Efficacy of a cry1Ab Gene for Control of Maruca vitrata (Lepidoptera: Crambidae) in Cowpea (Fabales: Fabaceae).

Cowpea [Vigna unguiculata (L) Walp.] is an important staple legume in the diet of many households in sub-Saharan Africa. Its production, however, is negatively impacted by many insect pests including bean pod borer, Maruca vitrata F., which can cause 20-80% yield loss. Several genetically engineered cowpea events that contain a cry1Ab gene from Bacillus thuringiensis (Bt) for resistance against M. vitrata were evaluated in Nigeria, Burkina Faso, and Ghana (West Africa), where cowpea is commonly grown. As part of the regulatory safety package, these efficacy data were developed and evaluated by in-country scientists. The Bt-cowpea lines were planted in confined field trials under Insect-proof netting and artificially infested with up to 500 M. vitrata larvae per plant during bud formation and flowering periods. Bt-cowpea lines provided nearly complete pod and seed protection and in most cases resulted in significantly increased seed yield over non-Bt control lines. An integrated pest management strategy that includes use of Bt-cowpea augmented with minimal insecticide treatment for protection against other insects is recommended to control pod borer to enhance cowpea production. The insect resistance management plan is based on the high-dose refuge strategy where non-Bt-cowpea and natural refuges are expected to provide M. vitrata susceptible to Cry1Ab protein. In addition, there will be a limited release of this product until a two-toxin cowpea pyramid is released. Other than South African genetically engineered crops, Bt-cowpea is the first genetically engineered food crop developed by the public sector and approved for release in sub-Saharan Africa.

Physiological responses related to increased grain yield under drought in the first biotechnology-derived drought-tolerant maize.

Maize (Zea mays ssp. mays L.) is highly susceptible to drought stress. This work focused on whole-plant physiological mechanisms by which a biotechnology-derived maize event expressing bacterial cold shock protein B (CspB), MON 87460, increased grain yield under drought. Plants of MON 87460 and a conventional control (hereafter 'control') were tested in the field under well-watered (WW) and water-limited (WL) treatments imposed during mid-vegetative to mid-reproductive stages during 2009-2011. Across years, average grain yield increased by 6% in MON 87460 compared with control under WL conditions. This was associated with higher soil water content at 0.5 m depth during the treatment phase, increased ear growth, decreased leaf area, leaf dry weight and sap flow rate during silking, increased kernel number and harvest index in MON 87460 than the control. No consistent differences were observed under WW conditions. This indicates that MON 87460 acclimated better under WL conditions than the control by lowering leaf growth which decreased water use during silking, thereby eliciting lower stress under WL conditions. These physiological responses in MON 87460 under WL conditions resulted in increased ear growth during silking, which subsequently increased the kernel number, harvest index and grain yield compared to the control.

Composition of grain and forage from insect-protected and herbicide-tolerant corn, MON 89034 × TC1507 × MON 88017 × DAS-59122-7 (SmartStax), is equivalent to that of conventional corn (Zea mays L.).

Monsanto Company and Dow AgroSciences LLC have developed the combined-trait corn product MON 89034 × TC1507 × MON 88017 × DAS-59122-7 (SmartStax, a registered trademark of Monsanto Technology LLC). The combination of four biotechnology-derived events into a single corn product (stacking) through conventional breeding provides broad protection against lepidopteran and corn rootworm insect pests as well as tolerance to the glyphosate and glufosinate-ammonium herbicide families. The purpose of the work described here was to assess whether the nutrient, antinutrient, and secondary metabolite levels in grain and forage tissues of the combined-trait product are comparable to those in conventional corn. Compositional analyses were conducted on grain and forage from SmartStax, a near-isogenic conventional corn hybrid (XE6001), and 14 conventional reference hybrids, grown at multiple locations across the United States. No statistically significant differences between SmartStax and conventional corn were observed for the 8 components analyzed in forage and for 46 of the 52 components analyzed in grain. The six significant differences observed in grain components (p < 0.05) were assessed in context of the natural variability for that component. These results demonstrate that the stacked product, SmartStax, produced through conventional breeding of four single-event products containing eight proteins, is compositionally equivalent to conventional corn, as previously demonstrated for the single-event products.

Comments on the paper "A statistical assessment of differences and equivalences between genetically modified and reference plant varieties" by van der Voet et al. 2011.

van der Voet et al. (2011) describe statistical methodology that the European Food Safety Authority expects an applicant to adopt when making a GM crop regulatory submission. Key to their proposed methodology is the inclusion of reference varieties in the experimental design to provide a measure of natural variation amongst commercially grown crops. While taking proper account of natural variation amongst commercial varieties in the safety assessment of GM plants makes good sense, the methodology described by the authors is shown here to be fundamentally flawed and consequently cannot be considered fit for purpose in its current form.

