Monsanto Company 09-SY-201U Page 2 of 471

RELEASE OF INFORMATION 

Monsanto is submitting the information in this petition for review by the USDA as part of 

the regulatory process. By submitting this information, Monsanto does not authorize its 

release to any third party. In the event the USDA receives a Freedom of Information 

Act request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of this 

information, Monsanto expects that, in advance of the release of the document(s), USDA 

will provide Monsanto with a copy of the material proposed to be released and the 

opportunity to object to the release of any information based on appropriate legal 

grounds, e.g., responsiveness, confidentiality, and/or competitive concerns. Monsanto 

understands that a copy of this information may be made available to the public in a 

reading room and by individual request as part of a public comment period. Except in 

accordance with the foregoing, Monsanto does not authorize the release, publication or 

other distribution of this information (including website posting) without Monsanto's 

prior notice and consent. 

© 2009 Monsanto Company. All Rights Reserved. 

This document is protected under copyright law. This document is for use only by the 

regulatory authority to which it has been submitted by Monsanto Company and only in 

support of actions requested by Monsanto Company. Any other use of this material, 

without prior written consent of Monsanto, is strictly prohibited. By submitting this 

document, Monsanto does not grant any party or entity any right to license or to use the 

information or intellectual property described in this document. 

Monsanto Company 09-SY-201U Page 4 of 471

EXECUTIVE SUMMARY 

The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of 

Agriculture (USDA) has responsibility under the Plant Protection Act (Title IV Pub. L. 

106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and 

dissemination of plant pests into the U.S. APHIS regulation 7 CFR Part 340.6 provides 

that an applicant may petition APHIS to evaluate submitted data to determine that a 

particular regulated article does not present a plant pest risk and no longer should be 

regulated. If APHIS determines that the regulated article does not present a plant pest 

risk, the petition is granted, thereby allowing unrestricted introduction of the article. 

Monsanto Company is submitting this request to APHIS for a determination of 

nonregulated status in whole for the new biotechnology-derived soybean product, 

MON 87705, any progeny derived from crosses between MON 87705 and conventional 

soybean, and any progeny derived from crosses of MON 87705 with other 

biotechnology-derived soybean that has been granted nonregulated status under 7 CFR 

Part 340. 

Product Description

Monsanto Company has developed biotechnology-derived soybean MON 87705 with an 

improved fatty acid (FA) profile that results in an oil that has enhanced nutritional 

characteristics, and improved suitability and stability for food and industrial uses. 

Currently commodity soybean oil requires hydrogenation to improve its stability for use 

in many foods given its high proportion of polyunsaturated fatty acids. Hydrogenation 

results in the formation of trans fatty acids that pose known coronary health risks. As 

food companies reformulated foods to replace trans fat-containing hydrogenated oils with 

healthier alternatives, they have faced challenges in finding high stability oils that are 

also relatively low in saturated fat. 

Using the extensive information known regarding the fatty acid biosynthetic pathway in 

soybean, MON 87705 was developed to selectively down-regulate, in seed, two key 

enzymes involved in fatty acid biosynthesis. As a result, MON 87705 soybean oil is 

lower in saturated fats (6% vs. 15% of total fatty acids) and higher in monounsaturated 

18:1 oleic acid (76% vs. 23% FA), with an associated decrease in the polyunsaturated 

18:2 linoleic acid levels (10% vs. 53% FA) relative to commodity soybean. 

Consequently, MON 87705 soybean oil, with improved oxidative stability and lower 

saturated fats than currently available commodity soybean oil, is suitable for a range of 

food and industrial applications. In addition, soybean meal derived from MON 87705, 

which contains very low residual oil, is unchanged in composition relative to commodity 

soybean meal. MON 87705 also contains the 5-enolpyruvylshikimate-3-phosphate 

synthase gene derived from Agrobacterium sp. strain CP4 (cp4 epsps) encoding the CP4 

EPSPS protein that is expressed throughout the plant conferring tolerance to glyphosate, 

the active ingredient in the Roundup® family of agricultural herbicides. 

 

® Roundup is a registered trademark of Monsanto Technology LLC. 
Monsanto Company 09-SY-201U Page 5 of 471

The improved fatty acid profile in MON 87705 soybean oil is achieved through the use of 

endogenous soybean (Glycine max L.) gene segments configured to suppress FATB and 

FAD2 gene expression. MON 87705 contains FATB1-A and FAD2-1A gene segments 

under the control of a seed promoter, limiting oil composition modification to this tissue. 

The assembled gene transcript has an inverted repeat that produces double stranded RNA 

(dsRNA) that, via the RNA interference (RNAi) pathway, suppresses endogenous FATB 

and FAD2 gene expression, thereby producing the desired fatty acid phenotype (see 

Figure below). 

Schematic of the Soybean Fatty Acid Biosynthetic Pathway and Summary of 

Modified Fatty Acid Content in Soybean Oil Derived from MON 87705 

Panel A: Schematic of the soybean fatty acid biosynthetic pathway 

Panel B: MON 87705 soybean oil compared to commodity soybean oil and other vegetable oils 

indicates suppression of endogenous FATB and FAD2 in MON 87705 seeds. 

Acyl-acyl carrier protein (ACP) thioesterases (referred to herein as FATB enzymes) are 

localized in plastids and hydrolyze saturated fatty acids from the ACP-fatty acid moiety. 

The suppression of FATB results in a decrease in the transport of the saturated fats out of 

the plastid, thus retaining their availability for desaturation to 18:1 oleic acid (see Figure 

above). Therefore, suppression of FATB decreases saturated fat content in the oil as well 

as increasing oleic acid. Subsequently, this increased amount of oleic acid is either 

delivered to the oil body or endoplasmic reticulum for further desaturation. Delta-12 
Monsanto Company 09-SY-201U Page 6 of 471

desaturases (referred to as FAD2 enzymes) desaturate 18:1 oleic acid to 18:2 linoleic acid. 

The suppression of FAD2 in soybean seed causes reduced desaturation of oleic to linoleic 

acid thus contributing further to the increase in oleic while reducing linoleic acid content 

in the oil. Therefore, the overall result of the suppression of these two enzymes is a 

reduction in saturated 16:0 palmitic and 18:0 stearic fatty acids, an increase in 

monounsaturated 18:1 oleic acid, and lower levels of polyunsaturated 18:2 linoleic acid 

relative to commodity soybean. 

The MON 87705 soybean oil improved fatty acid profile provides new options for food 

companies interested in the formulation of lower saturated fat foods. Soybean oil is 

comprised primarily of five major fatty acids: saturated fatty acids 16:0 palmitic and 18:0 

stearic acids, monounsaturated 18:1 oleic acid, and the polyunsaturated fatty acids, 18:2 

linoleic, and 18:3 linolenic acids. These five major fatty acids have very different 

oxidative stabilities and chemical functionalities. Conventional soybean oil typically 

contains 60-65% polyunsaturated fatty acids, mostly in the form of linoleic acid. This 

composition makes soybean oil unsuitable for certain food applications since the high 

concentrations of polyunsaturated fatty acids in the oil are susceptible to oxidation and 

degradation at high temperature. Therefore, hydrogenation of soybean oil is necessary to 

reduce levels of polyunsaturated fatty acids by converting them to more saturated fatty 

acids resulting in higher stability oil suitable for a range of food uses. The hydrogenation 

process used to reduce polyunsaturated fatty acids and increase the stability of soybean 

oil produces trans fatty acids that are linked to increased cardiovascular risk due to the 

elevation of low-density lipoproteins (LDL) and reduced levels of high-density 

lipoproteins (HDL). Because MON 87705 soybean oil has a reduced level of 

polyunsaturated fatty acids, it has higher oxidative stability without the need for 

hydrogenation, while also containing a lower level of saturated fats. Saturated fats, 

notably palmitic acid, have also been shown to contribute to cardiovascular disease and 

other chronic diseases. As a result, the reduced saturated fat levels in MON 87705 

soybean oil can also positively impact the goal of keeping human dietary consumption of 

saturated fats below 10% of total energy intake1

. 

In addition to providing improved formulation options for food companies, the reduction 

in saturated fats and increased oxidative stability of MON 87705 soybean oil increases 

suitability for biodiesel and other industrial applications. Low saturated fats and high 

(>70%) oleic acid levels are key attributes for vegetable oils targeted for biodiesel and 

industrial uses because of improved cold weather performance, improved stability, and 

reduced nitrous oxide emissions. 

Therefore, the fatty acid profile of MON 87705 soybean oil, with an increase in 

monounsaturated oleic acid and decreases in saturated fats and polyunsaturated fatty 

acids (17% vs. 60% FA), significantly improves the soybean oil functionality and 

nutritional value. MON 87705 will be bred with current commercial low linolenic acid 

soybean varieties that will further enhance the oxidative stability of soybean oil. 

 

1

 Department of Health and Human Services, Dietary Guidelines for Americans, 2005. 

www.health.gov/dietaryguidelines [Accessed June 7, 2009] 
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Studies Confirm the Lack of Plant Pest Potential of MON 87705 

The data and information presented in this petition demonstrate MON 87705 is 

agronomically, phenotypically, and compositionally comparable to conventional soybean 

with the exception of the intended modifications to oil composition and tolerance to 

glyphosate. Moreover, the data presented show MON 87705 is unlikely to pose an 

increased plant pest risk, including weediness or adverse environmental impact, 

compared to conventional soybean. The food, feed and environmental safety of 

MON 87705 was confirmed based on multiple, well established lines of evidence: 

1. Soybean is a familiar crop that does not possess any of the attributes commonly 

associated with weeds, has a history of safe consumption, and serves as an 

appropriate basis of comparison. 

2. A detailed molecular characterization of the inserted DNA demonstrated a single, 

intact copy of the transgenic insert in a single locus within the soybean genome. 

This insert contains the FATB and FAD2 suppression cassette and the cp4 epsps

expression cassette. 

3. The inverted repeat encoded by the FATB and FAD2 suppression cassette in 

MON 87705 does not code for any protein. The RNA-based suppression of FATB 

and FAD2 soybean genes in MON 87705 is mediated by double stranded RNA 

(dsRNA) molecules. Double stranded RNAs are commonly used by eukaryotes, 

including plants, for endogenous gene suppression and pose no novel risks from a 

food, feed or environmental perspective. Nucleic acids, such as RNA, have a 

long history of safe consumption and are considered GRAS by the U.S. Food and 

Drug Administration. 

4. The only introduced protein produced in MON 87705 is CP4 EPSPS. Data 

confirmed the CP4 EPSPS protein in MON 87705 is unlikely to be a toxin or 

allergen based on extensive information collected and evaluations performed. 

The CP4 EPSPS protein in MON 87705 has the same functional and enzymatic 

activity as the CP4 EPSPS in other Roundup Ready® crops previously deregulated 

by USDA, and is structurally homologous to EPSPSs naturally present in other 

crops. 

5. A compositional assessment confirmed that, except for intended fatty acid 

changes, MON 87705 seed and forage are compositionally equivalent to seed and 

forage of conventional soybean. MON 87705 soybean oil does not contain any 

new fatty acids that are not already present in commodity soybean oil, and the 

fatty acid profile of MON 87705 soybean oil is similar to many other commercial 

oils currently available. 

6. An extensive evaluation of MON 87705 phenotypic and agronomic characteristics 

and environmental interactions demonstrate MON 87705 shows no increased 

plant pest risk potential compared to conventional soybean. 

 

® Roundup Ready is a registered trademark of Monsanto Technology LLC. 
Monsanto Company 09-SY-201U Page 8 of 471

7. An assessment of potential impact to nontarget organisms (NTOs) and 

endangered species indicates MON 87705 is unlikely to have adverse effects on 

these organisms compared to conventional soybean under normal agricultural 

practices. 

8. An evaluation of MON 87705 on current cultivation and management practices 

for soybean concluded deregulation of MON 87705 will not significantly impact 

current soybean agronomic practices and land use. 

Soybean is a Familiar Crop Lacking Weedy Characteristics 

There is a long standing history of safe consumption of conventional soybean and its oil, 

as soybean is the most prevalently grown oilseed in the world, with approximately 222.1 

million metric tons of harvested seed (MMT) produced in 2007, representing 56% of 

world oilseed seed production that year. Soybean is grown as a commercial crop in over 

35 countries and domestication of this crop can be traced back to approximately 1000 

B.C. A major food use for soybean is purified oil, for use in margarines, shortenings, 

cooking, and salad oils. 

The commercial Glycine species in the U.S. (Glycine max L.) does not exhibit weedy 

characteristics and is not effective in invading established ecosystems. Soybean is not 

listed as a weed in major weed references, nor is it present on the lists of noxious weed 

species distributed by the federal government. Soybean does not possess any of the 

attributes commonly associated with weeds, such as long persistence of seed in the soil, 

the ability to disperse, invade, and become a dominant species in new or diverse 

landscapes, or the ability to compete well with native vegetation. Due to a pronounced 

lack of dormancy, soybean seed can germinate quickly under adequate temperature and 

moisture and potentially can grow as a volunteer plant. However, a volunteer plant likely 

would be killed by frost during autumn or winter of the year it was produced. If it did 

become established, a volunteer plant would not compete well with the succeeding crop, 

and could be controlled readily by either mechanical or chemical means. In addition, 

since wild populations of Glycine species are not known to exist in the U.S., the potential 

does not exist for MON 87705 to outcross to wild or weedy relatives and alter their 

weediness potential. 

The genetic background of MON 87705 was matched with that of an appropriate control, 

so the effect of the genetic insertion and the presence of the CP4 EPSPS protein could be 

assessed in an unbiased manner. Since MON 87705 was derived from the A3525 

conventional variety, it was deemed appropriate to use the nontransformed A3525 as the 

control variety because its use would minimize the potential bias in subsequent 

comparative assessments. 

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

MON 87705 was produced by Agrobacterium-mediated transformation of soybean with 

the binary vector PV-GMPQ/HT4404 that contains two T-DNAs. T-DNA I and T-DNA 

II both contain DNA segments designed to suppress endogenous FAD2 and FATB genes 

which encode for two key enzymes in the soybean fatty acid biosynthetic pathway. TDNA I also contains a cp4 epsps expression cassette. The partial suppression cassette in 

T-DNA I contains the sense segments of the FAD2-1A intron and FATB1-A 5' 
Monsanto Company 09-SY-201U Page 9 of 471

untranslated region (UTR) which are under the regulation of the 7Sα' seed promoter. TDNA II contains a partial suppression cassette that contains the antisense segment of 

FAD2-1A and FATB1-A. 

During plant transformation, the two T-DNAs (T-DNA I and T-DNA II, respectively) cointegrated into one locus in the soybean genome. The cointegration of the T-DNAs in 

this configuration creates an insert containing a single cp4 epsps expression cassette and a 

single FAD2-1A and FATB1-A suppression cassette. The in planta assembled suppression 

cassette includes an inverted repeat that results in suppression of endogenous FAD2 and 

FATB RNA expression. 

Molecular characterization of MON 87705 by Southern blot demonstrates there is one 

copy of each T-DNA insert within the same locus of integration. Backbone sequences 

from plasmid PV-GMPQ/HT4404 were not detected in the genome of MON 87705. 

Additionally, the data confirm the organization and sequence of the insert, demonstrate 

the stability of the insert over several generations, and that the genomic DNA sequences 

flanking the 5' and 3' ends of the insert are native to the soybean genome. 

RNA-Based Suppression Technology in MON 87705 Does not Pose Unique Safety 

Risks

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is 

mediated by dsRNA molecules. Double stranded RNAs are commonly found in 

eukaryotes, including plants, for endogenous gene suppression and are composed of 

nucleic acids. Nucleic acids have a long history of safe consumption and are considered 

GRAS by the U.S. Food and Drug Administration. There is no evidence to suggest 

dietary consumption of RNA is associated with toxicity or allergenicity. Moreover, 

analysis of the DNA segments encoding this dsRNA showed they lack the sequences 

required for translation initiation and protein synthesis. The production of a protein from 

the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several 

biotechnology-derived plant products previously deregulated by APHIS were developed 

using RNA-based suppression mechanisms, including virus-resistant papaya and squash, 

high oleic soybean, FLAVR SAVR tomatoes, and plum trees resistant to Plum pox virus. 

Based on this information, it is concluded that the inserted DNA and resulting dsRNA are 

safe and unlikely to produce a protein or polypeptide. As a result, the RNA-based 

suppression technology used in MON 87705 poses no novel risks from a food, feed or 

environmental perspective.

Data Confirm CP4 EPSPS Protein Safety

A multistep approach was used to characterize and assess the safety of the CP4 EPSPS 

protein expressed in MON 87705. This detailed characterization confirms the CP4 

EPSPS protein is safe for human and animal consumption. The assessment involved: 1) 

characterizing the physicochemical and functional properties of the protein; 2) 

quantifying protein levels in MON 87705 plant tissues; 3) examining the similarity of the 

CP4 EPSPS protein to known allergens, toxins and other biologically-active proteins 

known to have adverse effects on mammals; 4) evaluating the digestibility of CP4 EPSPS 

protein in simulated gastrointestinal fluids; 5) documenting the history of safe 

consumption CP4 EPSPS protein; and 6) investigating potential mammalian toxicity 

through an oral gavage assay. Results confirm that CP4 EPSPS is expressed in all tissues 
Monsanto Company 09-SY-201U Page 10 of 471

collected, including root, forage, seed, and leaf tissues at four developmental stages. CP4 

EPSPS has no relevant amino acid sequence similarities with known allergens, gliadins, 

glutenins, or protein toxins which can have adverse effects on mammals. Studies 

utilizing the CP4 EPSPS protein revealed that it degrades rapidly in simulated gastric and 

intestinal fluids and mouse acute oral toxicity evaluations demonstrate the protein is not 

acutely toxic and does not cause any adverse effect, even at the highest dose tested. The 

safety assessment supports the conclusion that dietary exposure to CP4 EPSPS protein 

derived from MON 87705 poses no meaningful risk to human or animal health. 

MON 87705 is Compositionally Equivalent to Conventional Soybean, Except for the 

Intended Fatty Acid Changes 

Detailed compositional and nutritional comparisons were conducted to assess whether 

levels of nutrients, anti-nutrients, and key secondary metabolites in seed and forage 

derived from MON 87705 are comparable to those in a conventional soybean control and 

several commercially available reference varieties. The analysis included proximates 

(protein, fat, carbohydrates, fiber, ash, moisture) in seed and forage, and fatty acids, 

amino acids, vitamin E, and antinutrients in seed. Composition data were analyzed 

statistically for all components and their biological and nutritional significance were 

evaluated. Statistical analyses were conducted on data from a combination of all sites 

(combined-site) and data from each of five individual sites (individual-site). If a 

significant difference (p<0.05) in an analyte was detected between MON 87705 and its 

conventional control in the combined-site comparison, an analysis including 

reproducibility across individual sites, magnitude of differences, and comparisons of 

MON 87705 mean analyte values to the 99% tolerance interval for the population of 

commercial conventional soybean varieties grown concurrently at the same time and field 

sites and published values was made to assess whether the difference was biologically 

meaningful from a food and feed safety or nutritional perspective. The compositional 

analysis confirmed that MON 87705 seed had the intended fatty acid composition, while 

the other components analyzed in MON 87705 seed and forage were considered to be 

compositionally equivalent to conventional soybean. Moreover, no new fatty acids 

beyond those presently found in soybean were detected in MON 87705. 

Of the nine fatty acids that were analyzed statistically, significant differences (p<0.05) 

were observed for seven fatty acids in the combined-site analysis. As intended, 

MON 87705 seed had significantly (p<0.05) lower 16:0 palmitic and 18:0 stearic acid 

levels (combined saturates 5.7%), higher 18:1 oleic acid (76.5%) and an associated 

decrease in 18:2 linoleic acid (10.1%) compared to conventional soybean. Differences in 

these four fatty acids were consistently observed at each of the individual sites and levels 

fell within the intended fatty acid ranges. A combined-site statistical difference (p<0.05) 

between MON 87705 and the conventional control was observed in the levels of 18:3 

linolenic acid. The decrease in 18:3 linolenic acid is expected given that it is produced 

from 18:2 linoleic acid which was reduced by the suppression of the FAD2 gene. 

