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The dicamba-tolerant soybeans (Glycine max L.) MON 87708 and MON 87708 × MON 89788 are compositionally equivalent to conventional soybean Mary L. Taylor, Anna Bickel, Rhonda Mannion, Erin Bell, and George Harrigan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03844 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017
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Journal of Agricultural and Food Chemistry
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The dicamba-tolerant soybeans (Glycine max L.) MON 87708 and
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MON 87708 × MON 89788 are compositionally equivalent to conventional
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soybean
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Mary Taylor*†, Anna Bickel†, Rhonda Mannion†, Erin Bell†, and George G. Harrigan†**
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*
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[email protected] 7
†
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**
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Georgia, 30313
To whom correspondence should be addressed.
Tel: 314 694-8530. E-mail:
Monsanto Company, 800 North Lindbergh Boulevard; St. Louis, Missouri 63167 Current address: The Coca-Cola Company, One Coca Cola Plaza NW, Atlanta,
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ABSTRACT
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Herbicide-tolerant crops can expand both tools for and timing of weed control
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strategies. MON 87708 soybean has been developed through genetic modification and
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confers tolerance to the dicamba herbicide. As part of the safety assessment conducted
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for new genetically modified (GM) crop varieties, a compositional assessment of
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MON 87708 was performed. Levels of key soybean nutrients and anti-nutrients in
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harvested MON 87708 were compared with levels of those components in a closely
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related non-GM variety, as well as to levels measured in other conventional soybean
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varieties. From this analysis, MON 87708 was shown to be compositionally equivalent
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to its comparator. A similar analysis conducted for a stacked trait product produced by
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conventional breeding, MON 87708 × MON 89788, which confers tolerance to both
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dicamba and glyphosate herbicides, reached the same conclusion. These results are
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consistent with other results that demonstrate no compositional impact of genetic
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modification, except in those cases where an impact was an intended outcome.
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Keywords: Soybean (Glycine max); dicamba-tolerant; MON 87708; glyphosate-tolerant; MON 89788;
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exploratory data analysis; composition
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Introduction Herbicide-tolerant crops can expand both the tools for and timing of weed control
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strategies used by farmers. In addition, use of such crops may lead to a reduction in the
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need for tillage as a strategy for weed control, leading to increased soil conservation and
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reduction in greenhouse gas emissions.1 Herbicide-tolerant crops have been used for
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over 20 years and have primarily been developed either through breeding selection of
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naturally occurring tolerance alleles in the crop or through genetic modification.
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Monsanto Company has developed a genetically modified (GM) soybean, MON 87708,
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that is tolerant to dicamba (3,6-dichloro-2-methoxybenzoic acid) herbicide. To confer
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tolerance, MON 87708 contains a demethylase gene from Stenotrophomonas maltophilia
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that encodes for the dicamba monooxygenase (DMO) protein. The weight of evidence
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from protein safety studies supports the conclusion that the DMO protein introduced into
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the dicamba tolerant soybean is safe for food and feed consumption.2
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In addition, conventional breeding has been used to develop a combined trait product,
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MON 87708 × MON 89788, that is tolerant to both dicamba and glyphosate herbicides.
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The ability to apply dicamba herbicide to dicamba tolerant soybean provides an important
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additional integrated option for soybean producers to manage broadleaf weeds, including
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those that are glyphosate-resistant. Dicamba herbicides have been found safe for their
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intended uses by the U.S. Environmental Protection Agency (EPA) and are registered for
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agricultural, industrial, and residential use; consequently, over 400 dicamba-based
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formulations have been developed since the initial EPA approval in 1967.3,4
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Compositional assessment is a key component of the safety assessment paradigm for new GM crop varieties;5 therefore, assessment of MON 87708 was conducted to evaluate 3 ACS Paragon Plus Environment
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whether the composition of MON 87708 is as safe as that of conventional soybean
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varieties. Compositional assessment of the combined trait product MON 87708 ×
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MON 89788 was also conducted. Selection of components for compositional analysis
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followed considerations relevant to the nutritional quality of soybean and was based on
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internationally accepted guidelines for assessments of new crops recommended by the
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Organization of Economic Cooperation and Development (OECD).6 The laboratory
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methods used for component analysis were widely accepted methods that have been
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validated through sufficient testing to ensure the quality, reliability and consistency of
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results.