Compositional equivalence of insect-protected glyphosate-tolerant soybean MON 87701 × MON 89788 to conventional soybean extends across different world regions and multiple growing seasons.

The soybean product MON 87701 × MON 89788 expresses both the cry1Ac gene derived from Bacillus thuringiensis and the cp4 epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene derived from Agrobacterium sp. strain CP4. Each biotechnology-derived trait confers specific benefits of insect resistance and glyphosate tolerance, respectively. The purpose of this study was to compare the composition of seed and forage from this combined-trait product to those of conventional soybean grown in geographically and climatically distinct regions. Field trials were conducted in the United States during the 2007 growing season, in Argentina during the 2007-2008 growing season, and in the northern and southern soybean regions of Brazil during the 2007-2008 and 2008-2009 growing seasons. Results demonstrated that the compositional equivalence of MON 87701 × MON 89788 to the conventional soybean extended across all regions and growing seasons. Further evaluation of the data showed that natural variation (region and growing season) contributed more to compositional variability in soybean, particularly for such components as isoflavones, fatty acids, and vitamin E, than transgene insertion.

Compositional variability in conventional and glyphosate-tolerant soybean (Glycine max L.) varieties grown in different regions in Brazil.

The compositions of a diverse range of commercially available conventional and genetically modified (GM; glyphosate-tolerant) soybean varieties from maturity groups 8 and 5, respectively, grown in the northern and southern soybean regions of Brazil during the 2007-2008 and 2008-2009 growing seasons were compared. Compositional analyses included measurement of essential macro- and micronutrients, antinutrients, and selected secondary metabolites in harvested seed as well as measurement of proximates in both forage and harvested seed. Statistical comparisons utilized a mixed analysis of variance model to evaluate the relative contributions of growing season, soybean growing region, production site, phenotype (GM or conventional), and variety. The study highlighted extensive variability in the overall data set particularly for components such as fatty acids, vitamin E, and isoflavones. There were few differences between the GM and non-GM populations, and most of the variability in the data set could be attributed to regional and variety differences. Overall, the results were consistent with the expanding literature on the lack of any meaningful impact of transgene insertion on crop composition.

Stability in the composition equivalence of grain from insect-protected maize and seed from glyphosate-tolerant soybean to conventional counterparts over multiple seasons, locations, and breeding germplasms.

Insect-protected maize MON 810 and Roundup Ready soybean 40-3-2 represent major milestones in the adoption of genetically modified (GM) crops to enhance agricultural productivity. This study provides an assessment of the compositional stability of these products over multiple seasons, multiple germplasms, and diverse geographies encompassing North, Central, and South America and Europe. The compositional assessment evaluated levels of proximates in MON 810 and proximates, antinutrients, and isoflavones in 40-3-2. The means and range values for component levels in the GM crops and their conventional comparators were consistently similar to each other within each corresponding year from 2000 to 2009. To our knowledge, this study represents the first meta-analysis of comparative composition assessments of GM products. This approach, combined with graphical approaches, provided an effective summary of the overall data set and confirmed the continued compositional equivalence of these important crops to their conventional counterparts over time.

Evaluation of compositional equivalence for multitrait biotechnology crops.

Compositional analysis is an important tool in the evaluation of the safety and nutritional status of biotechnology-derived crops. As part of the comparative assessment of a biotechnology-derived crop, its composition is evaluated by quantitative measurement of the levels of key nutrients, antinutrients, and secondary metabolites and compared to that of conventional crops. To evaluate the effect of combining multiple biotech traits through conventional breeding, the forage and grain compositions of the double combinations MON 810 × NK603, MON 863 × MON 810, and MON 863 × NK603 and the triple combination MON 863 × NK603 × MON 810 were compared to their respective near-isogenic, conventional control hybrids. Overall, a total of 241 statistical comparisons between the multitrait biotechnology crop and its corresponding conventional controls were conducted. Of these comparisons 192 (79.7%) were not statistically significantly different (p > 0.05), and all 49 of the differences were within the 99% tolerance interval for commercial hybrids grown in the same field or related field trials. These data on combined trait biotechnology-derived products demonstrated that the forage and grain were compositionally equivalent to their conventional comparators, indicating the absence of any influence of combining insect protection and herbicide tolerance traits by conventional breeding on compositional variation.

Dietary intake of stearidonic acid-enriched soybean oil increases the omega-3 index: randomized, double-blind clinical study of efficacy and safety.

