Dicamba-Tolerant Soybeans (Glycine max L.) MON 87708 and MON 87708 × MON 89788 Are Compositionally Equivalent to Conventional Soybean.

Herbicide-tolerant crops can expand both tools for and timing of weed control strategies. MON 87708 soybean has been developed through genetic modification and confers tolerance to the dicamba herbicide. As part of the safety assessment conducted for new genetically modified (GM) crop varieties, a compositional assessment of MON 87708 was performed. Levels of key soybean nutrients and anti-nutrients in harvested MON 87708 were compared to levels of those components in a closely related non-GM variety as well as to levels measured in other conventional soybean varieties. From this analysis, MON 87708 was shown to be compositionally equivalent to its comparator. A similar analysis conducted for a stacked trait product produced by conventional breeding, MON 87708 × MON 89788, which confers tolerance to both dicamba and glyphosate herbicides, reached the same conclusion. These results are consistent with other results that demonstrate no compositional impact of genetic modification, except in those cases where an impact was an intended outcome.

Stacking transgenic event DAS-Ø15Ø7-1 alters maize composition less than traditional breeding.

The impact of crossing ('stacking') genetically modified (GM) events on maize-grain biochemical composition was compared with the impact of generating nonGM hybrids. The compositional similarity of seven GM stacks containing event DAS-Ø15Ø7-1, and their matched nonGM near-isogenic hybrids (iso-hybrids) was compared with the compositional similarity of concurrently grown nonGM hybrids and these same iso-hybrids. Scatter plots were used to visualize comparisons among hybrids and a coefficient of identity (per cent of variation explained by line of identity) was calculated to quantify the relationships within analyte profiles. The composition of GM breeding stacks was more similar to the composition of iso-hybrids than was the composition of nonGM hybrids. NonGM breeding more strongly influenced crop composition than did transgenesis or stacking of GM events. These findings call into question the value of uniquely requiring composition studies for GM crops, especially for breeding stacks composed of GM events previously found to be compositionally normal.

Evaluation of metabolomics profiles of grain from maize hybrids derived from near-isogenic GM positive and negative segregant inbreds demonstrates that observed differences cannot be attributed unequivocally to the GM trait.

INTRODUCTION: Past studies on plant metabolomes have highlighted the influence of growing environments and varietal differences in variation of levels of metabolites yet there remains continued interest in evaluating the effect of genetic modification (GM). OBJECTIVES: Here we test the hypothesis that metabolomics differences in grain from maize hybrids derived from a series of GM (NK603, herbicide tolerance) inbreds and corresponding negative segregants can arise from residual genetic variation associated with backcrossing and that the effect of insertion of the GM trait is negligible. METHODS: Four NK603-positive and negative segregant inbred males were crossed with two different females (testers). The resultant hybrids, as well as conventional comparator hybrids, were then grown at three replicated field sites in Illinois, Minnesota, and Nebraska during the 2013 season. Metabolomics data acquisition using gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) allowed the measurement of 367 unique metabolite features in harvested grain, of which 153 were identified with small molecule standards. Multivariate analyses of these data included multi-block principal component analysis and ANOVA-simultaneous component analysis. Univariate analyses of all 153 identified metabolites was conducted based on significance testing (α = 0.05), effect size evaluation (assessing magnitudes of differences), and variance component analysis. RESULTS: Results demonstrated that the largest effects on metabolomic variation were associated with different growing locations and the female tester. They further demonstrated that differences observed between GM and non-GM comparators, even in stringent tests utilizing near-isogenic positive and negative segregants, can simply reflect minor genomic differences associated with conventional back-crossing practices. CONCLUSION: The effect of GM on metabolomics variation was determined to be negligible and supports that there is no scientific rationale for prioritizing GM as a source of variation.

Metabolomic Assessment of Key Maize Resources: GC-MS and NMR Profiling of Grain from B73 Hybrids of the Nested Association Mapping (NAM) Founders and of Geographically Diverse Landraces.

