the behaviors of insect pollinators needs to be clarified in Korea.     

Effect of feeding genetically modified Bt MON810 maize to ∼40-day-old pigs for 110 days on growth and health indicators

A total of 72 male weaned pigs were used in a 110-day study to investigate the effect of feeding genetically modified (GM) Bt MON810 maize on selected growth and health indicators. It was hypothesised that in pigs fed Bt maize, growth and health are not impacted compared with pigs fed isogenic maize-based diets. Following a 12-day basal period, pigs (10.7 ± 1.9 kg body weight (BW); ∼40 days old) were blocked by weight and ancestry and randomly assigned to treatments: (1) non-GM maize diet for 110 days (non-GM), (2) GM maize diet for 110 days (GM), (3) non-GM maize diet for 30 days followed by GM maize diet up to day 110 (non-GM/GM) and (4) GM maize diet for 30 days followed by non-GM maize diet up to day 110 (GM/non-GM). BW and daily feed intake were recorded on days 0, 30, 60 and 110 (n = 15). Body composition was determined by dual energy X-ray absorptiometry (n = 10) on day 80. Following slaughter on day 110, organs and intestines were weighed and sampled for histological analysis and urine was collected for biochemical analysis (n = 10). Serum biochemistry analysis was performed on days 0, 30, 60, 100 and 110. Growth performance and serum biochemistry were analysed as repeated measures with time and treatment as main factors. The slice option of SAS was used to determine treatment differences at individual time points. There was no effect of feeding GM maize on overall growth, body composition, organ and intestinal weight and histology or serum biochemistry on days 60 and 100 and on urine biochemistry on day 110. A treatment × time interaction was observed for serum urea (SU; P < 0.05), creatinine (SC; P < 0.05) and aspartate aminotransferase (AST; P < 0.05). On day 30, SU was lower for the non-GM/GM treatment compared with the non-GM, GM and GM/non-GM treatments (P < 0.05). On day 110, SC was higher for the non-GM/GM and GM/non-GM treatments compared with non-GM and GM treatments (P < 0.05). Overall, serum total protein was lower for the GM/non-GM treatment compared with the non-GM/GM treatment (P < 0.05). The magnitude of change observed in some serum biochemical parameters did not indicate organ dysfunction and the changes were not accompanied by histological lesions. Long-term feeding of GM maize to pigs did not adversely affect growth or the selected health indicators investigated.     

家蝇介导的水稻基因逃逸风险评估(英文)

【目的】对转基因作物进行生态风险评估是大面积种植前的一个必要步骤,水稻Oryza sativa访花昆虫有上百种,包括家蝇Musca domestica。本研究旨在明确访花昆虫家蝇介导转基因水稻外源基因逃逸的风险。【方法】2010年,我们使用转基因水稻B1, B6和G8-7作为父本(花粉供体),用同源非转基因水稻Jiazao 935和Wuyunjing 7作为母本（花粉受体）,并用家蝇作为授粉昆虫,在浙江大学华家池和长兴试验基地开展了田间种植试验,对收割的后代水稻种子进行室内种植培养,对种苗用潮霉素B和草甘膦处理进行转基因杂交种检测,对存活植株进行潮霉素和草甘膦抗性基因PCR检测,测试家蝇介导的转基因水稻外源基因逃逸频率。【结果】对浙江两个测试基地3个转基因水稻品种共计超过216 500粒后代水稻种子进行的检测及结果表明,在毗邻区域杂交种少,家蝇授粉区和无家蝇授粉区转基因水稻外源基因向非转基因水稻逃逸频率均较低(0~0.64%)。【结论】家蝇介导的转基因水稻外源基因逃逸频率较低,家蝇没有增加转基因水稻外源基因逃逸的风险。     

Regulating coexistence of GM and non-GM crops without jeopardizing economic incentives