Examination of the reproducibility within sites shows the levels of 18:3 linolenic acid 

were significantly lower than the soybean control in four of five individual-site analyses, 

with the absolute magnitude of the differences being small (<1.5% total FA content). In 

addition, all the mean levels of 18:3 linolenic acid in MON 87705 seed from the 

combined-site and individual-site analyses were well within the 99% tolerance interval, 
Monsanto Company 09-SY-201U Page 11 of 471

and therefore these differences are not considered biologically relevant compositional 

changes. Combined-site statistical differences between MON 87705 and the 

conventional control were also observed in the levels of two minor fatty acids, 20:0 

arachidic acid, and 20:1 eicosenoic acid. Examination of the reproducibility within sites 

shows the absolute magnitude of the differences for these two minor fatty acids was very 

small (<0.19% of total FA) and mean values and ranges in MON 87705 seed were within 

either the 99% tolerance interval for the population of the conventional reference 

varieties or the values reported in International Life Science Institute-Crop Composition 

Database (ILSI-CCD) and published literature. No difference was observed between 

MON 87705 and the conventional control for the remaining two minor fatty acids (22:0 

behenic acid and 24:0 lignoceric acid) evaluated. Thus, apart from the intended or 

expected differences in fatty acid levels, there were no other biologically meaningful 

differences in the levels of other fatty acids. 

Combined-site analyses of both forage and seed samples for non-fatty acid analytes 

showed no statistically significant difference (p>0.05) between MON 87705 and the 

conventional control for 37 of 41 analyte comparisons. Statistical comparisons between 

MON 87705 and the conventional control for the presence of other components showed 

that three analytes in soybean seed (cystine, arginine and total fat) and one analyte in 

forage (ash) were significantly different (p<0.05) in the combined-site analysis. For these 

four analytes where differences were noted (p<0.05), the absolute magnitude of 

differences between MON 87705 and the conventional soybean control were generally 

low (<1.1% dw), were not observed consistently across individual sites (individual-site 

analyses), and mean values for MON 87705 were within the calculated 99% tolerance 

interval for the population of conventional reference varieties. Harvested seed and forage 

analytical component values also were comparable to values reported in the ILSI-CCD 

and/or published literature, further supporting the conclusion that harvested seed and 

forage from MON 87705 are compositionally equivalent to those of conventional 

soybean. Therefore, it is concluded that the statistical differences observed are not 

biologically meaningful. 

In addition to the compositional analysis of seed and forage, four soybean processed 

fractions (refined oil, meal, lecithin, and protein isolate) were produced from 

MON 87705 and conventional control seed and subjected to compositional analysis in 

accordance with OECD guidelines. As expected, and consistent with results obtained for 

seed fatty acid levels, the intended fatty acid changes were observed in the refined oil 

fraction. As in seed, levels of several other less abundant fatty acids were also 

significantly different (p<0.05) between the refined soybean oil fractions from 

MON 87705 and the conventional soybean control. For these analytes, MON 87705 

mean values fell within the 99% tolerance intervals for the reference varieties and/or 

were comparable to published literature ranges for conventional soybean oil. Differences 

in the levels of two other minor fatty acids, likely formed by spontaneous isomerization 

during the oil refining process, were also observed. Since these differences are not 

considered biologically relevant, as they likely are an artifact of the refining process. 

Apart from the intended fatty acid changes in the oil, the composition of the soybean 

processed fractions from MON 87705 is equivalent to the composition of the soybean 

processed fractions from the conventional soybean control. Thus, the processed fractions 
Monsanto Company 09-SY-201U Page 12 of 471

from MON 87705 are as safe and nutritious as the processed fractions from conventional 

soybean. 

The compositional analysis confirmed that MON 87705 seed had the intended fatty acid 

profile. In all compositional and nutritional comparisons of MON 87705 to its 

conventional control where a significant difference (p<0.05) was detected, other than for 

intended fatty acid changes, an analysis including reproducibility across individual sites, 

magnitude of differences, and comparisons of mean test analyte values to the 99% 

tolerance interval and published values, indicated that differences observed were not 

biologically meaningful from a food and feed safety or nutritional perspective. 

Therefore, except for the intended fatty acid changes, the compositional and nutritional 

assessment of MON 87705 supports the conclusion that seed and forage and key 

processed fractions produced from MON 87705 are compositionally equivalent to those 

of conventional soybean in accordance with OECD guidelines. 

MON 87705 Does Not Change Soybean Plant Pest Potential or Environmental 

Interactions

The phenotypic, agronomic, and environmental interaction assessment indicates that 

MON 87705 is comparable to the parental conventional soybean control, A3525, which 

has background genetics similar to MON 87705, but lacks the introduced traits. Thus, 

MON 87705 is unlikely to have changed plant pest risk potential or environmental impact 

compared to conventional soybean. An important element in assessing plant pest risk 

potential and environmental impact of MON 87705 is to compare MON 87705 to 

conventional soybean. The assessment is based initially on the concept of familiarity, 

which USDA recognizes plays an important role in these assessments. Familiarity is 

based on the fact that the biotechnology-derived plant is developed from a conventional 

plant variety whose biological properties and plant pest potential are known to experts. 

Familiarity considers the biology of the crop, the introduced trait, the receiving 

environment and the interactions among these factors, and provides a basis for 

comparative risk assessment between a biotechnology-derived plant and its appropriate 

conventional counterpart. The MON 87705 characteristics assessed include: seed 

dormancy and germination, pollen morphology, and symbiont interactions conducted in 

the laboratory and greenhouse, and plant phenotypic observations and environmental 

interaction evaluations conducted in the field. 

Seed dormancy and germination characterization indicated that MON 87705 seed had 

germination characteristics similar to that of the conventional soybean control. In 

particular, the lack of hard seed, a well-accepted characteristic of weediness affecting 

seed germination rate and viability, supports a conclusion of no increased weediness 

potential of MON 87705 compared to conventional soybean for germination and 

dormancy characteristics. For pollen characteristics and symbiont interactions, there 

were no statistically significant differences (p<0.05) observed for any of the parameters 

measured, including pollen viability, nodule dry weight, and shoot total nitrogen. 

Collectively, these results support the conclusion that MON 87705 is not likely to exhibit 

increased weed potential compared to conventional soybean. 

The field evaluation of phenotypic, agronomic, and ecological characteristics of 

MON 87705 also supports the conclusion that MON 87705 is not likely to pose an 
Monsanto Company 09-SY-201U Page 13 of 471

increased weed or plant pest potential compared to conventional soybean. The 

evaluations were conducted at 17 replicated field sites across U.S. soybean production 

regions. These assessments included 14 plant growth and development characteristics, as 

well as observations for plant-insect and plant-disease interactions and plant responses to 

abiotic stressors. The observed phenotypic characteristics were comparable between 

MON 87705 and the conventional soybean control. Across sites, data show no 

statistically significant differences between MON 87705 and the control for seedling 

vigor, plant height, lodging, pod shattering, seed moisture, test weight, or yield. Flower 

color and plant pubescence data were categorical and were not statistically analyzed; 

however, at each site, all plants of MON 87705 and the control had purple flowers and 

pubescence as expected. 

Four statistically significant differences (p<0.05) were detected between MON 87705 and 

the control in the combined-site analysis. MON 87705 was lower than the control for 

early stand count, final stand count, the weight of 100 seeds and flowered approximately 

one day later than the control. MON 87705 and the control were within the same range 

of plant growth stages for 113 out of the 114 growth stage observations among the sites, 

and the single different observation at one site, was within the range of growth stages 

observed for the reference varieties. None of these differences were considered 

biologically meaningful in terms of increased weed potential. 

In an individual site assessment of plant response to abiotic stress, disease damage, and 

arthropod damage, no differences were observed between MON 87705 and the control 

for 574 of 579 comparisons (including 167 abiotic stress response, 206 disease damage, 

and 206 arthropod damage comparisons) among all observations at the 17 sites. The five 

observed differences were in the disease and arthropod damage categories. For each of 

the five observed differences, the severity of damage in MON 87705 was within the 

range of the reference soybean varieties, and were not consistently observed across sites. 

Therefore, they were not considered biologically meaningful in terms of increased weed 

potential. 

In an assessment of pest and beneficial arthropod abundance, no statistically significant 

differences (p> 0.05) were detected between MON 87705 and the control for 95 out of 96 

comparisons (including 46 arthropod pest comparisons and 50 beneficial arthropod 

comparisons) among the collections at the four sites. The single statistically significant 

difference was for bean leaf beetle in a single collection time from a single site, where the 

mean abundance value from MON 87705 plots was lower than the conventional control, 

yet fell within the reference range. The differences in pest and beneficial arthropod 

abundance were not indicative of a consistent plant response associated with the traits 

and are unlikely to be biologically meaningful in terms of plant pest potential or 

environmental impact of MON 87705 compared to conventional soybean. 

The plant phenotypic and ecological interaction parameters evaluated were used to 

characterize the plant and its interactions with the environment, and to assess the plant 

pest or weed potential of MON 87705 compared to the conventional soybean control. An 

analysis based on the weight of the evidence, including reproducibility, magnitude and 

direction of a difference, and comparison to reference ranges of the detected differences 

(p<0.05) found in the evaluation of phenotypic, agronomic and environmental 

characteristics of MON 87705 compared to the conventional soybean control supports the 
Monsanto Company 09-SY-201U Page 14 of 471

conclusion that MON 87705 is not likely to increase weed or plant pest potential or to 

have a biologically meaningful change in terms of environmental impact potential. 

MON 87705 Will Not Adversely Affect NTOs or Threatened or Endangered Species

Evaluation of the impacts of MON 87705 on NTOs is a component of the plant pest risk 

assessment. Assessment of the expected differences between MON 87705 and 

conventional soybean included the presence of the inserted genes, the expression of the 

CP4 EPSPS protein, and the improved fatty acid profile in MON 87705 seed. The nature 

of MON 87705 as a product with no pesticidal activity leads to a conclusion that all 

exposed organisms are considered to be NTOs. The environmental assessment of 

MON 87705 indicates that MON 87705 poses no adverse effect on NTOs or endangered 

species under normal agricultural practices. The environmental interactions evaluation 

included data collected in the phenotypic studies on plant-insect, plant-disease, and plantenvironment interactions. The results of this assessment indicated the presence of the 

CP4 EPSPS protein and improved fatty acid profile in seed did not unexpectedly alter 

plant-insect interactions, including beneficial arthropods and insect pests, or alter disease 

susceptibility of MON 87705 compared to conventional soybean. 

The fatty acids present in MON 87705 seed are widely prevalent in the environment. 

Fatty acids play a key role in metabolic energy storage and as components of 

phospholipids, which are essential for cellular membrane formation and function. As 

natural components of the plant and animal world, these fatty acids are not expected to 

accumulate, persist or be detrimental to the environment. In addition, the improved fatty 

acid profile in MON 87705 seed results in an oil that is very similar to seed fatty acid 

profiles in widely cultivated crops, such as canola (Brassica napus L. and B. campestris

L.), thus establishing a history of safe environmental exposure. 

The naturally glyphosate-tolerant EPSPS protein from an Agrobacterium sp., CP4 

EPSPS, has been introduced into several conventional crops, such as soybean, corn and 

cotton, to provide tolerance to glyphosate, the active ingredient in the Roundup family of 

herbicides. There is no toxicity associated with this family of proteins, and since they are 

ubiquitous in plants and microorganisms, they have a history of safety in the 

environment. The CP4 EPSPS protein is nontoxic to animals including mammals, birds, 

and arthropods. Lack of hazard for MON 87705 was established through a combination 

of biochemical information and experimental data demonstrating the existence of no 

reasonable mechanism for harm. 

Therefore, the assessment considered pertinent product characterization information, 

information from the protein safety assessments, the history of environmental exposure to 

these fatty acids, and results from the environmental interaction assessment. Taken 

together, these data support the conclusion that MON 87705 is unlikely to have an 

adverse effect on NTOs or endangered species under normal agricultural practices in U.S. 

soybean production. 

The potential for MON 87705 to outcross with sexually compatible species, including 

threatened or endangered species, is unlikely in the U.S., since no known wild Glycine

species related to cultivated soybean are known to be present in North America. In those 

world areas where sexually compatible species do exist, the potential to outcross is 

concluded to be low because soybean is a highly self-pollinated species, with cross-
Monsanto Company 09-SY-201U Page 15 of 471

pollination to other soybean varieties occurring at very low frequencies (0 to 6.3%) in 

adjacent plants. Furthermore, in the rare event when cross-pollination does occur, 

MON 87705 and its progeny would not have a significant environmental impact, because 

evaluations have shown the improved fatty acid profile and glyphosate-tolerance traits in 

MON 87705 have not enhanced weediness or plant-pest potential relative to conventional 

soybean. Therefore, the environmental consequence of pollen transfer from MON 87705 

to other Glycine species is considered negligible. 

Deregulation of MON 87705 Will Not Significantly Impact Soybean Agronomic

Practices or Land Use

Soybean fields are typically highly managed agricultural areas that can be expected to be 

dedicated to crop production for many years. MON 87705 likely would be used in 

common rotations on land previously used for agricultural purposes. No significant 

impact would be expected following the introduction of MON 87705 on current 

cultivation and management practices for soybean. Except for the intended modification 

in fatty acid profile and glyphosate tolerance, MON 87705 is no different from 

conventional soybean in its agronomic, phenotypic, ecological, characteristics and has the 

same levels of resistance to insects and diseases as current commercial soybean. The 

introduction of MON 87705 provides growers the means to produce a highly valued oil 

for use in multiple food and industrial applications. Based on these considerations, there 

is no apparent potential for significant impact on agronomic practices or land use.

Conclusion

Based on the data and information presented in this petition, it is concluded that 

MON 87705 is not likely to be a plant pest. Therefore, Monsanto Company requests a 

determination from APHIS that MON 87705 and any progeny derived from crosses 

between MON 87705 and conventional soybean or deregulated biotechnology-derived 

soybean be granted nonregulated status under 7 CFR Part 340. 

 
Monsanto Company 09-SY-201U Page 16 of 471

TABLE OF CONTENTS 

RELEASE OF INFORMATION ........................................................................................ 2

CERTIFICATION .............................................................................................................. 3

EXECUTIVE SUMMARY ................................................................................................ 4

TABLE OF CONTENTS .................................................................................................. 16

LIST OF FIGURES .......................................................................................................... 23

ABBREVIATION AND DEFINITIONS ......................................................................... 24

I. RATIONALE FOR THE DEVELOPMENT OF MON 87705 ..................................... 32

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR Part 340.6............................................................................................. 32

I.B. Rationale for the Development of Nutritionally Improved Soybean 

MON 87705 .................................................................................................... 32

I.C. Gene Suppression of FATB and FAD2 and Resulting Fatty Acid 

Composition of the Seed ................................................................................. 34

I.D. Submissions to Other Regulatory Agencies ................................................... 41

II.  THE SOYBEAN FAMILY ......................................................................................... 43

II.A. Soybean as a Crop ........................................................................................... 43

II.B. History of Soybean ......................................................................................... 44

II.C. Taxonomy and Phylogenetics of Soybean ...................................................... 45

II.D. The Genetics of Soybean ................................................................................ 47

II.E. Pollination of Cultivated Soybean .................................................................. 47

II.F. Cultivated Soybean as a Volunteer ................................................................. 48

II.G. Characteristics of the Recipient Plant ............................................................. 48

II.H. Soybean as a Test System in Product Safety Assessment .............................. 48

III.  DESCRIPTION OF THE TRANSFORMATION SYSTEM .................................... 49

IV.  GENETIC ELEMENTS ............................................................................................. 51

IV.A. Vector GMPQ/HT4404 ................................................................................... 51

IV.B. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA I) ..... 51

IV.C. The FAD2-1A and FATB1-A segments Sequence (T-DNA I and II) .............. 52

IV.D. Regulatory Sequences ..................................................................................... 52

IV.E. T-DNA Borders .............................................................................................. 52

IV.F. Genetic Elements Outside of the T-DNA Borders ......................................... 53

V.  GENETIC ANALYSIS ............................................................................................... 59

V.A. Copy Number of T-DNA I and T-DNA II in MON 87705 ............................ 65

V.B. Southern Blot Analysis to Determine the Presence or Absence of Plasmid 

PV-GMPQ/HT4404 Backbone ....................................................................... 72

V.C. Organization and Sequence of the Insert and Adjacent Genomic DNA in 

MON 87705 .................................................................................................... 76

V.D. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 87705 ............................................................................ 76
Monsanto Company 09-SY-201U Page 17 of 471

V.E. Inheritance of the Genetic Insert in MON 87705 ........................................... 80

V.F. Conclusion of Molecular Characterization ..................................................... 84

VI.    CHARACTERIZATION OF THE INTRODUCED CP4 EPSPS PROTEIN IN 

MON 87705 .................................................................................................... 85

VI.A. Identity and Function of the CP4 EPSPS Protein from MON 87705 ............. 85

VI.B. Characterization of the Full Length CP4 EPSPS Protein from 

MON 87705 .................................................................................................... 86

VI.C. Expression Levels of CP4 EPSPS Protein in MON 87705 ............................ 87

VI.D. Assessment of Potential Allergenicity of the CP4 EPSPS Protein from 

MON 87705 .................................................................................................... 90

VI.E. Safety Assessment Summary of CP4 EPSPS Protein ..................................... 90

VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENT OF MON 87705 ..... 93

VII.A. Overall Assessment of the Composition of Forage and Seed from 

MON 87705 Compared to the Conventional Soybean Control ...................... 95

VII.A.1 Intended Changes to Fatty Acid Levels in MON 87705 Seed ........................ 96

VII.A.2 Fatty Acid Levels in Soybean Seed ................................................................ 97

VII.A.3. Levels of Non-Fatty Acid Nutrients in Soybean Seed .................................... 99

VII.A.4. Naturally Occurring Anti-Nutrient Levels in Soybean Seed ........................ 100

VII.A.5. Proximate and Fiber Levels in Forage .......................................................... 101

VII.B. Compositional Equivalence of MON 87705 Seed and Forage to 

Conventional Soybean .................................................................................. 101

VII.C. Compositional Comparison of Processed Fractions from Soybean 

Seed of MON 87705 and the Conventional Control ..................................... 112

VII.D Safety and Nutritional Assessment of the Intended Changes in 

MON 87705 .................................................................................................. 113

VII.E Safety and Nutrition Assessment Conclusion ............................................... 114

VIII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL INTERACTIONS 

ASSESSMENT ............................................................................................. 115

VIII.A. Characteristics Measured for Assessment .................................................... 115

VIII.B. Interpretation of Phenotypic and Environmental Interaction Data ............... 117

VIII.C. Interpretation of Detected Differences Criteria ............................................ 118

VIII.D. Phenotypic, Agronomic and Environmental Interactions 

Characteristics ............................................................................................... 119

VIII.E. Overall Conclusions for Phenotypic, Agronomic, and Environmental 

Interactions Evaluation ................................................................................. 130

IX. U.S. AGRONOMIC PRACTICES ....................................................................... 131

IX.A. Introduction ................................................................................................... 131

IX.B. Overview of U.S. Soybean Production ......................................................... 132

IX.C. Production Management Considerations ...................................................... 144

IX.D. Weed Management ....................................................................................... 149

IX.E. Management of Insects ................................................................................. 152

IX.F. Management of Diseases and Other Pests .................................................... 152

IX.G. Crop Rotation Practices in Soybean ............................................................. 154

IX.H. Soybean Volunteer Management .................................................................. 166

IX.I. Stewardship of MON 87705 ......................................................................... 168
Monsanto Company 09-SY-201U Page 18 of 471

IX.J. Impact of the Introduction of MON 87705 on Agricultural Practices .......... 169

X. ENVIRONMENTAL CONSEQUENCES AND IMPACT ON AGRONOMIC 

PRACTICES ................................................................................................. 170

X.A. Introduction ................................................................................................... 170

X.B. Plant Pest Assessment of the MON 87705 Insert and Expressed 

Substances ..................................................................................................... 171

X.C. Weediness Potential of MON 87705 ............................................................ 176

X.D. Potential for Pollen mediated Gene Flow ..................................................... 177

