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.     

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).     

Fate of genetically modified maize and conventional rapeseed,and endozoochory in wild boar (Sus scrofa)

Feeding experiments were carried out to investigate the digestive fate of transgenic DNA and novel protein in wild boar applying polymerase chain reaction (PCR) and immunodiagnostic techniques. Furthermore, the dispersal of viable maize and rapeseed (endozoochory) was studied. A diet containing conventional rapeseed, and either genetically modified (GM) maize expressing Cry1Ab protein (Bt176) or non-GM isogenic maize was offered. By conventional and quantitative PCR both chloroplast-specific plant DNA (rubisco) and cry1Ab gene fragments were detected only in gastrointestinal content. Using an enzyme-linked immunosorbent assay (ELISA) positive signals of immunoactive Cry1Ab protein were detected in digesta samples. Analysis of endozoochory showed that excreted maize seeds retain their germination capacity only in extremely rare cases and no intact rapeseed was found in faeces. A possible dispersal of viable seeds by wild boars is highly unlikely.     

Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure

A glasshouse experiment was undertaken to provide baseline data on the variation between conventional maize (Zea mays L.) varieties and genetically modified maize plants expressing the insecticidal Bacillus thuringiensis protein (Bt, Cry1Ab). The objective was to determine whether the variation in soil parameters under a range of conventional maize cultivars exceeded the differences between Bt and non-Bt maize cultivars. Variations in plant growth parameters (shoot and root biomass, percentage carbon, percentage nitrogen), Bt protein concentration in shoots, roots and soil, soil nematode abundance and soil microbial community structure were determined. Eight paired varieties (i.e. varieties genetically modified to express Bt protein and their near-isogenic control varieties) were investigated, together with a Bt variety for which no near-isogenic control was available (NX3622, a combined transformant expressing both Bt and herbicide tolerance) and a conventional barley (Hordeum vulgare L.) variety which was included as a positive control. The only plant parameter which showed a difference between Bt varieties and near-isogenic counterparts was the shoot carbon to nitrogen ratio; this was observed for only two of the eight varieties, and so was not attributable to the Bt trait. There were no detectable differences in the concentration of Bt protein in plant or soil with any of the Bt-expressing varieties. There were significant differences in the abundance of soil nematodes, but this was not related to the Bt trait. Differences in previously published soil nematode studies under Bt maize were smaller than these varietal effects. Soil microbial community structure, as determined by phospholipid fatty acid (PLFA) analysis, was strongly affected by plant growth stage but not by the Bt trait. The experimental addition of purified Cry1Ab protein to soil confirmed that, at ecologically relevant concentrations, there were no measurable effects on microbial community structure.     

加工产品中转基因玉米Bt11成分实时荧光PCR定量(性)检测

实验在玉米自身基因和外源基因的边界序列之间设计了具有品种和品系特异性的引物和探针 ,并以实时荧光PCR技术 ,建立了加工产品中转基因玉米Bt1 1成分品系鉴定检测和定量检测的方法。实验对加热条件和时间对检测转基因成分的影响作了探讨 ,并检测了部分市售食品和饲料。检测结果发现 ,加热时间温度越高、时间越长 ,对转基因成分定量检测的影响越大 ;在所检测的样品中可以检测出转基因玉米Bt1 1成分 ,有些样品还同时检出其他转基因成分。本研究实验建立的方法 ,可以用于加工产品中转基因成分的定量检测 ,也可以用于定性检测 ,或作为常规PCR定性检测后的确证实验方法。     

Testing coexistence and genetic containment for an autogamous crop

Is there any risk that the threshold for admixture of genetically modified seeds in the harvest of a conventional cultivar, 0.9% in Europe, will be exceeded in the case of inbreeder crops? Using herbicide-resistant foxtail millet, Setaria italica, as a model of a preferentially autogamous crop, such as wheat and rice, field experiments show that genotype admixture due to pollen flow between adjacent fields is about 0.03% on average for the 10 adjacent meters, and 10 times less in the next 20-m lane. In the case of a maternally inherited resistance gene, the admixture rate is at least 100 times lower. Recessive herbicide resistance has also been tested but would be efficient only if the agreed coexistence rules were based on phenotype detection.     