The present study expands metabolomic assessments of maize beyond commercial lines to include two sets of hybrids used extensively in the scientific community. One set included hybrids derived from the nested association mapping (NAM) founder lines, a collection of 25 inbreds selected on the basis of genetic diversity and used to investigate the genetic basis of complex plant traits. A second set included 24 hybrids derived from a collection of landraces representative of native diversity from North and South America that may serve as a source of new alleles for improving modern maize hybrids. Metabolomic analysis of grain harvested from these hybrids utilized gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) and (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) techniques. Results highlighted extensive metabolomic variation in grain from both hybrid sets, but also demonstrated that, within each hybrid set, subpopulations could be differentiated in a pattern consistent with the known genetic and compositional variation of these lines. Correlation analysis did not indicate a strong association of the metabolomic data with grain nutrient composition, although some metabolites did show moderately strong correlations with agronomic features such as plant and ear height. Overall, this study provides insights into the extensive metabolomic diversity associated with conventional maize germplasm.

Global Adoption of Genetically Modified (GM) Crops: Challenges for the Public Sector.

Advances in biotechnology continue to drive the development of a wide range of insect-protected, herbicide-tolerant, stress-tolerant, and nutritionally enhanced genetically modified (GM) crops, yet societal and public policy considerations may slow their commercialization. Such restrictions may disproportionately affect developing countries, as well as smaller entrepreneurial and public sector initiatives. The 2014 IUPAC International Congress of Pesticide Chemistry (San Francisco, CA, USA; August 2014) included a symposium on "Challenges Associated with Global Adoption of Agricultural Biotechnology" to review current obstacles in promoting GM crops. Challenges identified by symposium presenters included (i) poor public understanding of GM technology and the need for enhanced communication strategies, (ii) nonharmonized and prescriptive regulatory requirements, and (iii) limited experience with regulations and product development within some public sector programs. The need for holistic resistance management programs to enable the most effective use of insect-protected crops was also a point of emphasis. This paper provides details on the symposium discussion and provides background information that can be used in support of further adoption of beneficial GM crops. Overall, it emphasizes that global adoption of modern agricultural biotechnology has not only provided benefits to growers and consumers but has great potential to provide solutions to an increasing global population and diminishing agricultural land. This potential will be realized by continued scientific innovation, harmonized regulatory systems, and broader communication of the benefits of the high-yielding, disease-resistant, and nutritionally enhanced crops attainable through modern biotechnology.

Maize hybrids derived from GM positive and negative segregant inbreds are compositionally equivalent: any observed differences are associated with conventional backcrossing practices.

In this study, we show that compositional differences in grain harvested from genetically modified (GM) maize hybrids derived from near-isogenic trait-positive and trait-negative segregant inbreds are more likely related to backcrossing practices than to the GM trait. To demonstrate this, four paired GM trait-positive (NK603: herbicide tolerance) and trait-negative near-isogenic inbred male lines were generated. These were crossed with two different females (testers) to create a series of trait-positive and trait-negative hybrid variants. The hypothesis was, that compositional variation within the hybrid variants would reflect differences associated with backcrossing practices and provide context to any observed differences between GM and non-GM hybrids. The F1 hybrids, as well as corresponding conventional comparator hybrids, were grown concurrently at four field sites across the United States during the 2013 season. Grain was harvested for compositional analysis; proximates (protein, starch, and oil), amino acids, fatty acids, minerals, tocopherols (α-, δ-, γ-), ß-carotene, phytic acid, and raffinose were measured. Statistical analysis showed that within each hybrid tester set, there were very few significant (p < 0.05) differences between the paired trait-positive and trait-negative hybrids or between the conventional comparators and the trait-positive or trait-negative hybrids. Assessments of the magnitudes of differences and variance component analysis highlighted that growing location, and the tester used in hybrid formation, had a markedly greater effect on composition than did the GM trait. Significantly, for each tester set, compositional differences within the trait-positive and trait-negative hybrid variants were greater than differences between the GM and non-GM comparators. Overall, GM trait insertion is not intrinsically a meaningful contributor to compositional variation, and observed differences between GM and non-GM comparators typically reflect incidental changes associated with conventional breeding practices. These results contribute to ongoing discussions on the relevance of negative segregants as comparators in GM assessments.

Compositional assessments of key maize populations: B73 hybrids of the Nested Association Mapping founder lines and diverse landrace inbred lines.