X.E. Summary of Plant Pest Assessments ............................................................ 179

XI. ADVERSE CONSEQUENCES OF INTRODUCTION ....................................... 182

REFERENCES .............................................................................................................. 183

Appendix A. USDA Notification ................................................................................... 200

Appendix B. Materials and Methods Used for Molecular Analyses of 

MON 87705 .................................................................................................. 203

Appendix C. Materials, Methods and Results for Characterization of CP4 EPSPS 

Protein Produced in MON 87705 ................................................................. 208

Appendix D. Materials and Methods Used for the Analysis of the Levels of CP4 

EPSPS Protein in MON 87705 ..................................................................... 228

Appendix E. Materials and Methods Used for Compositional Analysis of 

MON 87705 Soybean Seed, Forage and Processed Fractions ...................... 231

Appendix F. Materials and Methods for Seed Dormancy and Germination 

Analyses of MON 87705 .............................................................................. 335

Appendix G. Material, Methods and Individual Site Results from Phenotypic, 

Agronomic and Ecological Interactions Analyses of MON 87705 .............. 341

Appendix H. Materials and Methods for Pollen Morphology and Viability 

Evaluation ..................................................................................................... 363

Appendix I. Materials and Methods for Symbiont Evaluation ................................ 365

Appendix J. Materials and Methods for Analysis of FAD2-1A and FATB1-A 

RNA levels in MON 87705 immature seeds ................................................ 367

Appendix K. Manufacturing Process for Soybean .................................................... 370

Appendix L. Glyphosate Resistance ......................................................................... 372

Appendix M. Nutritional Impact Evaluation of MON 87705 Soybean Oil ............... 390

Appendix N. Glyphosate-Tolerant Crops: Potential Impacts on Land Use and 

Agricultural Practices .................................................................................... 397

Appendix O. Potential Impact of Glyphosate on Human Health and the 

Environment .................................................................................................. 414

Appendix P. Petitioner’s Environmental Assessment .............................................. 442

 
Monsanto Company 09-SY-201U Page 19 of 471

LIST OF TABLES 

Table I-1. Summary of Product Concept Fatty Acid Levels for Test 

(MON 87705) vs. the Conventional Control (A3525) and 

Commercial Tolerance Interval ................................................................ 41

Table II-1. World Soybean Production in 2007/2008 ................................................. 43

Table II-2. List of Species in the Genus Glycine Willd., 2n Chromosome 

Number, Genome Symbol, and Distribution ............................................ 46

Table IV-1. Summary of Genetic Elements in Plasmid Vector 

PV- GMPQ/HT4404 ................................................................................. 56

Table V-1. Summary Chart of the Expected DNA Segments Based on 

Hybridizing Probes and Restriction Enzymes Used in MON 87705 

Analysis..................................................................................................... 62

Table V-2. Summary of Genetic Elements in MON 87705 ........................................ 63

Table V-3. Segregation of the H6 3’UTR Gene During the Development of 

MON 87705 .............................................................................................. 83

Table VI-1. Tissues collected for MON 87705 ............................................................ 88

Table VI-2. Summary of CP4 EPSPS Protein Levels in Leaf, Seed, Root, 

and Forage Tissues from MON 87705 Grown in 2007/2008 Chile 

Field Trials ................................................................................................ 89

Table VII-1. Summary of Intended Changes in Fatty Acid Levels for 

MON 87705 vs. the Conventional Soybean Control (A3525) in the 

Combined-Site Analysis ........................................................................... 97

Table VII-2. Summary of Differences (p<0.05) for the Comparison of Soybean 

Component Levels for MON 87705 vs. the Conventional Control 

(A3525) and Commercial Reference Varieties ....................................... 102

Table VIII-1. Phenotypic, Agronomic and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials or Greenhouse Studies ... 116

Table VIII-2. Germination Characteristics of MON 87705 and A3525 ........................ 121

Table VIII-3. Field Phenotypic Evaluation Sites for MON 87705 during 2007 ........... 124

Table VIII-4. Plant Growth and Development Data across 17 Locations 

during 2007 ............................................................................................. 125

Table VIII-5. Pollen Grain Diameter and Viability Analyses ....................................... 128

Table IX-1. Soybean Production in the U.S., 1999 – 2008 ........................................ 134

Table IX-2. U.S. Soybean Production by Region and State in 2008 .......................... 135

Table IX-3. U.S. Soybean Production Costs and Returns in 2006 ............................. 136

Table IX-4. Common Weeds in Soybean Production: Midwest Region ................... 150

Table IX-5. Common Weeds in Soybean Production: Mid-South Region ............... 150

Table IX-6. Common Weeds in Soybean Production: Eastern Coastal Region ........ 151

Table IX-7. Rotational Practices in the U.S. Following Soybean Production ............ 157

Table IX-8. Rotational Practices Following Soybean Production in the Midwest 

Region ..................................................................................................... 158

Table IX-9. Rotational Practices Following Soybean Production in the 

Mid-South Region ................................................................................... 162

Table IX-10. Rotational Practices Following Soybean Production in the Eastern 

Coastal Region ........................................................................................ 164
Monsanto Company 09-SY-201U Page 20 of 471

Table IX-11. Ratings for Control of Volunteer Glyphosate-Tolerant Soybean 

in Labeled Rotational Crops ................................................................... 167

Table X-1. Summary of Published Literature on Soybean Cross Pollination ........... 180

Table A-1. USDA Notifications Approved for MON 87705 and Status of 

Trials Conducted under These Notifications .......................................... 201

Table C-1. Molecular Weight Difference Between the MON 87705- and 

E. coli -produced CP4 EPSPS Proteins................................................... 215

Table C-2. Comparison of Immunoreactive Signal between MON 87705- 

Produced and E. coli -produced CP4 EPSPS Proteins ............................ 219

Table C-3. Summary of the Tryptic Masses Identified for the MON 87705- 

Produced CP4 EPSPS Using MALDI-TOF MS. .................................... 221

Table C-4. N-Terminal Sequence of the MON 87705-produced CP4 EPSPS. ........ 223

Table C-5. CP4 EPSPS Functional Assay ................................................................ 226

Table D-1. Protein Extraction Methods for Tissue Samples ..................................... 229

Table E-1. Statistical Summary of Site CdT Soybean Forage Fiber and 

Proximate Content for MON 87705 vs. the Conventional Control 

(A3525) ................................................................................................... 242

Table E-2. Statistical Summary of Site CdT Soybean Seed Amino Acid, 

Fatty Acid, Fiber, Proximate, Vitamin, Antinutrient and Isoflavone 

Content for MON 87705 vs. the Conventional Control (A3525) ........... 244

Table E-3. Statistical Summary of Site MEL Soybean Forage Fiber and 

Proximate Content for MON 87705 vs. the Conventional Control 

(A3525) ................................................................................................... 252

Table E-4. Statistical Summary of Site MEL Soybean Seed Amino Acid, 

Fatty Acid, Fiber, Proximate, Vitamin, Antinutrient and Isoflavone 

Content for MON 87705 vs. the Conventional Control (A3525) ........... 254

Table E-5. Statistical Summary of Site QUI Soybean Forage Fiber and 

Proximate Content for MON 87705 vs. the Conventional Control 

(A3525) ................................................................................................... 262

Table E-6. Statistical Summary of Site QUI Soybean Seed Amino Acid, 

Fatty Acid, Fiber, Proximate, Vitamin, Antinutrient and Isoflavone 

Content for MON 87705 vs. the Conventional Control (A3525) ........... 264

Table E-7. Statistical Summary of Site RAN Soybean Forage Fiber and 

Proximate Content for MON 87705 vs. the Conventional Control 

(A3525) ................................................................................................... 272

Table E-8. Statistical Summary of Site RAN Soybean Seed Amino Acid, 

Fatty Acid, Fiber, Proximate, Vitamin, Antinutrient and Isoflavone 

Content for MON 87705 vs. the Conventional Control (A3525) ........... 274

Table E-9. Statistical Summary of Site SFR Soybean Forage Fiber and 

Proximate Content for MON 87705 vs. the Conventional Control 

(A3525) ................................................................................................... 282

Table E-10. Statistical Summary of Site SFR Soybean Seed Amino Acid, 

Fatty Acid, Fiber, Proximate, Vitamin, Antinutrient and Isoflavone 

Content for MON 87705 vs. the Conventional Control (A3525) ........... 284
Monsanto Company 09-SY-201U Page 21 of 471

Table E-11. Statistical Summary of Combined Site Soybean Forage Fiber and 

Proximate Content for MON 87705 vs. the Conventional Control 

(A3525) ................................................................................................... 292

Table E-12. Statistical Summary of Combined Site Soybean Seed Amino Acid, 

Fatty Acid, Fiber, Proximate, Vitamin, Antinutrient and Isoflavone 

Content for MON 87705 vs. the Conventional Control (A3525) ........... 294

Table E-13. Literature and ILSI-CCD Ranges for Components in Soybean Forage 

and Seed .................................................................................................. 303

Table E-14. Estimation of 17:1 9c Heptadecenoic Acid Intake in Selected Foods 

and MON 87705 ..................................................................................... 308

Table E-15. Summary of Differences (p<0.05) for the Comparison of Soybean 

Processed Fraction Component Levels for MON 87705 vs. the 

Conventional Control (A3525) and Reference Varieties ........................ 311

Table E-16. Comparison of Amino Acid, Fiber, Proximate, and Antinutrient 

Content from MON 87705 and Conventional Control (A3525) 

Soybean Meal Processed from a 2007 U.S. Field Production ................ 320

Table E-17. Comparison of Fatty Acids and Vitamin E from MON 87705 and 

Conventional Control (A3525) Soybean RBD Oil Processed from the 

2007 U.S. Field Production ..................................................................... 324

Table E-18. Comparison of Amino Acids and Moisture from MON 87705 and 

Conventional Control (A3525) Soybean Protein Isolate Processed 

from a 2007 U.S. Field Production ......................................................... 327

Table E-19. Comparison of Phosphatides from MON 87705 and Conventional 

Control (A3525) Soybean Lecithin Processed from a 2007 U.S. Field 

Production ............................................................................................... 330

Table E-20. Literature Ranges for Components in Soybean Meal ............................. 331

Table E-21. Literature Ranges for Components in Soybean Protein Isolate .............. 332

Table E-22. Literature Ranges for Components in Soybean Lecithin ........................ 332

Table E-23. Literature Ranges for Components in Soybean Oil ................................ 333

Table F-1. Starting Seed of MON 87705, Control and Commercial Reference 

Soybean Varieties Used in Dormancy Assessment ................................ 337

Table F-2. Comparison of MON 87705 to the Control for Dormancy and 

Germination Characteristics .................................................................... 338

Table G-1. Starting Seed for Phenotypic Assessments ............................................. 342

Table G-2. Field and Planting Information ............................................................... 343

Table G-3. Phenotypic Comparison of MON 87705 to the Conventional 

Soybean Control within Each Site .......................................................... 349

Table G-4. Growth Stage Monitoring of MON 87705, the Conventional Soybean 

Control, and the Reference Soybean Varieties ....................................... 352

Table G-5. Abiotic Stressor Evaluation Using Observational Severity Scale for 

MON 87705 and the Conventional Soybean Control ............................. 356

Table G-6. Disease Damage Evaluations Using an Observational Severity 

Scale for MON 87705 and the Conventional Soybean Control .............. 357

Table G-7. Arthropod Damage Evaluated Using an Observational Severity 

Scale for MON 87705 and the Conventional Soybean Control .............. 358
Monsanto Company 09-SY-201U Page 22 of 471

Table G-8. Abundance of Pest Arthropods in Beat Sheet Samples Collected 

from MON 87705, the Conventional Soybean Control, and the 

Reference Soybean Varieties .................................................................. 359

Table G-9. Abundance of Beneficial Arthropods in Beat Sheet Samples 

Collected from MON 87705, the Conventional Soybean Control, 

and the Reference Soybean Varieties ..................................................... 361

Table I-1 Starting seed of MON 87705, Control, and Commercial Soybean 

Reference Varieties Used in the Symbiont Evaluation ........................... 365

Table L-1. U.S. Glyphosate Resistant Weeds through June 2009 ............................ 377

Table L-2. Management Reccomendations for Control of Glyphosate Resistant 

Weeds ...................................................................................................... 382

Table M-1. Fatty Acid Profile for MON 87705 Used in the Intake Estimations ....... 391

Table M-2. Mean Per Capita Fatty Acid Intake from Target Foods Pre and Post 

MON 87705 Replacement, U.S.population ............................................ 392

Table M-3. 90th Percentile Per User Fatty Acid Intake from Target foods, 

Pre and Post MON 87705 Replacement, U.S.Population ....................... 392

Table M-4. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, 

U.S.Population (g/day)............................................................................ 393

Table M-5. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, 

U.S.Population (% en) ............................................................................ 393

Table M-6. Mean Per Capita and 90th Percentile Per User Fatty Acid Intake 

from Total Diet, Post MON 87705, Age and Gender Groups (g/day) .... 394

Table N-1. Herbicide-Tolerant Crops Undergoing Review or Previously Deregulated 

by USDA-APHIS. ................................................................................... 397

Table O-1. Established Glyphosate Tolerances Prior to Glyphosate-tolerant Crops 

(1993) ...................................................................................................... 420

Table O-3. Aggregate Exposure Assessment for Glyphosate ................................... 422

Table P-1. Deregulated or Submitted Biotechnology-derived Soybean 

Products with Altered Oil Profiles .......................................................... 445

Table P-2. Organic and Conventional Soybean Seed Sources ................................. 453

Table P-3. Comparison of Fatty Acid Profiles from Plant Sources .......................... 458

Table P-5. Deregulated or Submitted Biotechnology-derived Soybean 

Products................................................................................................... 465
Monsanto Company 09-SY-201U Page 23 of 471

LIST OF FIGURES 

Figure I-1. Schematic of the Soybean Fatty Acid Biosynthetic Pathway ................... 38

Figure I-2. Northern Blot Analysis of FAD2-1A RNA Level in MON 87705 ........... 39

Figure I-3. Northern Blot Analysis of FATB1-A RNA Level in MON 87705............ 40

Figure III-1. Schematic of the Development of MON 87705 ....................................... 50

Figure IV-1. Circular Map of Plasmid PV-GMPQ/HT4404 showing probes 1-6 ......... 54

Figure IV-2 Plasmid Map of Vector PV-GMPQ/HT4404 Showing Probes 7-10 ........ 55

Figure IV-3. Deduced Amino Acid Sequence of the Mature CP4 EPSPS Protein 

in PV-GMPQ/HT4404 .............................................................................. 58

Figure V-1. Schematic Representation of the Insert and Genomic Flanking 

Sequences in MON 87705 ........................................................................ 61

Figure V-2. Southern Blot Analysis of MON 87705: Probes 1, 4, and 6 .................... 69

Figure V-3. Southern Blot Analysis of MON 87705: Probes 2 and 5 ......................... 70

Figure V-4. Southern Blot Analysis of of MON 87705: Probe 3 ................................ 71

Figure V-5. Southern Blot Analysis of MON 87705:PV-GMPQ/HT4404 

Backbone Probes 7 and 9 .......................................................................... 74

Figure V-6. Southern Blot Analysis of MON 87705: PV-GMPQ/HT4404 

Backbone Probes 8 and 10 ........................................................................ 75

Figure V-7. Breeding History of MON 87705 ............................................................. 78

Figure V-8. Generational Stability of MON 87705: Probes 1 and 6 ............................ 79

Figure V-9. Breeding Path for Generating Segregation Data MON 87705 ................. 82

Figure VIII-1. Schematic Diagram of Agronomic and Phenotypic Data 

Interpretation Methods ............................................................................ 118

Figure B-1. Overlapping PCR Analysis across the Insert in MON 87705 ................ 207

Figure C-1. Molecular Weight and Purity Analysis of the MON 87705- 

Produced CP4 EPSPS Protein ................................................................. 216

Figure C-2. Western Blot Analysis of MON 87705-produced and E. coli -produced 

CP4 EPSPS Proteins ............................................................................... 218

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87705-Produced CP4 

EPSPS. .................................................................................................... 222

Figure C- 4. Glycosylation Analysis of the MON 87705-Produced CP4 EPSPS 

Protein ..................................................................................................... 225

Figure K-1. Schematic of Manufacturing Process for Soybean Oil. .......................... 370

Figure L-1. Weed Resistance to Various Herbicide Chemical Families ................... 375

Figure N-1. Total Acres of Canola Relative to Acres of Glyphosate-tolerant 

Varieties planted. .................................................................................... 399

Figure N-2. Total Acres of Corn and Soybean Relative to Acres of Herbicidetolerant (HT) Varieties Planted. .............................................................. 400

Figure N-3. Total Acres of Cotton Relative to Acres of Herbicide-tolerant (HT) 

Varieties Planted. .................................................................................... 401

Figure N-4. Percentage of Certified Organic Acres for Corn, Cotton, and 

Soybean. .................................................................................................. 403

Figure O-1. Tier I Endangered Species Assessment .................................................. 428

Figure P-1. Harvested U.S. Acres of Canola, Sunflower and Soybean ..................... 448

 
Monsanto Company 09-SY-201U Page 24 of 471

ABBREVIATIONS AND DEFINITIONS∗

~ Approximately 

2T-DNA Plasmid vector containing two separate T-DNA regions each 

surrounded by left and right borders of the Ti plasmid 

7S α' 3’ region of the Sphas1 gene of Glycine max encoding the 7Sα’ 

seed storage protein, β-conglycinin, including 35 nucleotides of the 

carboxylterminal β-conglycinin coding region with the termination 

codon and the polyadenylation sequence 

35S Enhancer sequences from the promoter of the Figwort Mosaic 

virus (FMV) 35S RNA 

aadA Bacterial promoter and coding sequence for an aminoglycosidemodifying enzyme, 3'(9)-O-nucleotidyltransferase from the 

transposon Tn7 

AA Amino acid 

ACP Acyl carrier protein 

AD_2009 Allergen gliadin and gluten proteins sequence database 

ADF Acid detergent fiber 

AI Adequate Intake 

ANOVA Analysis of Variance 

AOAC Association of Analytical Chemists 

AOCS American Oil Chemists Society 

AOSA Association of Official Seed Analysts 

AOSCA Association of Official Seed Certifying Agencies 

APHIS Animal and Plant Health Inspection Service 

APS Analytical protein standard 

ASA America Soybean Association 

bp Base pair 

B. japonicum Bradyrhizobium japonicum 

B-Left Border DNA region from Agrobacterium tumefaciens containing the left 

border sequence used for transfer of the T-DNA 

B-Right Border DNA region from Agrobacterium tumefaciens containing the right 

border sequence used for transfer of the T-DNA 

BSA Bovine serum albumin 

oC Degree Celsius 

CAST Council for Agricultural Science and Technology, USDA 

CBI Confidential business information 

 

∗

 Note: Standard abbreviations, e.g., units of measure, are used according to the format described in 

‘Instructions to Authors’ in the Journal of Biological Chemistry. 
Monsanto Company 09-SY-201U Page 25 of 471

CdT Calera de Tango Maipo Province, Chile 

CES Cooperative Extension Services 

CFIA Canadian Food Inspection Agency 

CFR Code of Federal Regulations 

CI Confidence interval 

COA Certificate of analysis 

COC Chain of custody 

CP(A) Buffer Containing 50 mM MES, pH 5.8, 10% glycerol (v:v), 1 mM 

benzamidine-HCl and 1 mM DTT 

CP4 Agrobacterium sp strain CP4 

cp4 epsps coding sequence for the CP4 EPSPS Protein 

CP4 EPSPS 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium

sp., strain CP4 

CPB Cartagena Protocol on Biosafety 

cpm Counts per minute 

CS-cp4 epsps Codon modified coding sequence of the aroA gene from 

Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein 

CS-rop Coding sequence for repressor of primer protein for maintenance 

of plasmid copy number in E. coli. 