The ongoing debate about the coexistence of genetically modified (GM) and non-GM crops in the European Union (EU) mainly focuses on preventive measures needed to keep the adventitious presence of GM material in non-GM products below established tolerance thresholds, as well as on issues covering questions of liability and the duty to redress the incurred economic harm once adventitious mixing in non-GM products has occurred. By contrast, the interplay between the economic incentives and costs of coexistence has attracted little attention. The current overemphasis on the technical aspects and cost of coexistence over its economic incentives might lead EU policy-makers to adopt too stringent and rigid regulations on coexistence. Therefore, we argue for flexible coexistence regulations that explicitly take into account the economic incentives for coexistence. Our arguments provide a timely and important framework for EU policy-makers, who are currently struggling to implement coherent coexistence regulations in all member states.     

Assessment of the influence of field size on maize gene flow using SSR analysis

One of the factors that may influence the rate of cross-fertilization is the relative size of the pollen donor and receptor fields. We designed a spatial distribution with four varieties of genetically-modified (GM) yellow maize to generate different sized fields while maintaining a constant distance to neighbouring fields of conventional white kernel maize. Samples of cross-fertilized, yellow kernels in white cobs were collected from all of the adjacent fields at different distances. A special series of samples was collected at distances of 0, 2, 5, 10, 20, 40, 80 and 120 m following a transect traced in the dominant down-wind direction in order to identify the origin of the pollen through SSR analysis. The size of the receptor fields should be taken into account, especially when they extend in the same direction than the GM pollen flow is coming. From collected data, we then validated a function that takes into account the gene flow found in the field border and that is very useful for estimating the % of GM that can be found in any point of the field. It also serves to predict the total GM content of the field due to cross fertilization. Using SSR analysis to identify the origin of pollen showed that while changes in the size of the donor field clearly influence the percentage of GMO detected, this effect is moderate. This study demonstrates that doubling the donor field size resulted in an approximate increase of GM content in the receptor field of 7%. This indicates that variations in the size of the donor field have a smaller influence on GM content than variations in the size of the receptor field.     

Gene flow in genetically modified wheat

Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow distances can be efficiently estimated with population-level PCR analyses.     

Safety assessment of lepidopteran insect-protected transgenic rice with <Emphasis Type="Italic">cry2A*</Emphasis> gene

Numerous genetically modified (GM) crops expressing proteins for insect resistance have been commercialized following extensive testing demonstrating that the foods obtained from them are as safe as that obtained from their corresponding non-GM varieties. In this paper, we report the outcome of safety studies conducted on a newly developed insect-resistant GM rice expressing the cry2A* gene by a subchronic oral toxicity study on rats. GM rice and non-GM rice were incorporated into the diet at levels of 30, 50, and 70 % (w/w), No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. These results demonstrate that the GM rice with cry2A* gene is as safe for food as conventional non-GM rice.     

Fate of recombinant DNA and Cry1Ab protein after ingestion and dispersal of genetically modified maize in comparison to rapeseed by fallow deer (Dama dama)

The fate of recombinant DNA in fallow deer (Dama dama) was investigated by feeding a diet of isogenic or genetically modified (GM) maize expressing Cry1Ab protein against the European corn borer (Ostrinia nubilalis). To study the degradability of ingested DNA, polymerase chain reaction (PCR) assays were introduced to detect fragments of the endogenous, highly abundant chloroplast-specific rubisco gene, the maize-specific zein gene and the recombinant cry1Ab gene. PCR analysis revealed that small chloroplast- and maize-specific DNA fragments were detectable in contents of rumen, abomasums, jejunum, caecum and colon and occasionally in visceral tissues. In contrast, no fragments of the recombinant cry1Ab gene were detectable in gastrointestinal (GI) contents. The Cry1Ab protein was analysed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique. Neither ELISA nor immunoblotting yielded positive signals of immunoactive Cry1Ab protein in GI contents and tissues of fallow deer fed with GM maize. In conclusion, after uptake of GM maize, neither cry1Ab-specific gene fragments nor Cry1Ab protein were detected in the GI tract of fallow deer, indicating complete digestion of the GM maize. Additional investigations on the germination capacity of conventional rapeseed and maize seed after ingestion by fallow deer and faecal excretion (endozoochory) were performed to draw conclusions regarding a potential spreading of germinable GM crop seed by deer. Germination tests revealed that germinable rapeseed kernels were detectable in faeces; in contrast, no intact maize seeds were found in faeces.     