The present study provides an assessment of the compositional diversity in maize B73 hybrids derived both from the Nested Association Mapping (NAM) founder lines and from a diverse collection of landrace accessions from North and South America. The NAM founders represent a key population of publicly available lines that are used extensively in the maize community to investigate the genetic basis of complex traits. Landraces are also of interest to the maize community as they offer the potential to discover new alleles that could be incorporated into modern maize lines. The compositional analysis of B73 hybrids from the 25 NAM founders and 24 inbred lines derived from landraces included measurements of proximates (protein, fat, ash, and starch), fibers, minerals, amino acids, fatty acids, tocopherols (α-, γ-, and δ-), ß-carotene, phytic acid, and raffinose. Grain was harvested from a replicated trial in New York, USA. For each data set (NAM and landrace) canonical discriminant analysis allowed separation of distinct breeding groups (tropical, temperate, flint, mixed/intermediate) within each data set. Overall, results highlighted extensive variation in all composition components assessed for both sets of hybrids. The variation observed for some components within the landraces may therefore be of value for increasing their levels in modern maize lines. The study described here provided significant information on contributions of conventional breeding to crop compositional variation, as well as valuable information on key genetic resources for the maize community in the development of new improved lines.

Application of (1)h NMR profiling to assess seed metabolomic diversity. A case study on a soybean era population.

(1)H NMR spectroscopy offers advantages in metabolite quantitation and platform robustness when applied in food metabolomics studies. This paper provides a (1)H NMR-based assessment of seed metabolomic diversity in conventional and glyphosate-resistant genetically modified (GM) soybean from a genetic lineage representing ∼35 years of breeding and differing yield potential. (1)H NMR profiling of harvested seed allowed quantitation of 27 metabolites, including free amino acids, sugars, and organic acids, as well as choline, O-acetylcholine, dimethylamine, trigonelline, and p-cresol. Data were analyzed by canonical discriminant analysis (CDA) and principal variance component analysis (PVCA). Results demonstrated that (1)H NMR spectroscopy was effective in highlighting variation in metabolite levels in the genetically diverse sample set presented. The results also confirmed that metabolite variability is influenced by selective breeding and environment, but not genetic modification. Therefore, metabolite variability is an integral part of crop improvement that has occurred for decades and is associated with a history of safe use.

Kernel compositions of glyphosate-tolerant and corn rootworm-protected MON 88017 sweet corn and insect-protected MON 89034 sweet corn are equivalent to that of conventional sweet corn (Zea mays).

Monsanto Co. has developed two sweet corn hybrids, MON 88017 and MON 89034, that contain biotechnology-derived (biotech) traits designed to enhance sustainability and improve agronomic practices. MON 88017 confers benefits of glyphosate tolerance and protection against corn rootworm. MON 89034 provides protection against European corn borer and other lepidopteran insect pests. The purpose of this assessment was to compare the kernel compositions of MON 88017 and MON 89034 sweet corn with that of a conventional control that has a genetic background similar to the biotech sweet corn but does not express the biotechnology-derived traits. The sweet corn samples were grown at five replicated sites in the United States during the 2010 growing season and the conventional hybrid and 17 reference hybrids were grown concurrently to provide an estimate of natural variability for all assessed components. The compositional analysis included proximates, fibers, amino acids, sugars, vitamins, minerals, and selected metabolites. Results highlighted that MON 88017 and MON 89034 sweet corns were compositionally equivalent to the conventional control and that levels of the components essential to the desired properties of sweet corn, such as sugars and vitamins, were more affected by growing environment than the biotech traits. In summary, the benefits of biotech traits can be incorporated into sweet corn with no adverse effects on nutritional quality.

Compositional differences between near-isogenic GM and conventional maize hybrids are associated with backcrossing practices in conventional breeding.

