Persistence of Genetically Modified Potatoes in the Field

Volunteers from genetically modified (GM) potatoes may pose an environmental problem if allowed to grow in the field after the annual crop is harvested. We tested whether they are more likely to produce volunteers than non-GM potatoes. Specifically, we compared the number of volunteers, number of tubers per plant, tuber size, and their vertical distribution in the soil. More volunteer plants came from non-GM potatoes than from GM potatoes, but the number and size of tubers were similar between the two. Vertical distribution of the tubers differed significantly, with most non-GM tubers being found in shallower soil (<2 cm deep). Our results suggest that spontaneous GM volunteers may emerge and produce tubers to a degree similar to that of the non-GM plants. No viable volunteers emerged from GM tubers in the next growing season, probably deterred by winter frost and a period of low soil temperatures (below −2°C) at our study site. However, in regions with warmer climates, such GM volunteers may survive Winter and produce more plants the following year.     

Impact of gene stacking on gene flow: the case of maize

To respect the European labelling threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, stakeholders mainly rely on real-time PCR analysis, which provides a measurement expressed as a percentage of GM-DNA. However, this measurement veils the complexity of gene flow, especially in the case of gene stacking. We have investigated the impact of gene stacking on adventitious GM presence due to pollen flow and seed admixture as well as its translation in terms of the percentage of GM-DNA in a non-GM maize harvest. In the case of varieties bearing one to four stacked events, we established a set of relationships between the percentage of GM kernels and the percentage of GM-DNA in a non-GM harvest as well as a set of relationships between the rate of seed admixture and the percentages of GM material in a non-GM harvest. Thanks to these relationships, and based on simulations with a gene flow model, we have been able to demonstrate that the number of events and the stacking structure of the emitting fields impact the ability of a non-GM maize producer to comply with given GM kernel or GM-DNA thresholds. We also show that a great variability in the rates of GM kernels, embryos and DNA results from seed admixture. Finally, the choice of a unit of measurement for a GM threshold in seed lots can have opposite effects on the ability of farmers to comply with a given threshold depending on whether they are crop or seed producers.     

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.     

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.     

Long-term persistence of GM oilseed rape in the seedbank

Coexistence between genetically modified (GM) and non-GM plants is a field of rapid development and considerable controversy. In crops, it is increasingly important to understand and predict the GM volunteer emergence in subsequent non-GM crops. Theoretical models suggest recruitment from the seedbank over extended periods, but empirical evidence matching these predictions has been scarce. Here, we provide evidence of long-term GM seed persistence in conventional agriculture. Ten years after a trial of GM herbicide-tolerant oilseed rape, emergent seedlings were collected and tested for herbicide tolerance. Seedlings that survived the glufosinate herbicide (15 out of 38 volunteers) tested positive for at least one GM insert. The resulting density was equivalent to 0.01 plants m-2, despite complying with volunteer reduction recommendations. These results are important in relation to debating and regulating coexistence of GM and non-GM crops, particularly for planting non-GM crops after GM crops in the same field.     

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.     

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.     

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 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.     

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.     

Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting

The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF.     

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.     

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.     

The effects of phytase transgenic maize on the community components and diversity of arthropods

It is important to understand the effect of phytase transgenic (PT) maize on arthropod communities in natural ecosystems. In this study, a 2-year survey of arthropod community biodiversity in fields of PT maize (0 7 8) and non-genetically modified (RA119, non-GM) maize was performed using sweep-net sampling on the stems and leaves of the maize plants. The results showed that there was no significant difference in the individual number of herbivorous, predatory, neutral and parasitic groups in PT maize and non-GM maize. The species number of herbivorous group in PT maize was significantly lower than that in non-GM maize in 2013 (p < 0.05). The proportions of different arthropod groups were almost identical in the PT maize and non-GM maize in terms of both species and individual number. Moreover, there was no significant difference in the Shannon-Weiner diversity index (H'), evenness index (J), dominance index (D), richness (S), and species abundance (N) between the two types of maize. The similarity coefficient of the arthropod community suggested that the arthropod community composition of PT maize was similar to that of non-GM maize. Furthermore, PT maize had no significant effect on the relative stability of the arthropod community. These results indicated that despite the presence of a relatively minor difference in arthropod community between the PT maize and non-GM maize, the PT maize had little effect on arthropod community biodiversity.     

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。     

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.     

Molecular analysis of bacterial community structures in paddy soils for environmental risk assessment with two varieties of genetically modified rice, Iksan 483 and Milyang 204

The impacts of planted transgenic rice varieties on bacterial communities in paddy soils were monitored using both cultivation and molecular methods. The rice field plot consisted of eighteen subplots planted with two genetically modified (GM) rice and four non-GM rice plants in three replicates. Analysis with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes revealed that the bacterial community structures were quite similar to each other in a given month, suggesting that there were no significant differences in bacterial communities between GM and non- GM rice soils. The bacterial community structures appeared to be generally stable with the seasons, as shown by a slight variation of microbial population levels and DGGE banding patterns over the year. Comparison analysis of 16S rDNA clone libraries constructed from soil bacterial DNA showed that there were no significant differences between GM and non-GM soil libraries but revealed seasonal differences of phyla distribution between August and December. The composition profile of phospholipid fatty acids (PLFA) between GM and non-GM soils also was not significantly different to each other. When soil DNAs were analyzed with PCR by using primers for the bar gene, which was introduced into GM rice, positive DNA bands were found in October and December soils. However, no bar gene sequence was detected in PCR analysis with DNAs extracted from both cultured and uncultured soil bacterial fractions. The result of this study suggested that, in spite of seasonal variations of bacterial communities and persistence of the bar gene, the bacterial communities of the experimental rice field were not significantly affected by cultivation of GM rice varieties.     

Immunological and metabolomic impacts of administration of Cry1Ab protein and MON 810 maize in mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab-induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab-specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight "cultivar" effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.