A Comparison of Soil Microbial Community Structure, Protozoa and Nematodes in Field Plots of Conventional and Genetically Modified Maize Expressing the Bacillus thuringiens is CryIAb Toxin

Field trials were established at three European sites (Denmark, Eastern France, South-West France) of genetically modified maize (Zea mays L.) expressing the CryIAb Bacillus thuringiensis toxin (Bt), the near-isogenic non-Bt cultivar, another conventional maize cultivar and grass. Soil from Denmark was sampled at sowing (May) and harvest (October) over two years (2002, 2003); from E France at harvest 2002, sowing and harvest 2003; and from SW France at sowing and harvest 2003. Samples were analysed for microbial community structure (2003 samples only) by community-level physiological-profiling (CLPP) and phospholipid fatty acid analysis (PLFA), and protozoa and nematodes in all samples. Individual differences within a site resulted from: greater nematode numbers under grass than maize on three occasions; different nematode populations under the conventional maize cultivars once; and two occasions when there was a reduced protozoan population under Bt maize compared to non-Bt maize. Microbial community structure within the sites only varied with grass compared to maize, with one occurrence of CLPP varying between maize cultivars (Bt versus a conventional cultivar). An overall comparison of Bt versus non-Bt maize across all three sites only revealed differences for nematodes, with a smaller population under the Bt maize. Nematode community structure was different at each site and the Bt effect was not confined to specific nematode taxa. The effect of the Bt maize was small and within the normal variation expected in these agricultural systems.     

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.     

转基因玉米Bt176液相芯片检测方法的建立

为建立转基因玉米Bt176的液相芯片检测方法,根据已公布的转基因玉米Bt176外源插入基因CaMV35S启动子序列,外源基因3’端与玉米基因组DNA连接区序列,同时以玉米特异Zein内源基因序列为参照,利用Primer Premier5.0等软件设计特异性引物和探针。将探针与荧光编码微球偶联后,与PCR产物杂交反应,用液相芯片检测仪(Bio-plex 200)检测荧光信号。检测结果显示,该方法具有高特异性及灵敏度,各条探针之间无交叉反应,最低检测限可达0.01%。初步建立了检测转基因玉米Bt176的液相芯片技术,为其他转基因作物的快速高通量检测提供了借鉴和经验。     

大田环境下转Bt基因玉米对土壤酶活性的影响

在大田自然条件下,比较研究了转Bt基因玉米和非转基因亲本玉米在种植和秸秆分解时对土壤酶活性影响的差异。结果表明,与亲本非转基因玉米相比,在各生育期内种植转Bt玉米对土壤蛋白酶和土壤脲酶活性均没有显著影响;在喇叭口期和抽雄期,土壤蔗糖酶和土壤酸性磷酸酶活性显著提高。在秸秆还田后,两种玉米秸秆对土壤酸性磷酸酶活性的影响没有显著差异,但使用转Bt玉米秸秆的土壤蔗糖酶、土壤脲酶和土壤蛋白酶的活性则有显著提高。与亲本玉米相比,在所有观测期内,种植Bt玉米及秸秆还田对土壤酶活性的影响,在影响的幅度及趋势上随玉米生育期和土壤酶种类的不同而产生差异,但没有观测到显著不利影响;商业化Bt玉米的环境释放仍有待长期定位观测和评价。     

Effect of Stacked Insecticidal Cry Proteins from Maize Pollen on Nurse Bees (Apis mellifera carnica) and Their Gut Bacteria

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees.     

The Cry1Ab Protein Has Minor Effects on the Arbuscular Mycorrhizal Fungal Communities after Five Seasons of Continuous Bt Maize Cultivation

The cultivation of genetically modified plants (GMP) has raised concerns regarding the plants’ ecological safety. A greenhouse experiment was conducted to assess the impact of five seasons of continuous Bt (Bacillus thuringiensis) maize cultivation on the colonisation and community structure of the non-target organisms arbuscular mycorrhizal fungi (AMF) in the maize roots, bulk soils and rhizospheric soils using the terminal restriction fragment length polymorphism (T-RFLP) analysis of the 28S ribosomal DNA and sequencing methods. AMF colonisation was significantly higher in the two Bt maize lines that express Cry1Ab, 5422Bt1 (event Bt11) and 5422CBCL (MON810) than in the non-Bt isoline 5422. No significant differences were observed in the diversity of the AMF community between the roots, bulk soils and rhizospheric soils of the Bt and non-Bt maize cultivars. The AMF genus Glomus was dominant in most of the samples, as detected by DNA sequencing. A clustering analysis based on the DNA sequence data suggested that the sample types (i.e., the samples from the roots, bulk soils or rhizospheric soils) might have greater influence on the AMF community phylotypes than the maize cultivars. This study indicated that the Cry1Ab protein has minor effects on the AMF communities after five seasons of continuous Bt maize cultivation.     