BACKGROUND: The benefits of omega-3 (n-3) long-chain polyunsaturated fatty acids to heart health are well established. Stearidonic acid (SDA, 18:4n-3) may contribute to these benefits. OBJECTIVE: The objective was to evaluate the ability of SDA-containing soybean oil to increase the omega-3 index [erythrocyte eicosapentaenoic acid (EPA) + docosahexaenoic acid, as a percentage of total fatty acids] and to affect other cardiovascular disease risk markers compared with EPA and regular soy oil (control). DESIGN: This was a randomized, placebo-controlled, double-blind multicenter study in which 252 overweight subjects were randomly assigned to 1 of 3 treatments for 12 wk: 1 g encapsulated soybean oil/d plus 14.7 g liquid soybean oil/d to be mixed in food (control group), 1 g encapsulated EPA/d plus 14.7 g liquid soybean oil/d (EPA group), and 1 g encapsulated soybean oil/d plus 14.7 g liquid SDA-enriched soybean oil/d, providing 4.2 g SDA (SDA group). Subjects consumed treatment oils in exchange for other oils in their diet. RESULTS: The mean (±SE) baseline omega-3 index was similar between treatments, but after 12 wk of treatment values for this index were 4.15 ± 0.12%, 4.84 ± 0.13%, and 4.69 ± 0.15% for control, EPA, and SDA groups, respectively. Values for the EPA and SDA groups were greater than those for control subjects in the intent-to-treat population (P < 0.001 and P = 0.006, respectively). No adverse treatment-related effects of SDA-enriched soybean oil were reported. CONCLUSIONS: SDA-enriched soybean oil increased the omega-3 index by raising erythrocyte EPA concentrations. SDA-enriched soybean oil is a land-based n-3 fatty acid that is a sustainable approach to increasing tissue concentrations of long-chain polyunsaturated n-3 fatty acids.

Compositions of forage and seed from second-generation glyphosate-tolerant soybean MON 89788 and insect-protected soybean MON 87701 from Brazil are equivalent to those of conventional soybean (Glycine max).

Brazil has become one of the largest soybean producers. Two Monsanto Co. biotechnology-derived soybean products are designed to offer benefits in weed and pest management. These are second-generation glyphosate-tolerant soybean, MON 89788, and insect-protected soybean, MON 87701. The second-generation glyphosate-tolerant soybean product, MON 89788, contains the 5-enolpyruvylshikimate-3-phosphate synthase gene derived from Agrobacterium sp. strain CP4 (cp4 epsps). MON 87701 contains the cry1Ac gene and expression of the Cry1Ac protein providing protection from feeding damage caused by certain lepidopteran insect pests. The purpose of this assessment was to determine whether the compositions of seed and forage of MON 89788 and MON 87701 are comparable to those of conventional soybean grown in two geographically and climatically distinct regions in multiple replicated sites in Brazil during the 2007-2008 growing season. Overall, results demonstrated that the seed and forage of MON 89788 and MON 87701 are compositionally equivalent to those of conventional soybean. Strikingly, the results also showed that differences in mean component values of forage and seed from the two controls grown in the different geographical regions were generally greater than that observed in test and control comparisons. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of compositional data generated on MON 89788, MON 87701, and their respective region-specific controls provide a graphical illustration of how natural variation contributes more than biotechnology-driven genetic modification to compositional variability in soybean. Levels of isoflavones and fatty acids were particularly variable.

Compositions of seed, forage, and processed fractions from insect-protected soybean MON 87701 are equivalent to those of conventional soybean.

Monsanto Co. has developed biotechnology-derived, insect-protected soybean MON 87701 that produces the Cry1Ac insecticidal crystal (delta-endotoxin) protein derived from Bacillus thuringiensis (Bt) subsp. kurstaki. Cry1Ac provides protection from feeding damage caused by certain targeted lepidopteran pests. The purpose of this work was to assess whether the compositions of seed, forage, and processed fractions (meal, oil, protein isolate, and lecithin) of MON 87701 are comparable to those of conventional soybean. Compositional analyses were conducted on seed and forage tissues harvested from MON 87701 and conventional soybean grown in multiple replicated sites in the United States during the 2007 growing season and in Argentina during the 2007-2008 growing season. Seed, forage, and processed fractions from conventional soybean varieties currently in the marketplace were included in the analyses to establish a range of natural variability for each compositional component; the range of variability was defined by a 99% tolerance interval. Additional seed was collected from soybean grown in a separate U.S. production during the 2007 season. This seed and processed fractions (meal, oil, protein isolate, and crude lecithin) derived from it were also subjected to compositional analyses. Forage samples were analyzed for levels of proximates (ash, fat, moisture, and protein), carbohydrates by calculation, and fiber. Seed samples were analyzed for proximates, carbohydrates by calculation, fiber, amino acids, fatty acids, antinutrients, and vitamin E. Toasted, defatted (TD) meal was analyzed for proximates, fiber, amino acids, and antinutrients. Refined, bleached, and deodorized (RBD) oil was analyzed for fatty acids and vitamin E. Protein isolate was analyzed for amino acids and moisture. Crude lecithin was analyzed for phosphatides. Overall, results demonstrated that the seed, forage, and processed fractions of MON 87701 are compositionally equivalent to those of conventional soybean.