CSFII Continuing Survey of Food Intakes by Individuals 

CTAB Hexadecyltrimethylammonium bromide 

CTP Chloroplast transit peptide 

α-Cyano α-cyano-4-hydroxycinnamic acid 

Da Dalton 

DAP Days after planting 

dATP Deoxyadenosine triphosphate 

dCTP Deoxycytidine triphosphate 

DEEM-FCID Dietary exposure evaluation model-food commodity intake 

database 

DHB 2,5-Dihydroxybenzoic Acid 

DNA Deoxyribonucleic acid 

dNTP Deoxynucleotide triphosphate 

dsRNA Double-stranded RNA 

DTT Dithiothreitol 

DW Dry weight 

DWCF Dry weight conversion factor 

dwt Dry weight of tissue 

ECL Enhanced chemiluminescence 

E. coli Escherichia coli 
Monsanto Company 09-SY-201U Page 26 of 471

EDTA Ethylenediaminetetraacetic acid 

EFSA European Food Safety Authority 

ELISA Enzyme-linked immunosorbent assay 

EPA U.S. Environmental Protection Agency 

EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase 

E-Score Expectation score 

EU European Union 

FA Fatty acid 

FAARP Food Allergy Research and Resource Program Database 

FAD2 Glycine max gene for Δ-12 desaturase 

FAD2-1A The Glycine max FAD2-1A gene encoding the delta-12 desaturase 

FAD2-1AP

 Partial sequence from intron #1 of the Glycine max FAD2-1A gene 

encoding the delta-12 desaturase which forms part of the 

suppression cassette 

FAME fatty acid methyl ester 

FASTA Algorithm used to find local high scoring alignments between a 

pair of protein or nucleotide sequences 

FATB Glycine max gene for Palmitoyl-ACP Thioesterase 

FATB1-A The Glycine max FATB1-A gene encoding the palmitoyl acyl 

carrier protein thioesterase 

FATB1-AP

 Partial sequence from the 5' untranslated region and the plastid 

targeting sequence from Glycine max FATB1-A gene that encodes 

the palmitoyl acyl carrier protein thioesterase which forms part of 

the suppression cassette 

FDA U.S. Food and Drug Administration 

FFDCA Federal Food, Drug, and Cosmetic Act 

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FMV Figwort mosaic virus 

FONSI Finding of No Significant Impact 

FW Fresh weight 

fwt Fresh weight of tissue 

GenBank A public genetic database maintained by the National Center for 

Biotechnology Information at the National Institutes of Health, 

Bethesda, MD, USA 

GI Gene sequence identification number 

GLP Good Laboratory Practice 

GLP-T GLP Technologies 

GRAS Generarlly Recognized As Safe 

GRR Monsanto Company Guidelines for Keeping Research Records 

HCl Hydrochloric Acid 
Monsanto Company 09-SY-201U Page 27 of 471

HDL High-density lipoprotein 

HRP Horseradish peroxidase 

H.U. hemagglutinating unit 

IDP Identity preserved 

IgG Immunoglobulin G 

ILSI International Life Sciences Institute 

ILSI-CCD International Life Sciences Institute-Crop Composition Database 

ISO International Organization for Standardization 

I-Tsf1 Intron from the Tsf1 gene of Aradidopsis thaliana encoding 

elongation factor EF-1 alpha 

kb Kilo bases 

kDa Kilodaltons 

KCl Potassium Chloride 

LB Laemmli buffer 

LDL Low-density lipoprotein 

Left Border DNA region from Agrobacterium tumefaciens containing the left 

border sequence used for transfer of the T-DNA 

LOD Limit of detection 

LOQ Limit of quantitation 

L-Tsf1 5′ untranslated leader (exon1) from the Tsf1 gene of Aradidopsis 

thaliana encoding elongation factor EF-1 alpha 

MAFF Ministry of Agriculture, Forestry and Fisheries, Japan 

MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight 

MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight mass 

spectrometry 

MEEC Maximum expected environmental concentration 

MEL Melipilla, Melipilla Province, Chile 

MES 2-[N-Morpholino] Ethanesulfonic Acid 

MH+

 Protonated mass ion 

MHLW Ministry of Health, Labor and Welfare, Japan 

mM Millimolar 

MMT Million metric tones 

MOE Margin of exposure 

MRMP Monsanto Resistance Management Plan 

MS Mass Spectrometry 

MSL Monsanto Scientific Literature 

MTSA Monsanto Technology Stewardship Agreement 

MUFA Monounsaturated fatty acid 

MW Molecular weight 
Monsanto Company 09-SY-201U Page 28 of 471

MWCO Molecular weight cutoff 

MWM Molecular weight marker 

N/A Not applicable 

NCBI National Center of Biotechnology Information at the National 

Institutes of Health, Bethesda, MD, USA 

NDF Neutral detergent fiber 

NEPA National Environmental Policy Act 

NFDM Non-fat dried milk 

NHANES National Health and Nutrition Examination Survey 

NIST National Institute of Standards and Technology 

NMWC Nominal molecular weight cut-off 

NOAEL No observed adverse effect level 

NOP National organic program 

nt Nucleotide 

NTO(s) Nontarget organism(s) 

OECD Organization for Economic Co-operation and Development 

OR Origin of replication 

OR-ori-PBR322 Origin of replication from pBR322 for maintenance of plasmid in 

E. coli

OR-ori-V Origin of replication for Agrobacterium derived from the broad 

host range plasmid RK2 

OSL Overseason leaf 

P-7Sα′ Promoter and leader from the Sphas1 gene of Glycine max

encoding beta-conglycinin storage protein (alpha′-bcsp) that directs 

transcription in seed 

PAGE Polyacrylamide gel electrophoresis 

PBS Phosphate buffered saline 

PBST Phosphate buffered saline containing 0.05% (v/v) Tween-20 

PCR Polymerase chain reaction 

PEG Polyethylene glycol 

PEP Phosphoenolpyruvate 

P-FMV/Tsf1 Chimeric promoter consisting of enhancer sequences from the 

promoter of Figwort Mosaic Virus (FMV) 35S RNA combined 

with promoter from the Tsf1 gene of Arabidopsis thaliana that 

encodes elongation factor EF-1 alpha 

Pi Inorganic Phosphate 

PIP Plant-incorporated protectant 

PMSF Phenylmethanesulfonyl fluoride 

polyA+

 RNA polyA enriched RNA 

PPA Plant Protection Act 
Monsanto Company 09-SY-201U Page 29 of 471

PRESS predicted residual sums of squares 

PS(A) Buffer Containing 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10% 

glycerol (v:v), 1.5 M (NH4)2SO4

PRT_2009 A protein sequence database derived from GenBank release 169 

PTH Phenylthiohydantoin 

PUFA Polyunsaturated fatty acid 

PVDF Polyvinylidene difluoride 

PVPP Polyvinylpolypyrrolidone 

PV-GMPQ/HT4404 Plasmid used to transform the soybean genome to produce 

MON 87705 

QS(A) Buffer Containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM 

benzamidine-HCl, 4 mM DTT) 

QAU quality assurance unit 

QUI Quilapilun, Cachapoal Province, Chile 

RAN Rancagua, Cachapoal Province, Chile 

RBD refined, bleached, and deodorized 

RCB Randomized complete block 

Right Border DNA region from Agrobacterium tumefaciens containing the right 

border sequence used for transfer of the T-DNA 

RK2 Broad host range plasmid of Inc-P1 originally isolated in 

Klebsiella pneumonia 

RNAi RNA interference 

rop Coding sequence for repressor of primer protein for maintenance 

of plasmid copy number in E. coli

S3P Shikimate-3-phosphate 

SAP Shrimp Alkaline Phosphatase 

SAS Statistical Analysis System 

SCN Soybean cyst nematode 

SD Standard deviation 

SDS Sodium dodecyl sulfate 

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis 

SE Standard error 

SFR San Fernando, Colchagua Province, Chile 

SGF Simulated gastric fluid 

SIF Simulated intestinal fluid 

SOP Standard operating procedure 

T/C/R Test/Control/Reference 

TD toasted and defatted 

TDF Total dietary fiber 
Monsanto Company 09-SY-201U Page 30 of 471

T-DNA I Transfer DNA I 

T-DNA II Transfer DNA II 

T-DNA Transferred DNA 

T-E9 3′ untranslated region of the pea rbcS2 gene which functions to 

direct polyadenylation of the mRNA 

TFA Trans fatty acid(s) 

T-H6 3′ untranslated sequence of the H6 gene from Gossypium 

barbadense encoding a protein involved in secondary cell wall 

TES Threatened or endangered species

TIU Trypsin inhibitor units 

TMB 3,3’,5,5’-tetramethylbenzidene 

TOX_2009 Toxin protein sequence database 

Tris Tris (hydroxymethyl) aminomethane 

tRNA Transfer RNA 

TS-CTP2 Targeting sequence from the gene shkG encoding the transit 

peptide region of Arabidopsis thaliana EPSPS that functions to 

direct transport of the CP4 EPSPS protein to the chloroplasts 

TSSP Tissue-specific site pool 

TUG Technology Use Guide 

Tween-20 Polyoxyethylenesorbitan monolaurate 

Tz Tetrazolium 

U Unit (of enzyme activity) 

U.S. United States of America 

USDA United States Department of Agriculture 

USDA-APHIS U.S. Department of Agriculture – Animal and Plant Health 

Inspection Service 

USDA-ARS United States Department of Agriculture – Agricultural Research 

Service 

USDA-ERS U.S. Department of Agriculture – Economic Research Service 

USDA-GRIN United States Department of Agriculture – Germplasm Resources 

Information Network 

USDA-NASS U.S. Department of Agriculture – National Agricultural Statistics 

Service 

USDA-NSHS U.S. Department of Agriculture – National Seed Health System 

USFWS U.S. Fish and Wildlife Service 

UTR Untranslated region 

V Volts 

VOI Verification of Identity 

WPEP Weed Performance Evaluation Program 

v/v Volume per volume 
Monsanto Company 09-SY-201U Page 31 of 471

wt weight 

w/v Weight per volume 
Monsanto Company 09-SY-201U Page 32 of 471

I. RATIONALE FOR THE DEVELOPMENT OF MON 87705 

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR Part 340.6 

The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of 

Agriculture (USDA) has responsibility, under the Plant Protection Act (7 U.S.C. § 7701-

7772) to prevent the introduction and dissemination of plant pests into the U.S. The 

APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to 

evaluate submitted data to determine that a particular regulated article does not present a 

plant pest risk and no longer should be regulated. If APHIS determines that the regulated 

article does not present a plant pest risk, the petition is granted, thereby allowing 

unrestricted introduction of the article. 

Monsanto Company is submitting this request to APHIS for a determination of 

nonregulated status in whole for the new biotechnology-derived, nutritionally improved 

soybean product, MON 87705, any progeny derived from crosses between MON 87705 

and conventional soybean, and any progeny derived from crosses of MON 87705 with 

other biotechnology-derived soybean that has been granted nonregulated status under 7 

CFR Part 340. 

I.B. Rationale for the Development of Nutritionally Improved Soybean 

MON 87705 

Monsanto Company has developed biotechnology-derived soybean MON 87705 with an 

improved fatty acid profile to enhance the suitability of soybean oil for food and 

industrial uses. The fatty acid (FA) levels in MON 87705 soybean oil are lower for 

saturated fats (6% vs. 15% FA) and higher for oleic acid (76% vs. 23%). The increase in 

monounsaturated fatty acid (oleic) is accompanied by an overall decrease in 

polyunsaturated fatty acids (17% vs 60% FA). Conventional soybean oil typically 

contains 60-65% polyunsaturated fatty acids (PUFA’s), mostly in the form of linoleic 

acid. This high PUFA content makes soybean oil unsuitable for many food applications 

since the high concentrations of PUFA’s in the oil are susceptible to oxidation and 

degradation at high temperature. 

To improve the stability of soybean oil, the polyunsaturated fatty acids in the oil can be 

decreased through a process called hydrogenation that reduces the number of unstable 

double bonds found in fatty acids such as linolenic and linoleic, and converts them to 

saturated fats. Although hydrogenation produces oil with excellent thermal and oxidative 

stability, it also results in the production of significant levels of trans fatty acids in the 

oil. Trans fatty acids contribute to cardiovascular risk by elevating LDL (bad cholesterol) 

and reducing HDL (good cholesterol) (Kris-Etherton, 1995; Hu et al., 1997). 

Soybean oil oxidative stability can be improved because the stability of vegetable oils is 

drastically influenced by the proportion of monounsaturates to polyunsaturates. High 

oleic soybean oils are estimated to have improved oxidative stability up to 17.5 times 

greater than conventional soybean oil (Frankel, 2005). Oil from MON 87705 would 

similarly have an enhanced oxidative stability (approximately three-fold) relative to 
Monsanto Company 09-SY-201U Page 33 of 471

conventional soybean oil due to its increase in monounsaturated fatty acid and decrease 

in PUFA’s, without the need for hydrogenation. Therefore, MON 87705 soybean oil can 

provide the food industry with options of enhanced stability for food formulation. 

Numerous global health authorities recognize that diets high in total fat and saturated fat 

are associated with increased risk of chronic disease (FAO/WHO, 2002; Lichtenstein et 

al., 2006; IO, 2002; USHHS, 1988), and health experts, including the American Heart 

Association (Eckel et al., 2007), have recognized that an unintended consequence of a 

shift to oils with no or lower trans fat levels, may be an increase in levels of saturated fats 

in foods. With a fatty acid profile lower in saturated fats than commodity soybean oil, 

and a reduced need for hydrogenation, MON 87705 soybean oil can help address this 

concern as MON 87705 soybean oil contains less than 7% saturated fatty acids (palmitic 

+ stearic acid). Based on U.S. Food and Drug Administration (FDA) guidance, a low 

saturated fat food contains less than 1 g of fat per serving (www.fda.gov) and the typical 

serving size for soybean oil is 14g (www.thumboilseed.com/soy-oil.htm). As a result, to 

qualify as a low saturate soybean oil the maximum amount of saturated fats allowed is 

7% (0.98 g per serving). Therefore, under FDA guidance MON 87705 soybean oil can 

be classified as a low saturate oil. 

The fatty acid profile of MON 87705 soybean oil is also well suited for industrial 

applications. Soybean oils have very good lubricating properties, and are highly 

biodegradable compared to mineral oils, but typically lack the stability needed to meet 

industrial requirements. Hydrogenation of soybean oil is not acceptable for most 

industrial uses because it leads to formation of saturated and trans fatty acids which can 

cause the oil to be solid at lower temperatures, resulting in excessive wear and tear of 

machinery (Kinney, 1998). The fatty acid profile of MON 87705 provides an industrial 

oil with improved stability that could serve as a lubricant without needing hydrogenation. 

In addition, soybean oil with elevated oleic acid is an attractive source for other industrial 

applications, such as petrochemical-derived plasticizers (Kinney, 1998). The higher oleic 

acid and lower saturated fat levels of MON 87705 also make it much more suitable for 

use in biodiesel due to its greater stability, improved cold weather performance, and 

reduced nitrous oxides emissions (Knothe 2005; Bringe, 2005; Graef, 2009) 

As with all new biotechnology-derived traits, MON 87705 will also be bred into soybean 

varieties with diverse genetic backgrounds. These varieties will include commercial 

varieties with low linolenic acid levels which can further enhance the oxidative stability 

of the soybean oil. In addition, MON 87705 will be combined using traditional breeding 

methods with other biotechnology-derived traits, including glyphosate tolerance 

(MON 89788), to deliver the best agronomic platform to farmers. 

To summarize, MON 87705 was developed to improve soybean oil’s oxidative stability 

profile, without the need for hydrogenation, and lower the saturated fat content of the oil. 

Due to the compositional improvement in MON 87705 soybean oil, MON 87705 could 

expand the food market applicability of soybean oil, without contributing further to 

known dietary health risks or sacrificing food flavor. Similarly, MON 87705 soybean oil 

could also serve as an improved source for industrial and biofuel products. 
Monsanto Company 09-SY-201U Page 34 of 471

I.C. Gene Suppression of FATB and FAD2 and Resulting Fatty Acid Composition 

of the Seed 

Suppression of FATB and FAD2 RNAs in soybean decreases saturated fats (16:0 

palmitic acid and 18:0 stearic acid), increases oleic acid (18:1), and decreases linoleic 

acid (18:2). 

The improved fatty acid profile in MON 87705 soybean oil is achieved through the use of 

endogenous soybean (Glycine max L.) gene segments configured to suppress FATB and 

FAD2 gene expression. MON 87705 contains FATB1-A and FAD2-1A gene segments 

under the control of a seed promoter, limiting oil composition modification to this tissue. 

The assembled gene transcript has an inverted repeat that produces double stranded RNA 

(dsRNA) that, via the RNA interference (RNAi) pathway, suppresses endogenous FATB

and FAD2 gene expression, thereby producing the desired fatty acid phenotype. Acylacyl carrier protein (ACP) thioesterases (referred to herein as FATB enzymes) are 

localized in plastids and hydrolyze saturated fatty acids from the ACP-fatty acid moiety. 

The suppression of FATB results in a decrease in the transport of the saturated fats out of 

the plastid, thus retaining their availability for desaturation to 18:1 oleic acid (see Figure 

I-1). Therefore, suppression of FATB decreases saturated fat content in the oil as well as 

increasing oleic acid (Kinney, 1996). Subsequently, this increased amount of oleic acid 

is either delivered to the oil body or endoplasmic reticulum for further desaturation 

(Kinney, 1996). Delta-12 desaturases (referred to as FAD2 enzymes) desaturate 18:1 

oleic acid to 18:2 linoleic acid. The suppression of FAD2 in soybean seed causes 

reduced desaturation of oleic to linoleic acid thus contributing further to the increase in 

oleic while reducing linoleic acid content in the oil (Dyer and Mullen, 2005). Therefore, 

the overall result of the suppression of these two enzymes is a reduction in saturated 16:0 

palmitic and 18:0 stearic fatty acids, an increase in monounsaturated 18:1 oleic acid, and 

lower levels of polyunsaturated 18:2 linoleic acid relative to commodity soybean. 

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is 

mediated by dsRNA molecules. Double stranded RNAs are commonly found in 

eukaryotes, including plants, for endogenous gene suppression and are composed of 

nucleic acids. Nucleic acids have a long history of safe consumption and are considered 

GRAS by the U.S. Food and Drug Administration. There is no evidence to suggest 

dietary consumption of RNA is associated with toxicity or allergenicity. Moreover, 

analysis of the DNA segments encoding this dsRNA showed that they lack the sequences 

required for translation initiation and protein synthesis. The production of a protein from 

the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several 

biotechnology-derived plant products previously deregulated by APHIS were developed 

using RNA-based suppression mechanisms, including virus-resistant papaya and squash, 

high oleic soybean, FLAVR SAVR tomatoes, and plum trees resistant to Plum pox virus 

(FDA, 1994; USDA-APHIS, 1994; USDA-APHIS, 1997; USDA-APHIS, 2006; USDAAPHIS, 2007). Based on this information, it is concluded that the inserted DNA and 

resulting dsRNA are safe and unlikely to produce a protein or polypeptide. As a result, 

the RNA-based suppression technology used in MON 87705 poses no novel risks from a 

food, feed or environmental perspective. 
Monsanto Company 09-SY-201U Page 35 of 471

To examine suppression of the RNA levels of the endogenous FAD2-1A and FATB1-A

genes, immature seed 2 at stage R5/6 were subjected to northern blot analyses and 

compared to a conventional control3 that has a genetic background similar to the test 

substance. The details of the materials and methods are described in Appendix J. 

To confirm the outcome of gene suppression on the seed fatty acid profile of 

MON 87705, fatty acid analysis was performed on MON 87705 compared to a 

conventional control. This analysis indicates the impact of the suppression of FATB and 

FAD2 genes on fatty acid profiles. The details of the full compositional analyses of 

MON 87705 are in Section VII, and the materials and methods for these analyses are 

described in Appendix E from Section VII. 

Northern blot analysis of FAD2-1A RNA level in MON 87705 confirms suppression. 

PolyA enriched RNA (PolyA+ RNA) isolated from approximately 50 μg total RNA from 

immature seed was resolved on a formaldehyde/agarose gel, blotted, and hybridized to 

the FAD2-1A probe (Figure I-2A). Approximately 200 pg of the FAD2-1A probe 

template was loaded on the gel to serve as a positive control, and the probe template 

produced a hybridization signal at approximately 0.4 kb (Figure I-2A, lane 9). 