Detection limits of the strip test and PCR for genetically modified corn in Brazil

Brazilian legislation establishes a labeling limit for products that contain more than 1% material from genetically modified organisms (GMOs). We assessed the sensitivity of the lateral flow strip test in detection of the GMO corn varieties Bt11 and MON810 and the specificity and sensitivity of PCR techniques for their detection. For the strip test, the GMO seeds were mixed with conventional seeds at levels of 0.2, 0.4 and 0.8% for Bt11, and 0.4, 0.8 and 1.6% for MON810. Three different methodologies were assessed and whole seeds, their endosperm and embryonic axis were used. For the PCR technique, the GMO seeds of each of the two varieties were mixed with conventional seeds at levels of 20, 10, 5, 2, 1, and 0.5%. The seeds were ground and the DNA extracted. For detection of the GMO material, specific primers were used for MON810 and Bt11 and maize zein as an endogenous control. The sensitivity of the strip test varied for both maize varieties and methodologies. The test was positive for Bt11 only at 0.8%, in contrast with the detection limit of 0.4% indicated by the manufacturer. In the multiplex PCR, the primers proved to be specific for the different varieties. These varieties were detected in samples with one GMO seed in 100. Thus, this technique proved to be efficient in detecting contaminations equal to or greater than 1%.     

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.     

Transgenic Bacillus thuringiensis (Bt) Rice Is Safer to Aquatic Ecosystems than Its Non-Transgenic Counterpart

Rice lines genetically modified with the crystal toxin genes from Bacillus thuringiensis (Bt) have experienced rapid development, with biosafety certificates for two Bt rice lines issued in 2009. There has still been no commercial release of these lines yet due to public concerns about human health and environmental risks. Some studies confirmed that Bt rice was as safe as conventional rice to non-target organisms when pesticides were not applied, however, pesticides are still required in Bt rice to control non-lepidopteran pests. In this study, we assessed the environmental effects of two Bt rice lines expressing either the cry1Ab/1Ac or cry2A genes, respectively, by using zooplanktons as indicator species under normal field management practices using pesticides when required. In the whole rice growing season, non-Bt rice was sprayed 5 times while Bt rice was sprayed 2 times, which ensured both rice achieved a normal yield. Field investigations showed that rice type (Bt and non-Bt) significantly influenced zooplankton abundance and diversity, which were up to 95% and 80% lower in non-Bt rice fields than Bt rice fields. Laboratory rearing showed that water from non-Bt rice fields was significantly less suitable for the survival and reproduction of Daphnia magna and Paramecium caudatum in comparison with water from Bt rice fields. Higher pesticide residues were detected in the water from non-Bt than Bt rice fields, accounting for the bad performance of zooplankton in non-Bt field water. Our results demonstrate that Bt rice is safer to aquatic ecosystems than non-Bt rice, and its commercialization will be beneficial for biodiversity restoration in rice-based ecosystems.     

Farm‐scale evaluation of the impact of Cry1Ab Bt maize on canopy nontarget arthropods: a 3‐year study

The cultivation of Cry1Ab‐expressing genetically modified MON810 (Bt maize) has led to public concern in Europe, regarding its impact on nontarget arthropods (NTAs). We have assessed the potential effects of DKC 6451 YG (MON810) maize on canopy NTAs in a farm‐scale study performed in Central Spain during 3 years. The study focused on hemipteran herbivores (leafhoppers and planthoppers) and hymenopteran parasitic wasps (mymarids) collected by yellow sticky traps, which accounted for 72% of the total number of insects studied. The dynamics and abundance of these groups varied among years, but no significant differences were found between Bt and non‐Bt maize, indicating that Bt maize had no negative effect on these taxa. Nonetheless, the Cry1Ab toxin was detected in 2 different arthropods collected from Bt maize foliage, the cicadellids Zyginidia scutellaris and Empoasca spp. A retrospective power analysis on the arthropod abundance data for our field trials has determined that Z. scutellaris and the family Mymaridae have high capacity to detect differences between the Bt maize and its isogenic counterpart. The use of these canopy NTAs as surrogates for assessing environmental impacts of Bt maize is discussed.     