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Materials and Methods
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Soybean samples for compositional analyses
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Seed samples were collected from MON 87708 grown in 2008 and treated with
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dicamba at five replicated sites (one location each in Iowa, Indiana and Pennsylvania and
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two locations in Illinois). A conventional soybean variety (A3525) was included as a
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near-isogenic conventional comparator at each location; other commercially available
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soybean varieties were also grown at each location as reference varieties (see Supporting
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Information, Table S1). Seed samples were also collected from
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MON 87708 × MON 89788 grown in 2009 at eight replicated sites representative of
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soybean growing regions across the U.S (one location each in Arkansas, Iowa, Kansas
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and Nebraska and two locations in both Illinois and Indiana). In addition, to meet a
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specific business need, MON 87708 was also grown at these locations. The conventional
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soybean variety (A3525) was grown at each location along with commercially available
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soybean reference varieties (see Supporting Information, Table S1). Each site was
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planted in a randomized complete block design with three (2008) or four (2009) blocks
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grown side by side, and grown under normal agronomic field conditions for their
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respective geographic region. All plants in the production were treated with maintenance
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pesticides per label directions, as customary for the growing location. In addition,
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MON 87708 and MON 87708 × MON 89788 plots were treated at the V2-V3 growth
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stage with dicamba herbicide at the target label rate (0.5 lb/acre a.e.), and
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MON 87708 × MON 89788 plots were also subjected to a glyphosate treatment at the
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V4-R1 growth stage at a rate of approximately 0.77 lb glyphosate/acre a.e. Seed was
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harvested from each of four replicated plots at maturity in a similar timeframe within and
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across locations. Harvested seed samples were ground with dry ice and were stored at
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approximately -20 °C after processing. The identity of the harvested seed was confirmed
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by chain of custody records and event-specific PCR analysis. Due to lack of availability
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of a suitable sample, a single A3525 replicate from each of two locations and a single
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MON 87708 replicate from one location in 2009 were omitted from the compositional
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analysis.
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Compositional analyses
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Selection of components for compositional analysis was based on recommendations
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found in OECD.6 Components were assessed at Covance Laboratories Inc. (Madison,
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Wisconsin) and included proximates (ash, fat, and protein), carbohydrates (by
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calculation), acid detergent fiber (ADF), neutral detergent fiber (NDF), amino acids, fatty
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acids, vitamin E, anti-nutrients (lectin, phytic acid, raffinose, stachyose, and trypsin
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inhibitors), and isoflavones (daidzein, genistein, glycitein). Brief descriptions of the
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analytical methods are provided in Lundry, et al.7; as cited there, the methods used have
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been validated through sufficient testing to ensure the quality, reliability and consistency
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of results. The use of validated methods, evaluated through determination of parameters
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such as specificity, accuracy, precision, linearity and robustness, is also a prerequisite for
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submission of composition data to the ILSI Crop Composition Database (ILSI-CCDB), a
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repository of over 15 years of crop composition data for conventional soybean varieties.8
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Statistical analysis of composition data Pre-analysis and ANOVA were conducted on the compositional data. A few data
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values that were below the assay LOQ were assigned a value equal to one-half the assay
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LOQ; four individual replicate values for vitamin E within the set of reference varieties
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were assigned, and one individual replicate value for lectin for A3525 was assigned.
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Compositional components were statistically analyzed using a mixed model analysis of
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variance as described in Lundry, et al.7 The data were analyzed as a combined-site
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analysis. For each component analysis, mean comparison tests of MON 87708 or
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MON 87708 × MON 89788 to the near-isogenic conventional comparator were
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conducted. Statistically significant differences were identified at the 5% level (α = 0.05).
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Statistical results were calculated using unrounded values. Subsequently, all values were
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rounded for consistent formatting. Relative magnitudes of difference were calculated
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from rounded values using the formula (|test value – control value|/control value) × 100.
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Results and Discussion
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The current regulatory framework for assessment of compositional equivalence for
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new GM crop varieties contains has two key elements.5 First, a statistical comparison of
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the new variety to a similar conventional variety is conducted. Second, an assessment of 6 ACS Paragon Plus Environment
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the compositional relevance of any observed significant differences is conducted using
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documented natural varibility for the crop as context. This second step is critical,
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because statistical significance does not imply biological relevance from a compositional
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perspective or from a safety and nutrition perspective. Many significant differences
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observed in compositional comparisons are small and are a consequence of high
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replication found in study designs associated with regulatory trials. 9,10,11,12 In addition, it
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has been shown the relatively minor differences in composition between individual plant
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selections can occur within a variety due to intra-cultivar variation.13 This is an
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important consideration because differences between near-isogenic GM and non-GM
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comparators may likewise arise due to minor genetic differences not directly related to
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the introduced trait(s).12,14
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A summary of the direct comparison of component values in MON 87708 or
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MON 87708 × MON 89788 to a close conventional comparator is presented below.
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Where statistical differences were observed, magnitudes of difference were evaluated
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based on known compositional variability of conventional soybean as evidenced in the
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scientific literature and data from the conventional reference varieties analyzed within the
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study. In addition, values were compared to those found in the ILSI-CCDB, which
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contains crop composition data obtained from studies conducted over many years at
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worldwide locations and provides insight into the natural variability of the nutritional
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composition of conventional crops. Overall, the assessment approach within the study
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highlighted that differences between the GM and near-isogenic comparators were
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inconsequential from a food and feed perspective and in terms of their impact on the
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charcteristics of the harvested materials. This observation also confirms a lack of any
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compositional impact of combining these GM traits through conventional breeding.
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Protein and amino acid composition
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A significant difference in mean levels of protein (p