Sources of uncertainty in the quantification of genetically modified oilseed rape contamination in seed lots

Testing of seed and grain lots is essential in the enforcement of GM labelling legislation and needs reliable procedures for which associated errors have been identified and minimised. In this paper we consider the testing of oilseed rape seed lots obtained from the harvest of a non-GM crop known to be contaminated by volunteer plants from a GM herbicide tolerant variety. The objective was to identify and quantify the error associated with the testing of these lots from the initial sampling to completion of the real-time PCR assay with which the level of GM contamination was quantified. The results showed that, under the controlled conditions of a single laboratory, the error associated with the real-time PCR assay to be negligible in comparison with sampling error, which was exacerbated by heterogeneity in the distribution of GM seeds, most notably at a small scale, i.e. 25 cm³. Sampling error was reduced by one to two thirds on the application of appropriate homogenisation procedures.     

Persistence of seeds from crops of conventional and herbicide tolerant oilseed rape (Brassica napus)

A series of rotation experiments at five sites over four years has explored the environmental and agronomic implications of growing herbicide tolerant oilseed rape and sugar beet. This paper reports on the population dynamics of volunteer rape (Brassica napus). The experiments compared four winter oilseed rape (WOSR) cultivars: a conventional cultivar (Apex) and three developmental cultivars either genetically modified (GM) to be tolerant to glyphosate or glufosinate, or conventionally bred to be tolerant to herbicides of the imidazolinone group. Seed losses at harvest averaged 3575 seeds m(-2) but ranged from less than 2000 up to more than 10000 seeds m(-2). There was a rapid decline in seed numbers during the first few months after harvest, resulting in a mean loss of seeds of 60%. In subsequent seasons, the seedbank declined much more slowly at four of the five sites (ca 20% per year) and the models predicted 95% seed loss after approximately 9 years. Seed decline was much faster at the fifth site. There were no clear differences between the four cultivars in either the numbers of seeds shed at harvest or in their subsequent persistence. The importance of the persistence of GM rape seeds, in the context of the coexistence of GM and non-GM crops and the role of good management practices that minimize seed persistence, are discussed.     

Comparison of growth and aflatoxin B1 production profiles of Aspergillus flavus strains on conventional and isogenic GM-maize-based nutritional matrices

Maize grown in both North and South America are now predominantly genetically modified (GM) cultivars with some resistance to herbicide, pesticide, or both. There is little information on the relative colonisation and aflatoxin B₁ (AFB₁) production with maize meal-based nutritional matrices based on kernels of non-GM maize and isogenic GM-ones by strains of Aspergillus flavus. The objectives were to examine the effect of interacting conditions of temperature (25–35 °C) and water availability (0.99–0.90 water activity, a_w) on (a) mycelial growth, (b) AFB₁ production and (c) develop contour maps of optimum and marginal conditions of these parameters for four strains of A. flavus on three different non-GM and isogenic GM-maize based nutritional media. The growth of the four strains of A. flavus (three aflatoxigenic; one non-aflatoxigenic) was relatively similar in relation to the temperature × a_w conditions examined on both non-GM and GM-based matrices. Optimum growth overall was at 30–35 °C and 0.99 a_w for all four strains. Under water stress (0.90 a_w) growth was optimum at 35 °C. Statistically: non-GM, GM cultivars, temperature and a_w all significantly affected growth rates. For AFB₁ production, all single and interacting factors were statistically significant except for non-GM × GM cultivar. In conclusion, colonisation of GM- and non-GM nutritional sources was similar for the different A. flavus strains examined. The contour maps will be very useful for understanding the ecological niches for both toxigenic and non-toxigenic strains in the context of the competitive exclusion of those producing aflatoxins.