Here, we show that differences between genetically modified (GM) and non-GM comparators cannot be attributed unequivocally to the GM trait, but arise because of minor genomic differences in near-isogenic lines. Specifically, this study contrasted the effect of three GM traits (drought tolerance, MON 87460; herbicide resistance, NK603; insect protection, MON 89034) on maize grain composition relative to the effects of residual genetic variation from backcrossing. Important features of the study included (i) marker-assisted backcrossing to generate genetically similar inbred variants for each GM line, (ii) high-resolution genotyping to evaluate the genetic similarity of GM lines to the corresponding recurrent parents and (iii) introgression of the different GM traits separately into a wide range of genetically distinct conventional inbred lines. The F1 hybrids of all lines were grown concurrently at three replicated field sites in the United States during the 2012 growing season, and harvested grain was subjected to compositional analysis. Proximates (protein, starch and oil), amino acids, fatty acids, tocopherols and minerals were measured. The number of statistically significant differences (α = 0.05), as well as magnitudes of difference, in mean levels of these components between corresponding GM variants was essentially identical to that between GM and non-GM controls. The largest sources of compositional variation were the genetic background of the different conventional inbred lines (males and females) used to generate the maize hybrids and location. The lack of any compositional effect attributable to GM suggests the development of modern agricultural biotechnology has been accompanied by a lack of any safety or nutritional concerns.

Compositional equivalence of grain from multi-trait drought-tolerant maize hybrids to a conventional comparator: univariate and multivariate assessments.

MON 87460 (D1) maize contains a gene that expresses the cold shock protein B (CSPB) from Bacillus subtilis to confer a yield advantage when yield is limited by water availability. This study evaluated the composition of grain from the D1-containing combined-trait maize hybrids D1 × NK603, D1 × MON 89034 × NK603, and D1 × MON 89034 × MON 88017. These stacks offer a combination of insect protection and herbicide tolerance traits. These hybrids were grown under well-watered and water-limited conditions at three replicated field sites across Chile during the 2006-2007 growing season. Compositional analyses included measurement of proximates, fibers, total amino acids, fatty acids, minerals, vitamins, raffinose, phytic acid, p-coumaric acid, and ferulic acid. The statistical analyses included an evaluation of the applicability of multiblock principal component analysis (MB-PCA) and ANOVA-simultaneous component analysis (ASCA) to studies when more than one experimental factor will contribute to compositional variability. Results from these multivariate procedures highlighted that water treatment was the greatest contributor to compositional variability and, as expected, confirmed that the grain of combined-trait drought-tolerant hybrids was compositionally equivalent to that of conventional comparators as established by traditional statistical significance testing.

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.

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.

Principal variance component analysis of crop composition data: a case study on herbicide-tolerant cotton.

Compositional studies on genetically modified (GM) and non-GM crops have consistently demonstrated that their respective levels of key nutrients and antinutrients are remarkably similar and that other factors such as germplasm and environment contribute more to compositional variability than transgenic breeding. We propose that graphical and statistical approaches that can provide meaningful evaluations of the relative impact of different factors to compositional variability may offer advantages over traditional frequentist testing. A case study on the novel application of principal variance component analysis (PVCA) in a compositional assessment of herbicide-tolerant GM cotton is presented. Results of the traditional analysis of variance approach confirmed the compositional equivalence of the GM and non-GM cotton. The multivariate approach of PVCA provided further information on the impact of location and germplasm on compositional variability relative to GM.

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.

Assessing compositional variability through graphical analysis and Bayesian statistical approaches: case studies on transgenic crops.

New transgenic (GM) crops are subjected to extensive safety assessments that include compositional comparisons with conventional counterparts as a cornerstone of the process. The influence of germplasm, location, environment, and agronomic treatments on compositional variability is, however, often obscured in these pair-wise comparisons. Furthermore, classical statistical significance testing can often provide an incomplete and over-simplified summary of highly responsive variables such as crop composition. In order to more clearly describe the influence of the numerous sources of compositional variation we present an introduction to two alternative but complementary approaches to data analysis and interpretation. These include i) exploratory data analysis (EDA) with its emphasis on visualization and graphics-based approaches and ii) Bayesian statistical methodology that provides easily interpretable and meaningful evaluations of data in terms of probability distributions. The EDA case-studies include analyses of herbicide-tolerant GM soybean and insect-protected GM maize and soybean. Bayesian approaches are presented in an analysis of herbicide-tolerant GM soybean. Advantages of these approaches over classical frequentist significance testing include the more direct interpretation of results in terms of probabilities pertaining to quantities of interest and no confusion over the application of corrections for multiple comparisons. It is concluded that a standardized framework for these methodologies could provide specific advantages through enhanced clarity of presentation and interpretation in comparative assessments of crop composition.

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.