A Sensitive Method to Monitor <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Streptomyces coelicor</Emphasis>-related Bacteria in Maize Rhizobacterial Communities: The Use of Genome-Wide Microarrays

Commercially available DNA microarrays containing genome-wide spotted oligonucleotides encompass the soil bacteria Bacillus subtilis or Streptomyces coelicolor genomes. These have been used to analyse potential differences in rhizobacterial communities of transgenic maize engineered to express the Bacillus thuringensis Cry toxin (Bt maize) in three different agricultural soils. No differences in hybridisation were observed between genetically and non-genetically modified maize rhizobacteria from two Bt lines with a detection sensitivity of five copies of a particular gene above the background. Soil-specific hybridisation results were obtained when rhizobacterial DNA was compared to the corresponding genomic DNA spotted in the microarrays suggesting that the use of genome-wide DNA arrays could serve as a useful tool for the molecular monitoring of rhizobacterial communities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Field-evolved resistance to Bt maize by western corn rootworm: predictions from the laboratory and effects in the field

Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) provide an effective management tool for many key insect pests. However, pest species have repeatedly demonstrated their ability to adapt to management practices. Results from laboratory selection experiments illustrate the capacity of pest species to evolve Bt resistance. Furthermore, resistance has been documented to Bt sprays in the field and greenhouse, and more recently, by some pests to Bt crops in the field. In 2009, fields were discovered in Iowa (USA) with populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, that had evolved resistance to maize that produces the Bt toxin Cry3Bb1. Fields with resistant insects in 2009 had been planted to Cry3Bb1 maize for at least three consecutive years and as many as 6years. Computer simulation models predicted that the western corn rootworm might evolve resistance to Bt maize in as few as 3years. Laboratory and field data for interactions between western corn rootworm and Bt maize indicate that currently commercialized products are not high-dose events, which increases the risk of resistance evolution because non-recessive resistance traits may enhance survival on Bt maize. Furthermore, genetic analysis of laboratory strains of western corn rootworm has found non-recessive inheritance of resistance. Field studies conducted in two fields identified as harboring Cry3Bb1-resistant western corn rootworm found that survival of western corn rootworm did not differ between Cry3Bb1 maize and non-Bt maize and that root injury to Cry3Bb1 maize was higher than injury to other types of Bt maize or to maize roots protected with a soil insecticide. These first cases of field-evolved resistance to Bt maize by western corn rootworm provide an early warning and point to the need to apply better integrated pest management practices when using Bt maize to manage western corn rootworm.     

The web-building spider Theridion impressum (Araneae: Theridiidae) is not adversely affected by Bt maize resistant to corn rootworms

The growth of genetically engineered maize that produces the insecticidal protein Cry3Bb1 from Bacillus thuringiensis ( Bt ) is an effective method to control corn rootworms ( Diabrotica spp.), which are threatening maize production in North America and Europe. In this study, the risk of Cry3Bb1-expressing maize for the predatory spider Theridion impressum , a common species in European maize fields, was assessed. Quantification of Cry3Bb1 in potential prey species collected in Bt maize plots and prey spectrum analysis revealed that T. impressum ingests Cry3Bb1 in the field. Exposure to the Bt protein, however, was highly variable because some potential prey species, such as phloem-feeding herbivores and predators, contained little or no Cry3Bb1, whereas leaf-feeding herbivores contained high concentrations. Adult and juvenile T. impressum spiders were fed with Cry3Bb1-containing food (prey or maize pollen) for 8 weeks in the laboratory to examine the toxicity of the Bt protein. No differences in mortality, weight development or offspring production were observed between spiders provided with food containing or not containing Cry3Bb1. Retrospective power analysis indicated that the bioassays were sufficiently sensitive to detect meaningful differences if present. Although Cry3Bb1 is ingested by the spider in the field, our data provide no evidence for toxicity. Consequently, the growth of corn rootworm-resistant Bt maize appears to pose no risk for T. impressum .     

Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods

A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in genetically modified soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the efficiency of all reactions. This multiplex PCR has constituted the basis for an efficient platform for genetically modified organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High specificity and sensitivity were obtained.