The forage and grain of MON 87460, a drought-tolerant corn hybrid, are compositionally equivalent to that of conventional corn.

MON 87460 contains a gene that expresses cold shock protein B (CSPB) from Bacillus subtilis. Expression of this gene confers a yield advantage when yield is limited by water availability. Compositional analyses of MON 87460 and a conventional corn variety with similar background genetics were conducted on forage and grain harvested from multiple replicated field sites across the United States during the 2006 growing season and across Chile during the 2006-2007 growing season. The U.S. field trials were conducted under typical agronomic practices, whereas the Chilean field trials incorporated a strip-plot design that included well-watered and water-limited treatments. Results demonstrated that levels of the components analyzed were comparable between MON 87460, the conventional control, and the commercially available corn hybrids.

Stearidonic acid-enriched soybean oil increased the omega-3 index, an emerging cardiovascular risk marker.

A plant source of omega-3 fatty acid (FA) that can raise tissue eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) is needed. A soybean oil (SBO) containing approximately 20% stearidonic acid [SDA; the delta-6 desaturase product of alpha-linolenic acid (ALA)] derived from genetically modified soybeans is under development. This study compared the effects of EPA to SDA-SBO on erythrocyte EPA+DHA levels (the omega-3 index). Overweight healthy volunteers (n=45) were randomized to SDA-SBO (24 ml/day providing approximately 3.7 g SDA) or to regular SBO (control group) without or with EPA ethyl esters (approximately 1 g/day) for 16 weeks. Serum lipids, blood pressure, heart rate, platelet function and safety laboratory tests were measured along with the omega-3 index. A per-protocol analysis was conducted on 33 subjects (11 per group). Compared to baseline, average omega-3 index levels increased 19.5% in the SDA group and 25.4% in the EPA group (p<0.05 for both, vs. control). DHA did not change in any group. Relative to EPA, SDA increased RBC EPA with about 17% efficiency. No other clinical endpoints were affected by SDA or EPA treatment (vs. control). In conclusion, SDA-enriched SBO significantly raised the omega-3 index. Since EPA supplementation has been shown to raise the omega-3 index and to lower risk for cardiac events, SDA-SBO may be a viable plant-based alternative for providing meaningful intakes of cardioprotective omega-3 FAs.

Composition of grain, forage, and processed fractions from second-generation glyphosate-tolerant soybean, MON 89788, is equivalent to that of conventional soybean (Glycine max L.).

Developments in biotechnology and molecular-assisted breeding have led to the development of a second-generation glyphosate-tolerant soybean product, MON 89788. The MON 89788 event was produced by direct transformation of a cp4 epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene cassette derived from Agrobacterium sp. strain CP4 into an elite soybean germplasm known for its superior agronomic characteristics and high yielding property. The purpose of this work was to assess whether the nutrient and antinutrient levels in seed and forage tissues of MON 89788 are comparable to those in the conventional soybean variety, A3244, which has background genetics similar to MON 89788 but does not contain the cp4 epsps gene cassette. Additional conventional soybean varieties currently in the marketplace were also included in the analysis to establish a range of natural variability for each analyte, where the range of variability is defined by a 99% tolerance interval for that particular analyte. Compositional analyses were conducted on forage, seed and four processed fractions from soybeans grown in ten sites across both the United States and Argentina during the 2004-2005 growing seasons. Forage samples were analyzed for levels of proximates (ash, fat, moisture, and protein) and fiber. Seed samples were analyzed for proximates, fiber, antinutrients, and vitamin E. Defatted, toasted (DT) meal was analyzed for proximates, fiber, amino acids, and antinutrients. Refined, bleached, and deodorized (RBD) oil was analyzed for fatty acids and vitamin E. Protein isolate was analyzed for amino acids and moisture. Crude Lecithin was analyzed for phosphatides. Results of the comparisons indicate that MON 89788 is compositionally and nutritionally equivalent to conventional soybean varieties currently in commerce.

