Matthew L. Breeze

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.

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.

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.

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.

Investigation of biochemical diversity in a soybean lineage representing 35 years of breeding.

This paper reports an assessment of seed biochemical and metabolite variability and diversity in a series of nine soybean varieties; all lines share the same genetic lineage but represent ∼35 years of breeding (launch years 1972-2008) and differing yield potentials. These varieties, including six conventional and three glyphosate-tolerant lines, were grown concurrently at two replicated field sites in the United States during the 2011 growing season, and seeds were harvested at maturity. A compositional assessment included measurement of proximates, amino acids, fatty acids, tocopherols, isoflavones, saccharides, organic acids, and selected phytohormones. Statistical analysis included application of principal variance component analysis (PVCA) to investigate the interrelationships among compositional components from these soybean varieties and the impacts of location (environment) and pedigree on variability of these components. Results demonstrated that (i) some biochemical analytes showed trends (either increased or decreased) with launch year and/or yield, (ii) some analytes varied according to variety but showed no trend with launch year and/or yield, and (iii) almost all analytes showed extensive variation within and across sites. In summary, varietal development of high-yielding soybean, as represented in this study, has been accompanied by compositional changes but these are typically modest relative to environmental factors.

Introduction to Bayesian statistical approaches to compositional analyses of transgenic crops 1. Model validation and setting the stage.

Statistical comparisons of compositional data generated on genetically modified (GM) crops and their near-isogenic conventional (non-GM) counterparts typically rely on classical significance testing. This manuscript presents an introduction to Bayesian methods for compositional analysis along with recommendations for model validation. The approach is illustrated using protein and fat data from two herbicide tolerant GM soybeans (MON87708 and MON87708×MON89788) and a conventional comparator grown in the US in 2008 and 2009. Guidelines recommended by the US Food and Drug Administration (FDA) in conducting Bayesian analyses of clinical studies on medical devices were followed. This study is the first Bayesian approach to GM and non-GM compositional comparisons. The evaluation presented here supports a conclusion that a Bayesian approach to analyzing compositional data can provide meaningful and interpretable results. We further describe the importance of method validation and approaches to model checking if Bayesian approaches to compositional data analysis are to be considered viable by scientists involved in GM research and regulation.

Compositional analysis of grain and forage from MON 87427, an inducible male sterile and tissue selective glyphosate-tolerant maize product for hybrid seed production.

Conventional maize hybrid seed production has historically relied upon detasseling using either manual methods or semiautomated processes to ensure the purity of the hybrid cross. Monsanto Co. has developed biotechnology-derived MON 87427 maize with tissue-selective glyphosate tolerance to facilitate the production of hybrid maize seed. MON 87427 utilizes a specific promoter and intron combination to drive expression of CP4 EPSPS protein in vegetative and female reproductive tissues, conferring tolerance to glyphosate. This specific combination of regulatory elements also results in limited or no production of CP4 EPSPS protein in two key male reproductive tissues: pollen microspores, which develop into pollen grains, and tapetum cells that supply nutrients to the pollen. Thus, MON 87427 induces a male sterile phenotype after appropriately timed glyphosate applications. To confer additional benefits of herbicide tolerance and/or insect resistance, MON 87427 was combined with MON 89034 and NK603 by conventional breeding to develop MON 87427 × MON 89034 × NK603. The work described here is an assessment of the nutrient, antinutrient, and secondary metabolite levels in grain and forage tissues of MON 87427 and MON 87427 × MON 89034 × NK603. Results demonstrated that MON 87427 is compositionally equivalent to a near-isogenic conventional comparator. Results from this analysis established that the compositional equivalence observed for the single-event product MON 87427 is extendable to the combined-trait product, MON 87427 × MON 89034 × NK603. With increasing global demand for food production, the development of more efficient seed production strategies is important to sustainable agriculture. The study reported here demonstrated that biotechnology can be applied to simplify hybrid maize seed production without affecting crop composition.

Measurement of Vitamin K1 in Commercial Canola Cultivars from Growing Locations in North and South America Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Plant oils, including canola oil, are considered to be major sources of vitamin K as the second most substantial contributor of vitamin K to the human diet. Green leafy vegetables are the largest source of vitamin K. However, the effects of environment and germplasm on vitamin K levels in harvested canola seed have not been extensively investigated. To better understand these relationships, harvested canola seed from a range of diverse cultivars grown in different geographical locations in North and South America was assessed for levels of vitamin K. The analytical method developed to perform this measurement was based on C30 reversed-phase HPLC that could distinguish the biologically active trans-vitamin K1 from the inactive cis-isomer. Results demonstrated that for the majority of the canola cultivars evaluated, those cultivated in the North American sites had higher average vitamin K1 levels than those cultivated in the South American sites. Not all of the cultivars exhibited differences in response to the environment, suggesting that individual cultivar genetics also played a role in the variability of vitamin K1 levels observed in canola seed. Results from this study suggest that cultivar and environmental effects influence vitamin K1 levels in canola seed and provide a context to assess compositional variability of new cultivars.

Bayesian statistical approaches to compositional analyses of transgenic crops 2. Application and validation of informative prior distributions

Bayesian approaches to evaluation of crop composition data allow simpler interpretations than traditional statistical significance tests. An important advantage of Bayesian approaches is that they allow formal incorporation of previously generated data through prior distributions in the analysis steps. This manuscript describes key steps to ensure meaningful and transparent selection and application of informative prior distributions. These include (i) review of previous data in the scientific literature to form the prior distributions, (ii) proper statistical model specification and documentation, (iii) graphical analyses to evaluate the fit of the statistical model to new study data, and (iv) sensitivity analyses to evaluate the robustness of results to the choice of prior distribution. The validity of the prior distribution for any crop component is critical to acceptance of Bayesian approaches to compositional analyses and would be essential for studies conducted in a regulatory setting. Selection and validation of prior distributions for three soybean isoflavones (daidzein, genistein, and glycitein) and two oligosaccharides (raffinose and stachyose) are illustrated in a comparative assessment of data obtained on GM and non-GM soybean seed harvested from replicated field sites at multiple locations in the US during the 2009 growing season.