Approximately 50 pg of actin probe template was loaded on the gel to serve as a negative 

control and, as expected, there is no hybridization signal produced (Figure I-2A, lane 10). 

The detection of the probe template hybridization control demonstrates that the probe is 

hybridizing only to the target DNA sequence. 

PolyA+ RNA from each of four replicates of the conventional soybean control immature 

seeds (Figure I-2A, lanes 1, 3, 5, and 7) produced a strong hybridization signal at 

approximately 1.5 kb which is the expected size of the FAD2-1A transcript, whereas 

polyA+ RNA isolated from each of the four replicates of MON 87705 immature seeds 

(Figure I-2A, lanes 2, 4, 6, and 8) produced a very faint hybridization signal of 

approximately 1.5 kb at a greatly reduced level relative to the conventional control RNA. 

There were faint, nonspecific hybridization signals observed at approximately 1.7 kb, 3.2 

kb, and 3.6 kb, which were detected in both the conventional soybean control and 

MON 87705. These data show a reduction in the levels of detectable FAD2-1A RNA in 

MON 87705 compared to conventional soybean. 

In order to confirm that the reduced FAD2-1A RNA levels in MON 87705 was not due to 

a reduced RNA loading on the gel or poor RNA quality, the FAD2-1A hybridization 

signal was removed and the blot was re-hybridized with the actin probe (Figure I-2B). 

The approximately 50 pg of actin probe template that was loaded on the gel as a positive 

hybridization control showed a band at approximately 0.9 kb (Figure I-2B, lane 10). 

There is a faint band at approximately 1.8 kb, which most likely resulted from 

dimerization of the actin probe template. In lane 9, there is a band at approximately 0.4 

kb, which likely results from the incomplete removal of the FAD2-1A probe template. 

 

2 For studies presented throughout this petition referring to the use of MON 87705, conventional control, or reference 

material seed, the term “seed” refers to the harvested seed.

3 For studies presented throughout this petition comparing MON 87705 to the conventional control, the conventional 

control was a nontransgenic parental soybean with genetics similar to MON 87705, but lacked the introduced traits
Monsanto Company 09-SY-201U Page 36 of 471

There is no hybridization signal with the actin probe at approximately 0.9 kb, as expected 

from a negative control (Figure I-2B, lane 9). The detection of the actin probe template 

hybridization control demonstrates that the probe is hybridizing only to the target DNA 

sequence. 

PolyA+ RNA from conventional (Figure I-2B, lanes 1, 3, 5, and 7) and MON 87705 

immature seeds (Figure I-2B, lanes 2, 4, 6, and 8) showed a strong hybridization signal at 

approximately 1.6 kb, which is expected for the actin transcript. The hybridization 

signals from conventional soybean and MON 87705 immature seeds have similar 

intensities, indicating that the RNA loading, RNA quality, and hybridization between 

conventional and MON 87705 are similar. Therefore, the difference in the FAD2-1A 

hybridization signals between conventional and MON 87705 reflects a large decrease in 

FAD2-1A RNA level. 

Northern blot analysis of FATB1-A RNA level in MON 87705 confirms suppression.

PolyA+ RNA isolated from approximately 100 μg total RNA from immature seed was 

resolved on a formaldehyde/agarose gel, blotted, and hybridized to the FATB1-A probe 

(Figure I-3A). Approximately 10 pg of the FATB1-A probe template was loaded on the 

gel to serve as a positive control. As expected, the probe template produced a 

hybridization signal at approximately 0.4 kb (Figure I-3A, lane 9). Approximately 50 pg 

of the actin probe template was loaded on the gel to serve as a negative control and, as 

expected, there is no signal produced (Figure I-3A, lane10). The detection of the 

FATB1-A probe template hybridization control demonstrates that the probe is 

hybridizing only to the target DNA sequence. 

PolyA+ RNA from four replicates of the immature seeds from conventional soybean 

(Figure I-3A, lanes 1, 3, 5, and 7) produced a strong hybridization signal at 

approximately 1.8 kb, which is expected for the FATB1-A transcript, whereas polyA+ 

RNA isolated from four replicates of MON 87705 immature seeds (Figure I-3A, lanes 2, 

4, 6, and 8) also produced a hybridization signal of approximately 1.8 kb, but at a much 

reduced level. In addition, there was a hybridization signal at approximately 1.5 kb, 

which was not present in the conventional soybean control. The approximately 1.5 kb 

signal is likely a degraded FATB1-A transcript. These data indicate a reduction in the 

RNA levels of the FATB1-A in MON 87705 compared to conventional soybean. 

In order to confirm that the difference in the hybridization of the FATB1-A probe in 

MON 87705 is not due to a reduced RNA loading on the gel or poor RNA quality, the 

FATB1-A hybridization signal was removed and the blot re-hybridized with the actin 

probe after stripping (Figure I-3B). The approximately 50 pg of actin probe template that 

was loaded on the gel as a positive control showed a band at approximately 0.9 kb 

(Figure I-3B, lane 10). The FATB1-A probe template did not produce any signal, as 

expected from a negative control (Figure I-3B, lane 9). The detection of the actin probe 

template control demonstrates that the probe is hybridizing only to the target DNA 

sequence. 

PolyA+ RNA from the conventional soybean control (Figure I-3B, lanes 1, 3, 5, and 7) 

and MON 87705 immature seed (Figure I-3B, lanes 2, 4, 6, and 8) showed a strong 

hybridization band at approximately 1.6 kb, which is expected for the actin transcript and 
Monsanto Company 09-SY-201U Page 37 of 471

the intensity of the bands are similar between the conventional control and MON 87705. 

These data indicate that the RNA loading, RNA quality, and hybridization between 

conventional soybean and MON 87705 are similar. Therefore the difference in the 

FATB1-A hybridization signals between conventional and MON 87705 reflects the 

difference in FATB1-A RNA level. 

Fatty Acid Composition of MON 87705shows predicted phenotype. 

An assessment of the seed fatty acid profile collected from five field sites in Chile during 

2007/2008 demonstrates MON 87705 has the intended fatty acid changes. MON 87705 

had a decrease in saturated fats (from 15.33% to 5.67% FA: 16:0 palmitic acid plus 18:0 

stearic acid), an increase in oleic acid (from 22.81% to 76.47% FA), and a decrease in 

linoleic acid (from 52.86% to 10.10% FA). Statistically significant differences reflecting 

intended seed fatty acid changes were observed in the combined-site analysis (Table I-1) 

and at each individual site (Appendix E). 

Conclusion 

The suppression of FATB in soybean seed decreases both of the major saturated fatty 

acids (16:0 palmitic acid and 18:0 stearic acid), and suppression of FAD2 in soybean 

seeds increases oleic acid (18:1) and subsequently decreases linoleic acid (18:2) in 

soybean oil (Dyer and Mullen, 2005). MON 87705 northern blot data confirms the 

suppression of endogenous FAD2-1A and FATB1-A RNAs. MON 87705 seed 

composition data demonstrates that suppression of these endogenous RNAs produces the 

intended alteration in fatty acid profile, which is a lower level of saturated fats (16:0 

palmitic acid and 18:0 stearic acid), a higher level of oleic acid (18:1), and an associated 

lower level of linoleic acid (18:2). 

 
Monsanto Company 09-SY-201U Page 38 of 471

Figure I-1. Schematic of the Soybean Fatty Acid Biosynthetic Pathway 

 indicates suppression of endogenous FATB and FAD2 in MON 87705 seeds
Monsanto Company 09-SY-201U Page 39 of 471

Figure I-2. Northern Blot Analysis of FAD2-1A RNA Level in MON 87705 

Panel A and Panel B are the same northern blot containing polyA+ RNA isolated from 

stage R5/6 immature seed tissue. Panel A is hybridized with the FAD2-1A probe. Panel B 

is hybridized with the actin probe after removal of the FAD2-1A signal. Arrow heads 

indicate FAD2-1A hybridization signals and stars indicate the actin hybridization signal. 

Lane designations are as follows: 

Lane 1: Conventional control (PolyA+ RNA replicate 1) 

 2: MON 87705 (PolyA+ RNA replicate 1) 

 3: Conventional control (PolyA+ RNA replicate 2) 

 4: MON 87705 (PolyA+ RNA replicate 2) 

 5: Conventional control (PolyA+ RNA replicate 3) 

 6: MON 87705 (PolyA+ RNA replicate 3) 

 7: Conventional control (PolyA+ RNA replicate 4) 

 8: MON 87705 (PolyA+ RNA replicate 4) 

 9: FAD2-1A probe template (200 pg) 

 10: Actin probe template (50 pg) 

 Symbol denotes size of RNA, in base pairs, obtained from MW markers on ethidium stained 

gel. 

 

4000

3000

2000

1500

1000

500

200

bp:

1 2 3 4 5 6 7 8 9 10

A B

*

*

6000

4000

3000

2000

1500

1000

500

200

bp:

6000

1 2 3 4 5 6 7 8 9 10

4000

3000

2000

1500

1000

500

200

bp:

1 2 3 4 5 6 7 8 9 10

A B

*

*

6000

4000

3000

2000

1500

1000

500

200

bp:

6000

1 2 3 4 5 6 7 8 9 10
Monsanto Company 09-SY-201U Page 40 of 471

Figure I-3. Northern Blot Analysis of FATB1-A RNA Level in MON 87705 

Panel A and Panel B is the same northern blot containing polyA+ RNA isolated from 

stage R5/6 immature seed tissue. Panel A is hybridized with the FATB1-A probe. Panel 

B is hybridized with the actin probe after removal of the FATB1-A signal. Arrow heads 

indicate FATB1-A hybridization signals and stars indicate the actin hybridization signal. 

Lane designations are as follows: 

Lane 1: Conventional control (PolyA+ RNA replicate 1) 

 2: MON 87705 (PolyA+ RNA replicate 1) 

 3: Conventional control (PolyA+ RNA replicate 2) 

 4: MON 87705 (PolyA+ RNA replicate 2) 

 5: Conventional control (PolyA+ RNA replicate 3) 

 6: MON 87705 (PolyA+ RNA replicate 3) 

 7: Conventional control (PolyA+ RNA replicate 4) 

 8: MON 87705 (PolyA+ RNA replicate 4) 

 9: FATB1-A probe template (10 pg) 

 10: Actin probe template (50 pg) 

Symbol denotes size of RNA, in base pairs, obtained from MW markers on ethidium stained 

gel 

 

6000

4000

3000

2000

1500

1000

500

200

bp:

1 2 3 4 5 6 7 8 9 10

6000

4000

3000

2000

1500

1000

500

200

bp:

1 2 3 4 5 6 7 8 9 10

A B

*

*
Monsanto Company 09-SY-201U Page 41 of 471

Table I-1. Summary of Product Concept Fatty Acid Levels for Test (MON 87705) 

vs. the Conventional Control (A3525) and Commercial Tolerance Interval 

Component (Units) 

MON 87705

Mean 

[Range] 

A3525 

Mean 

[Range] 

Commercial 

Tolerance 

Interval1

Statistical Differences Observed in Combined-Site Analysis Seed Fatty Acid 

(% Total FA)

16:0 Palmitic 2.36 

[2.25 - 2.44] 

10.83 

[10.51 – 11.08]

[7.62, 12.55] 

18:0 Stearic 3.31 

[3.07 - 3.82] 

4.50 

[4.24 – 4.85] 

[2.87, 7.15] 

18:1 Oleic 76.47 

[73.13 - 79.17]

22.81 

[21.41 – 25.08]

[18.40, 30.22] 

18:2 Linoleic 10.10 

[7.85 - 12.42] 

52.86 

[51.68 – 53.89]

[47.75, 56.46] 

1

With 95% confidence, interval contains 99% of the values expressed in the population of 

commercial soybean varieties 

I.D. Submissions to Other Regulatory Agencies 

Under the Coordinated Framework for Regulation of Biotechnology, the responsibility 

for regulatory oversight of biotechnology-derived crops that do not include plantincorporated protectants falls on two federal agencies: FDA and USDA. Deregulation of 

MON 87705 by USDA constitutes only one component of the overall regulatory 

oversight and review of this product. As a practical matter, MON 87705 cannot be 

released and marketed until FDA and USDA have completed their reviews and 

assessments under their respective jurisdictions. 

Submission to FDA 

MON 87705 falls within the scope of the 1992 U.S. Food and Drug Administration’s 

(FDA) policy statement concerning regulation of products derived from new plant 

varieties, including those developed through biotechnology (FDA, 1992). In compliance 

with this policy, Monsanto will initiate a consultation with the FDA on the food and feed 

safety and nutritional assessment summary for MON 87705. 

Submissions to Foreign Government Agencies 

To support commercial introduction of MON 87705 in the U.S., regulatory submissions 

will be made to countries that import significant quantities of soybean or its processed 

fractions from the U.S. and have established regulatory approval processes in place. 

These will include submissions to a number of foreign government regulatory authorities, 
Monsanto Company 09-SY-201U Page 42 of 471

including: Ministry of Agriculture, People’s Republic of China; Japan’s Ministry of 

Agriculture, Forestry, and Fisheries (MAFF) and the Ministry of Health, Labor, and 

Welfare (MHLW); the Canadian Food Inspection Agency (CFIA) and Health Canada; the 

Intersectoral Commission for Biosafety of Genetically Modified Organisms 

(CIBIOGEM), Mexico; the European Food Safety Authority (EFSA); and the regulatory 

authorities in other soybean importing countries with functioning regulatory systems. As 

appropriate, notifications of importation will be made to importing countries that do not 

have a formal approval process. 

 
Monsanto Company 09-SY-201U Page 43 of 471

II. THE SOYBEAN FAMILY 

This section summarizes the taxonomy, biology, and use of soybean based on: 1) the 

consensus document for Glycine max (L.) Merr. prepared by the Organization for 

Economic Co-operation and Development (OECD, 2000; OECD, 2001), and 2) a 

summary prepared by USDA-APHIS (2006) and a biology document published by 

Canadian Food Inspection Agency-Plant Biosafety Office (CFIA, 1996). 

II.A. Soybean as a Crop 

Soybean is the most prevalently grown oilseed in the world, with approximately 222.1 

million metric tons (MMT) of harvested seed produced in 2007, which represented 56% 

of world oilseed seed production that year (ASA, 2008; Soyatech, 2008). Soybean is 

grown as a commercial crop in over 35 countries. The major producers of soybean are 

the U.S., Brazil, Argentina, China, and India, which accounted for approximately 91% of 

the global soybean production in 2007 (Soyatech, 2008); also see Table II 1. 

Approximately one-third of the 2007 world soybean production was produced in the U.S. 

(Soyatech, 2008). The soybean produced in China and India are primarily for domestic 

use, while a significant portion of soybean produced in U.S., Brazil, and Argentina is 

traded globally in the form of soybean harvested seed, soybean meal, or soybean oil. 

Globally, the U.S. was the largest soybean seed export country, while Argentina led the 

soybean meal and soybean oil export markets in 2007 (ASA, 2008; Soyatech, 2008). 

Table II-1. World Soybean Production in 2007/2008 

Country Production (million metric tons)

U.S. 71.4 

Brazil 61.0 

Argentina 47.0 

China 15.6 

Other 8.9 

India 7.9 

Paraguay 6.2 

Canada 3.1 

EU 1.0 

Source: Soya and Oilseed Bluebook (Soyatech, 2008). 

Approximately 50% of the world soybean seed supply was crushed to produce soybean 

meal and oil in 2007 (ASA, 2008; Soyatech, 2008), and the majority was used to supply 

the feed industry for livestock use or the food industry for edible vegetable oil and 

soybean protein isolates. Another 34% of the world soybean seed supply was traded to 

other geographies, with China, EU, Japan, and Mexico being the top soybean seed import 

geographies (ASA, 2008). The remainder of the soybean seed produced was used as 

certified seed, feed, or stocks. 

Soybean is used in various food products, including tofu, soybean sauce, soymilk, energy 

bars, and meat products. A major food use for soybean is purified oil, for use in 

margarines, shortenings, cooking, and salad oils. Soybean oil generally has a smaller 
Monsanto Company 09-SY-201U Page 44 of 471

contribution to soybean’s overall value compared to soybean meal because the oil 

constitutes just 18 to 19% of the soybean's weight. Nonetheless, soybean oil accounted 

for approximately 30% of all the vegetable oils consumed globally, and was the second 

largest source of vegetable oil worldwide, slightly behind palm oil at approximately 32% 

share (Soyatech, 2008). 

Soybean meal is used as a supplement in feed rations for livestock. Soybean meal is the 

most valuable component obtained from processing the soybean, accounting for roughly 

50-75% of its overall value. By far, soybean meal is the world's most important protein 

feed, accounting for nearly 69% of world protein meal supplies (ASA, 2008). Industrial 

uses of soybean range from a carbon/nitrogen source in the production of yeasts via 

fermentation to the manufacture of soaps, inks, paints, disinfectants, and biodiesel. 

Industrial uses of soybean have been summarized by Cahoon (2003) and the American 

Soybean Association (ASA, 2008). 

Global soybean plantings reached 90.8 million hectares in 2007/08, an 8.9% increase 

over the previous four years, with an average of 82.3 million hectares planted from 

2002/03 to 2007/08 (Soyatech, 2008). Soybean production has realized, on average, a 

6.2% annual growth between 1995/96 to 2006/07. Increased planting flexibility, 

increased yield from narrow-row seeding practices, a higher rate of corn-soybean 

rotations, and low production costs favored expansion of soybean areas in the mid-1990s, 

and the expanded areas tended to be concentrated where soybean yields were highest. 

II.B. History of Soybean 

Domestication of soybean is thought to have taken place in China during the Shang 

dynasty (approximately 1500 to 1027 B.C.) or earlier (Hymowitz, 1970). However, 

historical and geographical evidence only could be traced back to the Zhou dynasty (1027 

to 221 B.C.) where the soybean was utilized as a domesticated crop in the northeastern 

part of China. By the first century A.D., the soybean probably reached Central and 

Southern China as well as peninsular Korea. The movement of soybean germplasms was 

probably associated with the development and consolidation of territories and the 

degeneration of Chinese dynasties (Ho, 1969; Hymowitz, 1970). 

From the first century A.D. to approximately the 15th and 16th centuries, soybean was 

introduced into several countries, with land races eventually developing in Japan, 

Indonesia, Philippines, Vietnam, Thailand, Malaysia, Myanmar, Nepal, and Northern 

India. The movement of soybean throughout this period was due to the establishment of 

sea and land trade routes, the migration of certain tribes from China, and the rapid 

acceptance of seeds as a staple food by other cultures (Hymowitz and Newell, 1981; 

Hymowitz et al., 1990). 

Starting in the late 16th century and throughout the 17th century, soybean was used by 

the Europeans, and in the 17th century, soybean sauce was a common item of trade from 

the East to the West. 

Soybean was introduced into North America in the 18th century. In 1851, soybean was 

introduced in Illinois and subsequently throughout the Corn Belt. In 1853, soybean seed 

were deposited at the New York State Agricultural Society, the Massachusetts 

Horticultural Society, and the Commissioner of Patents. The two societies and the 
Monsanto Company 09-SY-201U Page 45 of 471

Commissioner of Patents sent soybean seed to dozens of growers throughout the U.S. 

Soybean has been cultivated extensively and improved through conventional breeding 

following its introduction in the U.S. and subsequently has become a key source of 

nutrients for food and feed use in the U.S. (Hymowitz and Singh, 1987). 

II.C. Taxonomy and Phylogenetics of Soybean 

Cultivated soybean, Glycine max (L.) Merr., is a diploidized tetraploid (2n=40), which 

belongs to the family Leguminosae, the subfamily Papilionoideae, the tribe Phaseoleae, 

the genus Glycine Willd., and the subgenus Soja (Moench) F.J. Herm. 

Family: Leguminosae 

Subfamily: Papilionoideae 

Tribe: Phaseoleae 

Genus: Glycine

Subgenus: Soja (Moench) F.J. Herm. 