Composition of forage and grain from second-generation insect-protected corn MON 89034 is equivalent to that of conventional corn (Zea mays L.).

Insect-protected corn hybrids containing Cry insecticidal proteins derived from Bacillus thuringiensis have protection from target pests and provide effective management of insect resistance. MON 89034 hybrids have been developed that produce both the Cry1A.105 and Cry2Ab2 proteins, which provide two independent modes of insecticidal action against the European corn borer ( Ostrinia nubilalis ) and other lepidopteran insect pests of corn. The composition of MON 89034 corn was compared to conventional corn by measuring proximates, fiber, and minerals in forage and by measuring proximates, fiber, amino acids, fatty acids, vitamins, minerals, antinutrients, and secondary metabolites in grain collected from 10 replicated field sites across the United States and Argentina during the 2004-2005 growing seasons. Analyses established that the forage and grain from MON 89034 are compositionally comparable to the control corn hybrid and conventional corn reference hybrids. These findings support the conclusion that MON 89034 is compositionally equivalent to conventional corn hybrids.

Chemical composition of glyphosate-tolerant soybean 40-3-2 grown in Europe remains equivalent with that of conventional soybean (Glycine max L.).

The composition of glyphosate-tolerant (Roundup Ready) soybean 40-3-2 was compared with that of conventional soybean grown in Romania in 2005 as part of a comparative safety assessment program. Samples were collected from replicated field trials, and compositional analyses were performed to measure proximates (moisture, fat, ash, protein, and carbohydrates by calculation), fiber, amino acids, fatty acids, isoflavones, raffinose, stachyose, phytic acid, trypsin inhibitor, and lectin in grain as well as proximates and fiber in forage. The mean values for all biochemical components assessed for Roundup Ready soybean 40-30-2 were similar to those of the conventional control and were within the published range observed for commercial soybean. The compositional profile of Roundup Ready soybean 40-3-2 was also compared to that of conventional soybean varieties grown in Romania by calculating a 99% tolerance interval to describe compositional variability in the population of traditional soybean varieties already on the marketplace. These comparisons, together with the history of the safe use of soybean as a common component of animal feed and human food, lead to the conclusion that Roundup Ready soybean 40-3-2 is compositionally equivalent to and as safe and nutritious as conventional soybean varieties grown commercially.

Glyphosate-tolerant alfalfa is compositionally equivalent to conventional alfalfa (Medicago sativa L.).

Glyphosate-tolerant alfalfa (GTA) was developed to withstand over-the-top applications of glyphosate, the active ingredient in Roundup agricultural herbicides. As a part of the safety assessment, GTA (designated J101 x J163) was grown under controlled field conditions at geographically diverse locations within the United States during the 2001 and 2003 field seasons along with control and other conventional alfalfa varieties for compositional assessment. Field trials were conducted using a randomized complete block design with four replication blocks at each site. Alfalfa forage was harvested at the late bud to early bloom stage from each plot at five field sites in 2001 (establishment year) and from four field sites in 2003 (third year of stand). The concentration of proximate constituents, fibers, amino acids, coumestrol, and minerals in the forage was measured. The results showed that the forage from GTA J101 x J163 is compositionally equivalent to forage from the control and conventional alfalfa varieties.

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation.

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.

The value of short amino acid sequence matches for prediction of protein allergenicity.

Typically, genetically engineered crops contain traits encoded by one or a few newly expressed proteins. The allergenicity assessment of newly expressed proteins is an important component in the safety evaluation of genetically engineered plants. One aspect of this assessment involves sequence searches that compare the amino acid sequence of the protein to all known allergens. Analyses are performed to determine the potential for immunologically based cross-reactivity where IgE directed against a known allergen could bind to the protein and elicit a clinical reaction in sensitized individuals. Bioinformatic searches are designed to detect global sequence similarity and short contiguous amino acid sequence identity. It has been suggested that potential allergen cross-reactivity may be predicted by identifying matches as short as six to eight contiguous amino acids between the protein of interest and a known allergen. A series of analyses were performed, and match probabilities were calculated for different size peptides to determine if there was a scientifically justified search window size that identified allergen sequence characteristics. Four probability modeling methods were tested: (1) a mock protein and a mock allergen database, (2) a mock protein and genuine allergen database, (3) a genuine allergen and genuine protein database, and (4) a genuine allergen and genuine protein database combined with a correction for repeating peptides. These analyses indicated that searches for short amino acid sequence matches of eight amino acids or fewer to identify proteins as potential cross-reactive allergens is a product of chance and adds little value to allergy assessments for newly expressed proteins.