Species: max

The genus Glycine Willd. is of Asian and Australian origin and is divided into two 

subgenera, Glycine and Soja (Moench) F.J. Herm. The subgenus Glycine consists of 22 

wild perennial species, which are indigenous to Australia, West, Central and South 

Pacific Islands, China, Russia, Japan, Indonesia, Korea, Papua New Guinea, the 

Philippines, and Taiwan (Hymowitz, 2004). The subgenus Soja includes the cultivated 

soybean, G. max (L.) Merr. and its wild annual relatives from Asia, G. soja Sieb. and 

Zucc. The list of species in the genus Glycine Willd. is presented in Table II-2. 
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Table II-2. List of Species in the Genus Glycine Willd., 2n Chromosome Number, 

Genome Symbol, and Distribution 

Genus 2n Genome1 Distribution

Subgenus Glycine

 

1. G. albicans Tind. & Craven 40 I1 Australia

2. G. aphyonota B. Pfeil 40 --2 Australia

3. G. arenaria Tind. 40 HH Australia

4. G. argyrea Tind. 40 A2A2 Australia

5. G. canescens F.J. Herm. 40 AA Australia

6. G. clandestina Wendl. 40 A1A1 Australia

7. G. curvata Tind. 40 C1C1 Australia

8. G. cyrtoloba Tind. 40 CC Australia

9. G. dolichocarpa Tateishi and Ohashi 80 -- (Taiwan)

10. G. falcate Benth. 40 FF Australia

11. G. hirticaulis Tind. & Craven 40 H1H1 Australia

80 -- Australia

12. G. lactovirens Tind. & Craven. 40 I1I1 Australia

13. G. latifolia (Benth.) Newell & 

Hymowitz

40 B1B1 Australia

14. G. latrobeana (meissn.) Benth. 40 A3A3 Australia

15. G. microphylla (Benth.) Tind. 40 BB Australia

16. G. peratosa B. Pfeil & Tind. 40 -- Australia

17. G. pindanica Tind. & Craven 40 H3H2 Australia

18. G. pullenii B. Pfeil, Tind. & Craven 40 -- Australia

19. G. rubiginosa Tind. & B. Pfeil 40 -- Australia

20. G. stenophita B. Pfeil & Tind. 40 B3B3 Australia

21. G. tabacina (Labill.) Benth. 40 B2B2 Australia

80 Complex3 Australia, West Central and 

South Pacific Islands

22. G. tomentella Hayata 38 EE Australia

40 DD Australia, Papua New Guinea

78 Complex4 Australia, Papua New Guinea

80 Complex5 Australia, Papua New Guinea, 

Indonesia, Philippines, Taiwan

Subgenus Soja (Moench) F.J. Herm. 

23. G. soja Sieb. & Zucc. 40 GG China, Russia, Taiwan, Japan, 

Korea (Wild Soybean)

24. G. max (L.) Merr. 40 GG Cultigen (Soybean)

1 Genomically similar species carry the same letter symbols. 

2

 Genome designation has not been assigned to the species.

3

 Allopolyploids (A and B genomes) and segmental allopolyploids (B genomes). 

4

 Allopolyploids (D and E, A and E, or any other unknown combination). 

5

 Allopolyploids (A and D genomes, or any other unknown combination). 

Note: Table is adapted from Hymowitz (2004).

Glycine soja grows wild in China, Japan, Korea, the Russian Far East, and Taiwan, and is 

commonly found in fields, hedgerows, roadsides, and riverbanks (Lu, 2004). The plant is 
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an annual, slender in build with narrow trifoliolate leaves. The purple or very rarely 

white flowers are inserted on short, slender racemes. The pods are short and tawny with 

hirsute pubescence, producing oval-oblong seeds (Hermann, 1962). 

Glycine max (L.) Merr., the cultivated soybean, is an annual that generally exhibits an 

erect, sparsely branched, bush-type growth habit with trifoliolate leaves. The leaflets are 

broadly ovate, and the purple, pink, or white flowers are borne on short axillary racemes 

or reduced peduncles. The pods are either straight or slightly curved, and one to three 

ovoid to subspherical seeds are produced per pod. 

A third and unofficial species named G. gracilis is also described within the context of 

the Soja subgenus in addition to G. soja and G. max. The G. gracilis is known only from 

Northeast China, is intermediate in morphology between G. max and G. soja, and is 

sometimes considered a variant of G. max. The three species in the Soja subgenus can 

cross-pollinate, and the hybrid seed can germinate normally and subsequently produce 

fertile pollen and seed (Singh and Hymowitz, 1989). The taxonomic position of G. 

gracilis has been an area of debate, and neither ILDIS (International Legume Database 

and Information Service) nor USDA-GRIN (USDA Germplasm Resources Information 

Network) recognizes G. gracilis as a distinct species. The wild and weedy relatives (G. 

soja and G. gracilis) of soybean do not occur in the U.S., and, therefore, are not likely to 

contribute to the potential for outcrossing (USDA-APHIS, 2006). 

II.D. The Genetics of Soybean 

Glycine is the only genus in the tribe Phaseoleae where species have diploid chromosome 

numbers of 40 and 80, but not 20 (Lackey, 1981). The unique chromosome number of 

Glycine is probably derived from diploid ancestors with base number of 11. The 

ancestral species have undergone aneuploid reduction (loss of a specific chromosome), 

which is prevalent throughout the Papilionoideae, to a base number of 10 chromosomes 

(Lackey, 1981). Tetraploidization (2n = 2x = 40) through autopolyploidy or 

allopolyploidy of the progenitor species occurred either prior to or after dissemination 

from the ancestral region. The path of migration from a common progenitor is assumed 

by (Singh et al., 2001) as: wild perennial (2n = 4x = 40, unknown or extinct) to wild 

annual (2n = 4x = 40; G. soja) to soybean (2n = 4x = 40; G. max). Soybean should be 

regarded as a stable tetraploid with diploidized genome (Gurley, 1979; Lee and Verma, 

1984; Skorupska, 1989). 

II.E. Pollination of Cultivated Soybean 

Soybean is a self-pollinated species, propagated by seed (OECD, 2000). The 

papilionaceous flower consists of a tubular calyx of five sepals, a corolla of five petals, 

one pistil, and nine fused stamens with a single separate posterior stamen. The stamens 

form a ring at the base of the stigma and elongate one day before pollination, at which 

time the elevated anthers form a ring around the stigma (OECD, 2000). The soybean 

flower stigma is receptive to pollen approximately 24 hours before anthesis and remains 

receptive for 48 hours after anthesis. The anthers mature in the bud and directly pollinate 

the stigma of the same flower. As a result, soybean is considered to be a highly selfpollinated species, with cross-pollination to adjacent plants of other soybean varieties 
Monsanto Company 09-SY-201U Page 48 of 471

occurring at very low frequency (0 to 6.3%) in adjacent plants (Caviness, 1966; Ray et 

al., 2003; Yoshimura et al., 2006). Pollination typically takes place on the day the flower 

opens. The pollen naturally comes in contact with the stigma during the process of 

anthesis. Anthesis normally occurs in late morning, depending on the environmental 

conditions. The pollen usually remains viable for two to four hours, and no viable pollen 

can be detected by late afternoon. Natural or artificial cross-pollination can only take 

place during the short time when the pollen is viable. 

II.F. Cultivated Soybean as a Volunteer 

Cultivated soybean plants are annuals, and they reproduce solely by means of seeds. 

Volunteer soybean in rotational crops is typically not a concern in most environments 

where soybean is cultivated (CFIA, 1996; OECD, 2000). Soybean seed rarely exhibit 

any dormancy characteristics, and seed remaining in the field after harvest likely will 

readily imbibe water (Lersten and Carlson, 2004), germinate, and will be killed by frost 

or field cultivation. If they did become established, volunteer plants would not compete 

well with the succeeding crop, and could be controlled readily by either mechanical or 

chemical means (OECD, 2000). 

II.G. Characteristics of the Recipient Plant 

The soybean variety used as the recipient for the DNA insertion to create MON 87705 

was A3525, a nontransgenic conventional variety developed by Asgrow Seed Company. 

A3525 is a mid-maturity Group III soybean variety with very high yield potential. It has 

superior yields relative to lines of similar maturity and has excellent agronomic 

characteristics (Steffen, 2004). 

II.H. Soybean as a Test System in Product Safety Assessment 

In developing the data to support the safety assessment of MON 87705, A3525 was used 

as the nontransgenic conventional soybean comparator. In general, the genetic 

background of MON 87705 was matched with that of the control so the effect of the 

genetic insertion and the presence of the CP4 EPSPS protein could be assessed in an 

unbiased manner. Since MON 87705 was derived from the A3525 conventional variety, 

it was deemed appropriate to use the nontransformed A3525 as the control variety 

because its use would minimize the potential bias in subsequent comparative 

assessments. In addition, commercial conventional and Roundup Ready soybean (40-3-

2) varieties were used as reference materials to establish ranges of responses or values 

representative of commercial soybean varieties. The reference varieties used at each 

location were selected based on their availability and agronomic fit (Appendix F and 

Table F-1). 

 
Monsanto Company 09-SY-201U Page 49 of 471

III. DESCRIPTION OF THE TRANSFORMATION SYSTEM 

MON 87705 was developed through Agrobacterium-mediated transformation of soybean 

meristem tissue using the double-border, binary vector PV-GMPQ/HT4404 (Section IV, 

Figure IV-1). The vector, PV-GMPQ/HT4404, contains both the left and right border 

sequences flanking the transfer DNA (T-DNA) to facilitate transformation. 

This vector, PV-GMPQ/HT4404, is approximately 13.1 kb and contains two T-DNAs, 

each delineated by left and right border regions. The first T-DNA, designated T-DNA I, 

contains a cp4 epsps expression cassette and a partial suppression cassette. The cp4 

epsps expression cassette is under the regulation of FMV/Tsf1 chimeric promoter and E9

polyadenylation sequence. The partial suppression cassette in T-DNA I contains the 

sense segments of the FAD2-1A intron and FATB1-A 5' UTR, including the chloroplast 

targeting sequence, that are under the regulation of the seed 7Sα' promoter. The second 

T-DNA, designated T-DNA II, contains a partial suppression cassette that consists of the 

antisense segments of FAD2-1A intron and FATB1-A 5′ UTR, which is flanked by the H6

untranslated sequence. During plant transformation, the two T-DNAs co-integrated at 

one locus in the soybean genome, creating a DNA insert that contains a cp4 epsps cassette 

and a single FAD2-1A and FATB1-A suppression cassette. 

The Agrobacterium-mediated soybean transformation to produce MON 87705 was based 

on the method described by Martinell et al. (2002), which allows the generation of 

transformed plants without utilization of callus. Briefly, meristem tissues were excised 

from the embryos of germinated A3525 seed. After co-culturing with Agrobacterium

carrying the vector, the meristems were placed on selection medium containing 

glyphosate to inhibit the growth of untransformed plant cells, as well as carbenicillin and 

Claforan to inhibit excess Agrobacterium. The meristems were then placed in media 

conducive to shoot and root development, and only rooted plants with normal phenotypic 

characteristics were selected and transferred to soil for growth and further assessment.

The R0 plants generated through this transformation were self-pollinated, and the 

subsequent R1 plants were screened for the zygosity of inserted gene. Only the R1 plants 

that were homozygous for the insertion, as determined by quantitative PCR, and 

produced seeds with the desired fatty acid composition were advanced for development. 

Their progeny were subjected to further phenotypic assessments. MON 87705 was 

selected as the lead event based on superior phenotypic characteristics, agronomics, and 

molecular profile. Regulatory studies on MON 87705 were initiated to further 

characterize the genetic insertion and the expressed protein, and to establish the food, 

feed, and environmental safety relative to conventional soybean. The major steps 

involving the development of MON 87705 are depicted in Figure III-1. 
Monsanto Company 09-SY-201U Page 50 of 471

Figure III-1. Schematic of the Development of MON 87705 

 
Monsanto Company 09-SY-201U Page 51 of 471

IV. GENETIC ELEMENTS 

This section describes the vector, the donor genes and the regulatory elements used in the 

development of MON 87705 and the deduced amino acid sequence of the CP4 EPSPS 

protein produced in MON 87705. In this section, T-DNA refers to DNA that is 

transferred to the plant during transformation. An expression cassette is comprised of 

sequences to be transcribed and the regulatory elements necessary for the expression of 

those sequences. The suppression cassette refers to the sequences and regulatory 

elements necessary for the suppression of the endogenous FAD2 and FATB RNA 

transcripts. 

IV.A. Vector GMPQ/HT4404 

The PV-GMPQ/HT4404 vector used for the transformation of soybean to produce 

MON 87705 is shown in Figure IV-1 and its elements described in Table IV-1. This 

vector is approximately 13.1 kb and contains two T-DNAs, each delineated by left and 

right border regions. T-DNA I contains a cp4 epsps expression cassette and a partial 

suppression cassette. The cp4 epsps expression cassette is under the regulation of 

FMV/Tsf1 chimeric promoter and E9 polyadenylation sequence. The partial suppression 

cassette in T-DNA I contains the sense segments of the FAD2-1A intron and FATB1-A 5' 

UTR, including the chloroplast targeting sequence, which are under the regulation of the 

seed 7Sα' promoter. T-DNA II contains a partial suppression cassette, which consists of 

the antisense segment of FAD2-1A intron and FATB1-A 5′ UTR that is flanked by the H6

untranslated sequence. During plant transformation, a portion of the plants that were 

generated contained the two T-DNAs co-integrated at one locus in the soybean genome 

creating a DNA insert that contains a cp4 epsps cassette and a single FAD2-1A and 

FATB1-A suppression cassette. 

The vector backbone region outside of the T-DNA contains two origins of replication for 

maintenance of plasmid in bacteria (OR-oriV, OR-ori-PBR322), a bacterial selectable 

marker gene (aadA), and a coding sequence for repressor of primer protein for 

maintenance of plasmid copy number in E. coli (rop). A description of the genetic 

elements and their prefixes (e.g., P-, L-, I-, TS-, OR-, B-, CS-, and T-) in 

PV-GMPQ/HT4404 is provided in Table IV-1. 

IV.B. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA I) 

The cp4 epsps gene expression cassette is present in MON 87705. The cp4 epsps coding 

sequence encodes a 47.6 kDa CP4 EPSPS protein consisting of a single polypeptide of 

455 amino acids (Padgette et al., 1996b). The cp4 epsps gene expression cassette was 

used as a selectable marker during the transformation to produce MON 87705. The CP4 

EPSPS protein is similar and functionally identical to endogenous plant EPSPS enzymes, 

but has a much reduced affinity for glyphosate, the active ingredient in Roundup 

herbicides, relative to endogenous plant EPSPS (Padgette et al., 1996a). The CP4 EPSPS 

protein confers resistance to glyphosate and has been used safely and successfully in 

many Roundup Ready crops, such as canola, corn, cotton, soybean, and sugar beet. The 

deduced CP4 EPSPS protein full-length amino acid sequence is shown in Figure IV-3. 
Monsanto Company 09-SY-201U Page 52 of 471

IV.C. The FAD2-1A and FATB1-A segments Sequence (T-DNA I and II) 

MON 87705 contains a partial sequence of the soybean (Glycine max) FAD2-1A gene 

and FATB1-A gene. The FAD2-1A and FATB1-A gene segments are comprised of ~ 0.6 

kb of sequence from the FAD2-1A intron and the FATB1-A 5′ UTR, and form the 

MON 87705 suppression cassette. This suppression cassette expresses an inverted repeat 

of the FAD2-1A and FATB1-A gene segments. The assembled gene transcript has an 

inverted repeat that produces double stranded RNA (dsRNA) that, via the RNA 

interference (RNAi) pathway, suppresses endogenous FATB and FAD2 RNA, and, 

ultimately, an improved fatty acid composition in the seed. 

IV.D. Regulatory Sequences 

The cp4 epsps coding sequence that is located in T-DNA I, is under the regulation of the 

FMV/Tsf1 promoter, the Tsf1 leader and intron, the CTP2 targeting sequence and the E9 

3' untranslated sequence. The FMV/Tsf1 is a chimeric promoter consisting of enhancer 

sequences from the promoter of the Figwort Mosaic virus 35S RNA (Richins et al., 1987) 

combined with the promoter from the Tsf1 gene of Arabidopsis thaliana that encodes 

elongation factor EF-1 alpha (Axelos et al., 1989). The Tsf1 leader is the 5’ untranslated 

region from the Arabidopsis thaliana Tsf1 gene (Axelos et al, 1989) that encodes the 

elongation factor EF-1 alpha. The CTP2 targeting sequence is the sequence encoding the 

transit peptide from the ShkG gene of Arabidopsis thaliana (Klee et al., 1987) and is 

present to direct the CP4 EPSPS protein to the chloroplast. The E9 3′ untranslated region 

is the 3' untranslated sequence from the RbcS2 gene of Pisum sativum (Coruzzi et al., 

1984) and is present to direct polyadenylation of the cp4 epsps transcript. 

Also located in T-DNA I is a partial suppression cassette with a portion of the FAD2-1A 

intron and a portion of the FATB1-A 5' UTR, including the plastid targeting sequence 

under the regulation of the Glycine max 7Sα' seed storage gene promoter (designated 

Sphas 1) which drives expression in immature seeds (Doyle et al., 1986). 

T-DNA II contains another partial suppression cassette with the FAD2-1A and FATB1-A

gene segments followed by the H6 3' untranslated sequence. The H6 3' untranslated 

region is from the Gossypium barbadense cotton fiber gene and is present to terminate 

transcription (John and Keller, 1995). 

IV.E. T-DNA Borders 

Plasmid PV-GMPQ/HT4404 contains right border and left border regions (Figure IV-1 

and Table IV-1) that were derived from Agrobacterium tumefaciens plasmids (Barker et 

al., 1983; Zambryski et al., 1982). The border regions each contain a 24-25 bp nick site 

that is the site of DNA exchange during transformation. The border regions delineate the 

T-DNA and are involved in their efficient transfer into the soybean genome. Because 

PV-GMPQ/HT4404 is a two T-DNA vector, it contains two right border regions and two 

left border regions, where one set flank T-DNA I and the other set flank T-DNA II (see 

description above). 
Monsanto Company 09-SY-201U Page 53 of 471

IV.F. Genetic Elements Outside of the T-DNA Borders 

Genetic elements that exist outside of the T-DNA borders are those that are essential for 

the maintenance and selection of the vector PV-GMPQ/HT4404 in bacteria. The origin 

of replication OR-ori V is required for the maintenance of the plasmid in Agrobacterium

(Stalker et al., 1981) and is derived from the broad host plasmid RK2. The origin of 

replication OR-ori.pBR322 is required for the maintenance of the plasmid in E. coli and 

is derived from the plasmid pBR322 (Sutcliffe, 1979). CS-rop is the coding sequence of 

the repressor of primer (ROP) protein and is necessary for the maintenance of plasmid 

copy number in E. coli (Giza and Huang, 1989). The selectable marker aadA is a 

bacterial promoter and coding sequence for an enzyme from transposon Tn7 that confers 

spectinomycin and streptomycin resistance (Fling et al., 1985) in E. coli and 

Agrobacterium during molecular cloning. Because these elements are outside the border 

regions, they are not expected to be transferred into the soybean genome. The absence of 

the backbone sequence in MON 87705 has been confirmed by Southern blot analyses 

(see Section V.B.). 
Monsanto Company 09-SY-201U Page 54 of 471

Probe DNA Probe Start Position Stop Position 

Total Length 

(~kb) 

1 T-DNA I Probe 1A 7657 9406 1.8

2 T-DNA I Probe 2B 9270 10042 0.8

3 T-DNA I Probe 3C 9943 11325 1.4

4 T-DNA I Probe 4D 11151 160 2.1

5 T-DNA I Probe 5E 13080 973 1.0

6 T-DNA II Probe 1A 5570 6693 1.1

Figure IV-1. Circular Map of Plasmid PV-GMPQ/HT4404 showing probes 1-6 

Plasmid PV-GMPQ/HT4404 containing the T-DNAs used in Agrobacterium-mediated 

transformation to produce MON 87705. Genetic elements (depicted in the exterior of the 

map) and restriction sites for enzymes used in Southern analyses (with positions relative 

to the size of the plasmid vector) are shown on the exterior of the map. The T-DNA I and 

T-DNA II probes used in the Southern analyses (labeled 1-6 within the interior of the 

map) are detailed in the accompanying table. 

PV-GMPQ/HT4404

13088 bp

Nco I 9850

Spe I 12631

Xho I 13088

B-Right Border

aadA

OR-ori.pBR322

CS-rop

B-Left Border

B-Right Border T-H6

OR-ori V

B-Left Border

P-FMV/Tsf1

L-Tsf1

I-Tsf1

TS-CTP2

CS-cp4 epsps

T-E9

P-7Sα'

FAD2-1Ap FATB1Ap

FATB1Ap

FAD2-1Ap

1

2

3

4

5

6

T-DNA II

T-DNA I
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Probe DNA Probe Start Position Stop Position 

Total Length 

(~kb) 

7 Backbone Probe 1 974 2280 1.3

8 Backbone Probe 2 2140 4080 1.9

9 Backbone Probe 3 3631 5127 1.5

10 Backbone Probe 4 7025 7656 0.6

Figure IV-2 Plasmid Map of Vector PV-GMPQ/HT4404 Showing Probes 7-10 

Plasmid PV-GMPQ/HT4404 containing the T-DNAs used in Agrobacterium-mediated 

transformation to produce MON 87705. Genetic elements (depicted in the exterior of the 

map) and restriction sites for enzymes used in Southern analyses (with positions relative 

to the size of the plasmid vector) are shown on the exterior of the map. The backbone 

probes used in the Southern analyses (labeled 7-10 within the interior of the map) are 

detailed in the accompanying table. 

PV-GMPQ/HT4404

13088 bp

Nco I 9850 

Spe I 12631

Xho I 13088

B-Right Border

aadA

OR-ori.pBR322

CS-rop

B-Left Border

B-Right Border T-H6

OR-ori V

B-Left Border

P-FMV/Tsf1

L-Tsf1

I-Tsf1

TS-CTP2

CS-cp4 epsps

T-E9

P-7Sα'

FAD2-1A FATB1Ap p

FATB1Ap

FAD2-1Ap

T-DNA II

T-DNA I

7

8

9

10
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Table IV-1. Summary of Genetic Elements in Plasmid Vector PV- GMPQ/HT4404 

Genetic Element 

Location in 

Plasmid Function (Reference) 

T-DNA I

B1

-Left Border 7657 – 8098 

DNA region from Agrobacterium tumefaciens

containing the left border sequence used for 

transfer of the T-DNA (Barker et al., 1983) 

Intervening Sequence 8099 – 8134 Sequence used in DNA cloning 

P2

-FMV/Tsf1 8135 – 9174 

Chimeric promoter consisting of enhancer sequences 

from the promoter of the Figwort Mosaic virus 35S 

RNA (Richins et al., 1987) combined with the 

promoter from the Tsf1 gene of Arabidopsis thaliana

that encodes elongation factor EF-1alpha (Axelos et 

al., 1989) 

L3

-Tsf1 9175 – 9220 

5' untranslated leader (exon 1) from the 

Tsf1 gene of Arabidopsis thaliana that encodes 

elongation factor EF-1 alpha (Axelos et al., 1989) 

I

4

-Tsf1 9221 – 9842 

Intron with flanking exon sequence from the Tsf1

gene of Arabidopsis thaliana that encodes 

elongation factor EF-1alpha (Axelos et al., 1989) 

Intervening Sequence 9843 – 9851 Sequence used in DNA cloning 

TS5

-CTP2

9852 – 10079 

Targeting sequence from the ShkG gene encoding 

the transit peptide region of Arabidopsis thaliana

EPSPS (Klee et al., 1987) that directs transport of 

the CP4 EPSPS protein to the chloroplast 

CS6

-cp4 epsps 10080 –11447 

Codon modified coding sequence of the aroA gene 

from the Agrobacterium sp. strain CP4 encoding 

the CP4 EPSPS protein (Barry et al., 1997; 

Padgette et al., 1996a) 

Intervening Sequence 11448 – 11505 Sequence used in DNA cloning 

T7

-E9 11506 – 12148 

3′ untranslated region of the pea RbcS2 gene 

which functions to direct polyadenylation of the 

mRNA (Coruzzi et al., 1984) 

Intervening Sequence 12149 – 12236 Sequence used in DNA cloning 

P-7Sα′ 12237 – 13077 

Promoter and leader from the Sphas1 gene of 

Glycine max encoding beta-conglycinin storage 

protein (alpha'-bcsp) (Doyle et al., 1986) that 

directs transcription in seed 

B1 – Border; P2 – Promoter; L3– Leader; I4 – Intron; TS5 – Targeting Sequence; CS6 – Coding Sequence;

T7

 – 3' untranslated transcriptional termination sequence and polyadenylation signal sequences; 

 
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Table IV-1 (cont.). Summary of Genetic Elements in Plasmid Vector PVGMPQ/HT4404 

Intervening Sequence 13078 – 11 Sequence used in DNA cloning 

FAD2-1Ap

12 – 277 

Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 

desaturase (Fillatti et al., 2003) which forms part 

of the suppression cassette 

FATB1-Ap

278 – 578 

Partial sequence from the 5' untranslated region 

and the plastid targeting sequence from Glycine 

max FATB1-A gene that encodes the palmitoyl 

acyl carrier protein thioesterase (Fillatti et al., 

2003) which forms part of the suppression cassette 

Intervening Sequence 579 – 616 Sequence used in DNA cloning 

B-Right Border 617 – 973 

DNA region from Agrobacterium tumefaciens

containing the right border sequence used for 

transfer of the T-DNA (Zambryski et al., 1982) 

Vector Backbone 

Intervening Sequence 974 – 1109 Sequence used in DNA cloning 

aadA

1110 – 1998 

Promoter, coding sequence, and 3' UTR for an 

aminoglycoside-modifying enzyme, 3''(9)-Onucleotidyltransferase from the transposon Tn7 

(Fling et al., 1985) that confers spectinomycin and 

streptomycin resistance 

Intervening Sequence 1999 – 2528 Sequence used in DNA cloning 

OR8-ori-pBR322 2529 – 3117 

Origin of replication from pBR322 for 

maintenance of plasmid in E. coli (Sutcliffe, 1979) 

Intervening Sequence 3118 – 3544 Sequence used in DNA cloning 

CS-rop 3545 – 3736 

Coding sequence for repressor of primer protein 

from the ColE1 plasmid for maintenance of 

plasmid copy number in E. coli (Giza and Huang, 

1989) 

Intervening Sequence 3737 – 5127 Sequence used in DNA cloning 

T-DNA II 

B-Left Border 5128 – 5569 

DNA region from Agrobacterium tumefaciens

containing the left border sequence used for transfer 

of the T-DNA (Barker et al., 1983) P

 – Partial sequence; OR8 – Origin of Replication. 
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Table IV-1 (cont.). Summary of Genetic Elements in Plasmid Vector PVGMPQ/HT4404 

Intervening Sequence 5570 – 5667 Sequence used in DNA cloning 

T-H6 5668 – 6103 

3' UTR sequence of the H6 gene from Gossypium 

barbadense encoding a fiber protein involved in 

secondary cell wall assembly (John and Keller, 

1995) 

Intervening Sequence 6104 – 6115 Sequence used in DNA cloning 

FAD2-1Ap 6116 – 6381 

Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 desaturase 

(Fillatti et al., 2003) which forms part of the 

suppression cassette 

FATB1-Ap 6382 – 6682 

Partial sequence from the 5' untranslated region and 

the plastid targeting sequence from Glycine max

FATB1-A gene that encodes the palmitoyl acyl 

carrier protein thioesterase (Fillatti et al., 2003) 

which forms part of the suppression cassette 

Intervening Sequence 6683 – 6693 Sequence used in DNA cloning 

B-Right Border 6694 – 7024 

DNA region from Agrobacterium tumefaciens

containing the right border sequence used for transfer 

of the T-DNA (Zambryski et al., 1982) 

Vector Backbone 

Intervening Sequence 7025 – 7173 Sequence used in DNA cloning 

OR-ori V 7174 – 7570 

Origin of replication from the broad host range 

plasmid RK2 for maintenance of plasmid in 

Agrobacterium (Stalker et al., 1981). 

Intervening Sequence 7571 – 7656 Sequence used in DNA cloning 

 MLHG ASSRPATARK SSGLSGTVRI PGDKSISHRS FMFGGLASGE TRITGLLEGE DVINTGKAMQ AMGARIRKEG 

DTWIIDGVGN GGLLAPEAPL DFGNAATGCR LTMGLVGVYD FDSTFIGDAS LTKRPMGRVL NPLREMGVQV KSEDGDRLPV 

TLRGPKTPTP ITYRVPMASA QVKSAVLLAG LNTPGITTVI EPIMTRDHTE KMLQGFGANL TVETDADGVR TIRLEGRGKL 

TGQVIDVPGD PSSTAFPLVA ALLVPGSDVT ILNVLMNPTR TGLILTLQEM GADIEVINPR LAGGEDVADL RVRSSTLKGV 

TVPEDRAPSM IDEYPILAVA AAFAEGATVM NGLEELRVKE SDRLSAVANG LKLNGVDCDE GETSLVVRGR PDGKGLGNAS 

GAAVATHLDH RIAMSFLVMG LVSENPVTVD DATMIATSFP EFMDLMAGLG AKIELSDTKA A 

Figure IV-3. Deduced Amino Acid Sequence of the Mature CP4 EPSPS Protein in 

PV-GMPQ/HT4404 

The amino acid sequence of the CP4 EPSPS protein was deduced from the full-length 

coding nucleotide sequence present in vector PV-GMPV/HT4404. 

 
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V. GENETIC ANALYSIS 

A multi-faceted approach was taken to characterize the genetic modification made to 

produce MON 87705. The results confirm that MON 87705 contains a single insert that 

is stably integrated and is inherited according to Mendelian principles over multiple 

generations. These conclusions were based on several lines of evidence: 1) Southern blot 

analyses to assay the entire soybean genome for the presence of DNA derived from the 

transformation plasmid PV-GMPQ/HT4404, and to confirm that a single copy of the cp4 

epsps expression cassette and the FAD2-1A and FATB1-A suppression cassette was 

inserted at a single site and that the insert is stably inherited; 2) DNA sequencing 

analyses to determine the exact sequence of the inserted DNA and allow a comparison to 

the T-DNA sequence in the transformation vector to confirm that only the expected 

sequences were integrated; and 3) a comparison of the genomic DNA flanking the insert 

to the sequence of the insertion site in conventional soybean to identify any 

rearrangements that occurred at the insertion site during transformation. Taken together, 

the characterization of the genetic modification demonstrates that a single copy of the TDNA was inserted at a single locus of the genome. 

Southern blot analyses were used to determine the number of copies and the insertion 

sites of the integrated DNA as well as the presence or absence of plasmid backbone 

sequence. The Southern blot strategy was designed to ensure that all potential transgenic 

segments would have been identified. The entire soybean genome was assayed with 

probes that spanned the complete transformation plasmid to detect the presence of the 

insertion as well as confirm the absence of any backbone sequence. This was 

accomplished by using probes that were less than 2 kb in length ensuring a high level of 

sensitivity. This high level of sensitivity was demonstrated for each blot by detection of a 

positive control added at the equivalent of 1/10th of a genome. Two restriction enzyme 

sets were specifically chosen to independently confirm the presence of the insert. This 

two enzyme design also maximizes the possibility of detecting an insertion elsewhere in 

the genome which could be missed if that band comigrated with an expected band. 

Additionally, the restriction enzyme sets were chosen such that at least one enzyme from 

each set resides in the known 5′ or 3′ flanking sequence and that together the enzyme sets 

result in overlapping segments covering the entire insert. Therefore, at least one segment 

for each flank is of a predictable size and overlaps with another predictable size segment. 

This overlapping strategy confirms that the entire insert sequence is identified in a 

predictable hybridization pattern. 

The results of these analyses of MON 87705 show that a single copy of the T-DNA is 

inserted at a single locus of the genome. Generational stability analysis demonstrated 

that an expected Southern blot fingerprint of MON 87705 has been maintained through 

four generations of the breeding history, thereby confirming the stability of the insert. 

Results from segregation analyses show heritability and stability of the insert occurred as 

expected across multiple generations, which corroborates the molecular insert stability 

analysis and establishes the genetic behavior of the DNA insert at a single chromosomal 

locus. 

For each digest used to confirm copy number there were duplicated samples that 

consisted of equal amounts of digested DNA. One set of samples was run for a longer 
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period of time (long run) than the second set (short run). The long run allows for greater 

resolution of large molecular weight DNA, whereas the short run allows the detection of 

small molecular weight DNA. For estimating the sizes of bands present in the long run 

lanes of Southern blots, the molecular weight markers on the left of the figure were used. 

For estimating the sizes of bands present in the short run lanes, the molecular weight 

markers on the right of the figure were used. 

The DNA sequencing analyses complement the Southern analyses. Southern analyses 

determined that MON 87705 contains T-DNA I and T-DNA II-derived sequences at a 

single insertion site. Sequencing of the insert and the flanking genomic DNA confirmed 

the organization of the elements within the insert, determined the 5' and 3' insert-to-plant 

junctions, determined the complete DNA sequence of the insert and adjacent soybean 

genomic DNA, and confirmed that the genomic DNA sequences flanking the 5' and 3' 

ends of the insert in MON 87705 are native to the soybean genome. Each genetic element 

is intact and the sequence of the insert matches the corresponding sequence in 

PV-GMPQ/HT4404. In addition, genomic rearrangements at the insertion site were 

assessed by comparing the insert and flanking sequence to the insertion site in 

conventional soybean. 

The stability of the DNA insert across multiple generations (R3-R6) was also 

demonstrated by Southern blot fingerprint analyses. Four generations of MON 87705 

were digested with one of the enzyme sets used for the copy number analysis and were 

hybridized with probes that would detect restriction segments that encompass the entire 

insert (two hybridization bands). This fingerprint strategy consists of two border 

segments that assess not only the stability of the insert, but also the stability of genomic 

DNA directly adjacent to the insert. 

The Southern blot analysis confirmed the insert reported in Figure V-1 represents the 

only detectable insert in MON 87705. The genetic elements integrated in MON 87705 

are summarized in Table V-2. Maps of plasmid vector PV-GMPQ/HT4404, used in the 

transformation to produce MON 87705 and annotated with the probes used in the 

Southern analysis are presented in Figures IV-1 and IV-2. Figure V-1 shows a linear map 

depicting restriction sites within the insert as well as within the known soybean genomic 

DNA immediately flanking the insert in MON 87705. Based on the linear map of the 

insert and the plasmid map, a table summarizing the expected DNA segments for 

Southern analyses is presented in Table V-1. In some of the Southern blots, the 

migration of the genomic DNA is slightly different when compared to the migration of 

the molecular weight markers. These altered migrations are likely the result of different 

base pair composition and/or differences in salt concentration between the genomic DNA 

samples and the molecular weight marker (Sambrook, 1989). The generations used in 

these studies are depicted in the breeding history shown in Figure V-7. Materials and 

methods used for characterization of the insert for MON 87705 are found in Appendix B. 
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Figure V-1. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87705 A linear map of the insert and genomic DNA flanking the insert in MON 87705 is shown. Identified on the map 

within the insert, as well as restriction sites with positions relative to the size of the linear map for enzymes used analyses. Shown on the lower portion of the map are the expected sizes of the DNA segments after digestion witrestriction enzymes. Arrowheads ( ) indicate the end of the insert and the beginning of soybean genomic flanarrows (→) indicated the sequence direction of the elements in MON 87705. The * indicates partial sequences f

and Right Border sequences after integration into MON 87705 (Table V-2). Base pairs 906-3279 in the 5’ flanki

and 10535-12908 in the 3’ flanking genomic DNA represent duplicated bases from the 3′ end of the flanking soy

(see Section V.C). 
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Table V-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction Enz

MON 87705 Analysis 

Southern blot 

 Figure 

V-2 V-3 V-4 V-5 V-6 

Probes Used in 

Analysis 1, 4, 6 2, 5 3 7, 9 8, 10 

Positive Hybridization Controls  Xho I + Nco I Digested 

Plasmid ~3.2, ~9.9 kb ~3.2, ~9.9 kb ~3.2 kb ~9.9 kb ~9.9 kb 

Probe Templates 1 ~1.8, ~2.1, and ~1.1 kb ~0.8 and ~1.0 kb ~~2 ~1.3 and ~1.5 kb ~1.9 and ~0.6 kb ~MON 87705 DNA DigestionNco I ~4.0 and ~5.7 kb ~4.0 and ~5.7 kb ~5.7 kb No band No band ~Spe I > 8.1* and ~5.0kb > 8.1* and ~5.0kb > 8.1* kb No band No band 1 probe templates were added to predigested conventional soybean DNA when multiple probes are used in Southern blot analysis. 2 ‘~~’ indicates that the plasmid template was the only positive control used, because the Southern blot was hybridized with one probe.3 ‘--’ indicates that the particular restriction enzyme was not used in the analysis.*Southern analysis indicates this segment to be ~ 11 kb.
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Table V-2. Summary of Genetic Elements in MON 87705 

Genetic Element1

Location in 

Sequence2 Function (Reference) 

Unique 5' flanking 

sequence of the insert 1 – 905 Soybean genomic DNA 

Sequence flanking 5' end 

of the insert 906-3279 

2374 bp of soybean genomic DNA duplicated from 

the 3' end of the flanking sequence of the insert 

B3-Left Border 3280 – 3538 

259 bp sequence from the B-Left Border region 

remaining after integration (Barker et al., 1983) 

Intervening Sequence 3539 – 3574 Sequence used in DNA cloning 

P4

-FMV/Tsf1 3575 – 4614 

Chimeric promoter consisting of enhancer 

sequences from the promoter of the Figwort Mosaic 

virus 35S RNA (Richins et al., 1987) combined 

with the promoter from the Tsf1 gene of 

Arabidopsis thaliana that encodes elongation factor 

EF-1 alpha (Axelos et al., 1989) 

L5

-Tsf1 4615 – 4660 

5' untranslated leader (exon 1) from the 

Tsf1 gene of Arabidopsis thaliana that encodes 

elongation factor EF-1 alpha (Axelos et al., 1989) 

I

6

-Tsf1 4661 – 5282 

Intron with flanking exon sequence from the Tsf1

gene of Arabidopsis thaliana that encodes 

elongation factor EF-1 alpha (Axelos et al., 1989) 

Intervening Sequence 5283 – 5291 Sequence used in DNA cloning 

TS7

-CTP2 5292 – 5519 

Targeting sequence from the ShkG gene encoding 

the transit peptide region of Arabidopsis thaliana

EPSPS (Klee et al., 1987) that directs transport of 

the CP4 EPSPS protein to the chloroplast 

CS8

-cp4 epsps 5520 – 6887 

Codon modified coding sequence of the aroA gene 

from the Agrobacterium sp. strain CP4 encoding 

the CP4 EPSPS protein (Barry et al., 1997; 

Padgette et al., 1996c) 

Intervening Sequence 6888 – 6945 Sequence used in DNA cloning 

3

B – Border; P4 – Promoter; L5 – Leader;I

6 – Intron; TS7 – Targeting Sequence; CS8 – Coding Sequence; 

 
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Table V-2 (cont.) Summary of Genetic Elements in MON 87705

Genetic Element 

Location in 

Sequence Function (Reference) 

T9-E9 6946 – 7588 

3' untranslated region of the pea RbcS2 gene which 

functions to direct polyadenylation of the mRNA 

(Coruzzi et al., 1984) 

Intervening Sequence 7589 – 7676 Sequence used in DNA cloning 

P-7Sα′ 7677 – 8517 

Promoter and leader from the Sphas1 gene of 

Glycine max encoding beta-conglycinin storage 

protein (alpha'-bcsp) (Doyle et al., 1986) that 

directs transcription in seed 

Intervening Sequence 8518 – 8539 Sequence used in DNA cloning 

FAD2-1Ap 8540 – 8805 

Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 

desaturase (Fillatti et al., 2003) which forms part 

of the suppression cassette 

FATB1-Ap 8806 - 9106 

Partial sequence from the 5' untranslated region 

and the plastid targeting sequence from Glycine 

max FATB1-A gene that encodes the palmitoyl 

acyl carrier protein thioesterase (Fillatti et al., 

2003) which forms part of the suppression cassette 

Intervening Sequence 9107 - 9114 Sequence used in DNA cloning 

B-Right Border 9115 – 9134 

20 bp sequence from the B-Right Border region 

remaining after integration (Zambryski et al., 

1982) 

B-Left Border 9135 - 9172 

38 bp sequence from the B-Left Border region 

remaining after integration (Barker et al., 1983) 

FATB1-Ap1 9173 – 9443 

Partial sequence from the 5' untranslated region 

and the plastid targeting sequence from Glycine 

max FATB1-A gene that encodes the palmitoyl 

acyl carrier protein thioesterase (Fillatti et al., 

2003) which forms part of the suppression cassette 

FAD2-1Ap 9444 – 9709 

Partial sequence from intron #1 of the Glycine max

FAD2-1A gene that encodes the delta-12 

desaturase (Fillatti et al., 2003) which forms part 

of the suppression cassette 

Intervening Sequence 9710 – 9721 Sequence used in DNA cloning 

T9 – 3' untranslated transcriptional termination sequence and polyadenylation signal sequences; P

 – Partial 

sequence; P1 – Truncated partial sequence of FATB1-A. 

 
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Table V-2 (cont.) Summary of Genetic Elements in MON 87705

Genetic Element 

Location in 

Sequence Function (Reference) 

T-H6 9722 – 10157 

3' UTR sequence of the H6 gene from Gossypium 

barbadense encoding a fiber protein involved in 

secondary cell wall assembly (John and Keller, 

1995) 

Intervening Sequence 10158 –10255 Sequence used in DNA cloning 

B-Left Border 10256 - 10530 

275 bp sequence from the B-Left Border region 

remaining after integration (Adang et al., 1985) 

Sequence flanking 3' end 

of the insert 10531 – 12908 

Soybean genomic DNA including the 2374 bases 

duplicated at the 5' end of the flanking sequence 

of the insert 

Unique 3' flanking 

sequence of the insert 12909 – 13243 Soybean genomic DNA 

V.A. Copy Number of T-DNA I and T-DNA II in MON 87705 

The copy number and insertion sites of T-DNA I and T-DNA II were assessed by 

digesting test DNA with restriction enzymes Nco I or Spe I and hybridizing Southern 

blots with probes that span T-DNA I and T-DNA II (Figures IV-1 and IV-2). Each 

restriction digest is expected to produce a specific banding pattern on the Southern blots 

(Table V-1). Since each detected segment contains flanking genomic DNA, any 

additional integrated sites would produce a different banding pattern with additional 

bands. 

The restriction enzyme Nco I cuts once in the MON 87705 insert and once in each of the 

known 5' and 3' flanking sequences of MON 87705. Therefore, if T-DNA I and 

T-DNA II sequences are present at a single integration site in MON 87705, the digestion 

with Nco I was expected to generate two border segments with expected sizes of ~4.0 kb 

and ~5.7 kb (Figure V-1). The ~4.0 kb restriction segment contains genomic DNA 

flanking the 5' end of the insert, the Left Border, the FMV/Tsf1 promoter, the Tsf1 leader, 

and the Tsf1 intron. The ~5.7 kb restriction segment contains the CTP2 targeting 

sequence, cp4 epsps coding sequence, E9 3' untranslated sequence, 7Sα' promoter, FAD2-

1A and FATB1-A sense sequences, partial sequences of Right Border and Left Border, 

FATB1-A and FAD2-1A antisense sequences, H6 3' untranslated sequence, Left Border 

and genomic DNA flanking the 3' end of the insert. 

The restriction enzyme Spe I cuts once in the MON 87705 insert and once in the known 

3' flanking sequence of MON 87705. Therefore, if T-DNA I and T-DNA II sequences 

are present at a single integration site in MON 87705 digestion with Spe I is expected to 

release two border segments with expected sizes of ~5.0 kb and greater than 8.1 kb 

(Figure V-1). Since the Spe I site in the soybean genome flanking the 5' end of the insert 

lies outside of the known sequence, it was not possible to predict a precise segment size. 
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However, the segment size was determined by Southern blot analyses to be ~11 kb 

(Figures V-2, V-3, and V-4). The ~11 kb DNA segment contains genomic DNA flanking 

the 5' end of the insert, Left Border, FMV/Tsf1 promoter, Tsf1 leader, Tsf1 intron, CTP2

targeting sequence, cp4 epsps coding sequence, E9 3' untranslated sequence, and a 

portion of 7Sα' promoter. The ~5.0 kb restriction segment contains the remaining portion 

of the 7Sα' promoter, FAD2-1A and FATB1-A sense sequences, partial sequences of 

Right Border and Left Border, FATB1-A and FAD2-1A antisense sequences, H6 3' 

untranslated sequence, Left Border and genomic DNA flanking the 3' end of the insert. 

In the Southern blot analyses performed, each Southern blot contained a negative and 

several positive controls. Conventional soybean DNA digested with Nco I or Spe I was 

used as a negative control to determine if the probes hybridized to any endogenous 

soybean sequences. As a positive control on the Southern blots, digested plasmid and 

probe templates were used. Plasmid PV-GMPQ/HT4404 digested with a combination of 

Xho I and Nco I was mixed with predigested conventional soybean genomic DNA and 

loaded on the gel. For Southern blots hybridized with multiple probes, each probe 

template was mixed with predigested conventional soybean DNA. The positive 

hybridization control was spiked at 0.1 and 1 genome equivalent to demonstrate 

sufficient sensitivity of the Southern blot. Individual Southern blots were hybridized 

with the following probe sets: Probes 1, 4, and 6; Probes 2 and 5; Probe 3; Probes 7 and 

9; and Probes 8 and 10 (refer to Figures IV-1 and 2 and Table V-1). The results of this 

analysis are shown in Figures V-2 through V-6. 

V.A.1. Probes 1, 4 and 6 

Conventional soybean DNA digested with Nco I (Figure V-2, lanes 1 and 8) or Spe I 

(Figure V-2, lanes 3 and 10) and hybridized with the probes 1, 4, and 6 (Figure IV-1) 

produced several hybridization signals. These hybridization signals result from the 

probes (Probes 1, 4, and 6, Figure IV-1) hybridizing to endogenous sequences residing in 

the soybean genome and are not specific to the inserted DNA. These results were 

expected, because several genetic elements covered by probes 1, 4 and 6 are native to the 

soybean genome. These signals, as expected, were produced in both test and 

conventional soybean lanes, and therefore the bands are considered to be endogenous 

background hybridization. 

Probe template spikes (Probes 1, 4 and 6, Figure IV-1) generated from plasmid 

PV-GMPQ/HT4404 were mixed with the conventional soybean DNA predigested with 

Spe I and produced the expected bands at ~1.8, ~2.1, and ~1.1 kb, respectively, (Figure 

V-2, lanes 5-6) in addition to the endogenous background hybridization observed in the 

conventional soybean DNA (Figure V-2, lane 10). Plasmid PV-GMPQ/HT4404 digested 

with a combination of Xho I and Nco I and mixed with conventional soybean DNA 

predigested with Spe I (Figure V-2, lane 7) produced the expected size bands of ~3.2 and 

~9.9 kb (refer to Figure V-1) in addition to the endogenous background hybridization 

observed in the conventional soybean DNA (Figure V-2, lane 10). These results indicate 

that the probes are hybridizing to their target sequences. 

MON 87705 DNA digested with Nco I (Figure V-2, lanes 2 and 9) produced two unique 

bands of ~4.0 and ~5.7 kb in addition to the endogenous background hybridization 
Monsanto Company 09-SY-201U Page 67 of 471

observed in the conventional soybean DNA (Figure V-2, lanes 1 and 8). The ~4.0 kb 

band is the expected size for the border segment containing the 5' end of the inserted 

DNA (T-DNA I) along with the adjacent genomic DNA flanking the 5' end of the insert 

(Figure V-1). The ~5.7 kb band is the expected size for the border segment containing 

the 3' end of the inserted DNA (T-DNA I and II) along with the adjacent genomic DNA 

flanking the 3' end of the insert (Figure V-1). 

MON 87705 DNA digested with Spe I (Figure V-2, lanes 4 and 11) produced two unique 

bands of ~5.0 and ~11 kb, in addition to the endogenous background hybridization 

observed in the conventional soybean DNA (Figure V-2, lanes 3 and 10). The ~11 kb 

segment is consistent with the expected band being greater than 8.1 kb. This band in the 

short run appears slightly larger, at ~13 kb, than the corresponding band in the long run. 

This border segment contains the 5' end of the inserted DNA (T-DNA I) along with the 

adjacent genomic DNA flanking the 5' end of the insert (Figure V-1). The ~5.0 kb band 

(Figure V-2, lane 4) is consistent with the expected band of 5.0 kb; however, the 

migration of the segment is slightly higher at ~5.2 kb in the short run (Figure V-2, 

lane 11) as indicated by the molecular weight marker. The ~5.0 kb band is the expected 

size for the border segment containing the 3' end of the inserted DNA (T-DNA I and II) 

along with the adjacent genomic DNA flanking the 3' end of the insert (Figure V-1). 

There were no additional bands detected using the probes 1, 4, and 6. Based on the 

results presented in Figure V-2, it was concluded that T-DNA sequences covered by 

probes 1, 4, and 6 reside at a single integration locus in MON 87705. 

V.A.2. Probes 2 and 5 

Conventional soybean DNA digested with Nco I (Figure V-3, lanes 1 and 8) or Spe I 

(Figure V-3, lanes 3 and 10) and hybridized with probes 2 and 5 (Figure IV-1) produced 

several hybridization signals. These hybridization signals result from the probes (Probes 

2 and 5, Figure IV-1) hybridizing to endogenous sequences residing in the soybean 

genome and are not specific to the inserted DNA. These results were expected, because 

several genetic elements covered by probes 2 and 5 are native to the soybean genome. 

These signals, as expected, were produced in both test and conventional soybean lanes, 

and therefore the bands are considered to be endogenous background. 

Probe template spikes (Probes 2 and 5, Figure IV-1) generated from plasmid 

PV-GMPQ/HT4404 mixed with the conventional soybean DNA predigested with Spe I 

produced the expected bands at ~0.8 and ~1.0 kb (Figure V-3, lanes 5-6) in addition to 

the endogenous background hybridization observed in the conventional soybean DNA 

(Figure V-3, lane 10). Plasmid PV-GMPQ/HT4404 digested with a combination of Xho I 

and Nco I and mixed with conventional soybean DNA predigested with Spe I (Figure V3, lane 7) produced the expected size bands of ~3.2 and ~9.9 kb (refer to Figure V-1) in 

addition to the endogenous background hybridization observed in the conventional 

soybean DNA (Figure V-3, lane 10). These results indicate that the probes are 

hybridizing to their target sequences. 

MON 87705 DNA digested with Nco I (Figure V-3, lanes 2 and 9) produced two unique 

bands of ~4.0 and ~5.7 kb (Figure V-3, lanes 1 and 8). The ~4.0 kb band is the expected 

size for the border segment containing the 5' end of the inserted DNA (T-DNA I) along 
Monsanto Company 09-SY-201U Page 68 of 471

with the adjacent genomic DNA flanking the 5' end of the insert (Figure V-1). The ~5.7 

kb band is the expected size for the border segment containing the 3' end of the inserted 

DNA (T-DNA I and T-DNA II) along with the adjacent genomic DNA flanking the 3' 

end of the insert (Figure V-1). 

MON 87705 DNA digested with Spe I (Figure V-3, lanes 4 and 11) produced two unique 

bands of ~5.0 and ~11 kb (Figure V-3, lanes 3 and 10). The ~11 kb segment is consistent 

with the expected band being greater than 8.1 kb and with the ~11 kb segment seen with 

probes 1, 4, and 6 (Figure V-2, lanes 4 and 11). The ~11 kb band is the expected size for 

the border segment containing the 5' end of the inserted DNA (T-DNA I) along with the 

adjacent genomic DNA flanking the 5' end of the insert (Figure V-1). The ~5.0 kb border 

segment contains the 3' end of the inserted DNA (T-DNA I and T-DNA II) along with the 

adjacent genomic DNA flanking the 3' end of the insert (Figure V-1). There were no 

additional bands detected using probes 2 and 5. Based on the results presented in Figure 

V-3, it was concluded that sequence covered by probes 2 and 5 resides at a single 

integration locus in MON 87705. 

V.A.3. Probe 3 

Conventional soybean DNA digested with Nco I (Figure V-4, lanes 1 and 7) or Spe I 

(Figure V-4, lanes 3 and 9) and hybridized with probe 3 (Figure IV-1) showed no 

detectable hybridization bands, as expected for the negative control. 

Plasmid PV-GMPQ/HT4404 digested with a combination of Xho I and Nco I and mixed 

with conventional soybean DNA predigested with Spe I (Figure V-4, lanes 5-6) produced 

the expected size band of ~3.2 kb (refer to Figure V-1). This hybridization indicates that 

the probe is hybridizing to its target sequence. 

MON 87705 DNA digested with Nco I (Figure V-4, lanes 2 and 8) produced the expected 

band of ~5.7 kb. The ~5.7 kb band is the expected size for the border segment containing 

the 3' end of the inserted DNA (T-DNA I and T-DNA II) along with the adjacent 

genomic DNA flanking the 3' end of the insert (Figure V-1). 

MON 87705 DNA digested with Spe I (Figure V-4, lanes 4 and 10) produced the 

expected band of ~11 kb. The ~11 kb band is consistent with the expected band being 

greater than 8.1 kb and with the ~11 kb segment seen in Figures V-2 and 3 (lanes 4 and 

11). The ~11 kb band represents the border segment containing the 5' end of the inserted 

DNA (T-DNA I) along with the adjacent genomic DNA flanking the 5' end of the insert 

(Figure V-1). 

There were no additional bands detected using probe 3. Based on the results presented in 

Figure V-4, it was concluded that sequence covered by probe 3 resides at a single 

detectable integration locus in MON 87705. 

Taken together, the data presented in Figures V-2, V-3, and V-4 indicate that a single 

copy of the T-DNA I and T-DNA II sequences integrated into the soybean genome at a 

single detectable site in MON 87705. 
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Figure V-2. Southern Blot Analysis of MON 87705: Probes 1, 4, and 6 

The blot was hybridized with 32P-labeled probes that span a portion of T-DNA I and T-DNA II sequences 

(probes 1, 4, and 6, Figure IV-1). Each lane contains ~10 μg of digested genomic DNA isolated from leaf 

tissue. Lane designations are as follows:

Lane 1: Conventional soybean (Nco I) 

 2: MON 87705 (Nco I)

 3: Conventional soybean (Spe I)

 4: MON 87705 (Spe I)

 5: Conventional soybean (Spe I) spiked with probe templates [~0.1 genomic equivalent]

 6: Conventional soybean (Spe I) spiked with probe templates [~1 genomic equivalent]

 7: Conventional soybean (Spe I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~1 genomic equivalent]

 8: Conventional soybean (Nco I) 

 9: MON 87705 (Nco I)

 10: Conventional soybean (Spe I)

 11: MON 87705 (Spe I) 

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 

 
Monsanto Company 09-SY-201U Page 70 of 471

Figure V-3. Southern Blot Analysis of MON 87705: Probes 2 and 5 

The blot was hybridized with 32P-labeled probes that span a portion of T-DNA I sequences (probes 2 and 5, 

Figure IV-1). Each lane contains ~10 μg of digested genomic DNA isolated from leaf tissue. Lane 

designations are as follows:

Lane 1: Conventional soybean (Nco I) 

 2: MON 87705 (Nco I)

 3: Conventional soybean (Spe I)

 4: MON 87705 (Spe I)

 5: Conventional soybean (Spe I) spiked with probe templates [~0.1 genomic equivalent]

 6: Conventional soybean (Spe I) spiked with probe templates [~1 genomic equivalent]

 7: Conventional soybean (Spe I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~1 genomic equivalent]

 8: Conventional soybean (Nco I) 

 9: MON 87705 (Nco I)

 10: Conventional soybean (Spe I)

 11: MON 87705 (Spe I) 

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 
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Figure V-4. Southern Blot Analysis of of MON 87705: Probe 3 

The blot was hybridized with a 32P-labeled probe that span a portion T-DNA I sequences (probe 3, Figure 

IV-1). Each lane contains ~10 μg of digested genomic DNA isolated from leaf tissue. Lane designations 

are as follows: 

Lane 1: Conventional soybean (Nco I) 

 2: MON 87705 (Nco I)

 3: Conventional soybean (Spe I)

 4: MON 87705 (Spe I)

 5: Conventional soybean (Spe I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~0.1 genomic equivalent]

 6: Conventional soybean (Spe I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~1 genomic equivalent]

 7: Conventional soybean (Nco I) 

 8: MON 87705 (Nco I)

 9: Conventional soybean (Spe I)

 10: MON 87705 (Spe I) 

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.
Monsanto Company 09-SY-201U Page 72 of 471

V.B. Southern Blot Analysis to Determine the Presence or Absence of Plasmid 

PV-GMPQ/HT4404 Backbone 

MON 87705 and conventional soybean genomic DNA were digested with the restriction 

enzymes Nco I or Spe I. Probe template spikes (probes 7-10, Figure IV-2) generated 

from plasmid PV-GMPQ/HT4404 were mixed with the predigested conventional soybean 

genomic DNA to serve as positive hybridization controls. Additionally, plasmid 

PV-GMPQ/HT4404 DNA previously digested with the combination of Xho I and Nco I 

was mixed with conventional soybean DNA digested with Spe I and loaded on the gel to 

serve as a positive hybridization control. The blots were hybridized with probes 7-10 

(Figure IV-2) that covered the entire backbone sequence of PV-GMPQ/HT4404. If 

backbone sequences are present in MON 87705, then probing with backbone sequence 

should result in unique hybridizing bands. The results are shown in Figures V-5 and V-6. 

V.B.1. Plasmid Backbone Probes 7 and 9 

Conventional soybean DNA digested with the restriction enzyme Nco I (Figure V-5, 

lanes 1 and 8) or Spe I (Figure V-5, lanes 3 and 10) and hybridized with probes 7 and 9 

(Figure IV-2) showed no detectable hybridization bands, as expected for the negative 

control. 

Probe template spikes (Probes 7 and 9, Figure IV-2) generated from plasmid 

PV-GMPQ/HT4404 and mixed with conventional soybean DNA predigested with Spe I 

produced the expected bands at ~1.3 and ~1.5 kb (Figure V-5, lanes 5 and 6). In 

addition, there is an unexpected faint band at ~3.0 kb (Figure V-5, lane 6). Based on size, 

this band is likely derived from dimers of the probe template (Qiagen at 

www.qiagen.com). Since this extra band only is present in the positive control and is not 

present in the conventional or MON 87705 DNA, it was concluded that the presence of 

this extra band did not impact or alter the final results for MON 87705. Plasmid 

PV-GMPQ/HT4404 digested with Xho I/Nco I and mixed with conventional soybean 

DNA digested with Spe I (Figure V-5, lane 7) produced an expected band that migrated at 

~9.4 kb. This band is consistent with the expected band at ~9.9 kb; however, the 

migration of the ~9.4 kb segments is slightly lower than indicated by molecular weight 

marker most likely due to differences in salt concentrations between sample and marker. 

Overall, these results indicate that the probes are hybridizing to their target sequences. 

MON 87705 DNA digested with either Nco I (Figure V-5, lanes 2 and 9) or Spe I 

(Figure V-5, lanes 4 and 11) showed no detectable hybridization signal, indicating that 

MON 87705 does not contain any detectable backbone sequence from the transformation 

vector PV-GMPQ/HT4404 that is covered by probes 7 and 9. 

V.B.2. Plasmid Backbone Probes 8 and 10 

Conventional soybean DNA digested with the restriction enzyme Nco I (Figure V-6, 

lanes 1 and 8) or Spe I (Figure V-6, lanes 3 and 10) and hybridized with probes 8 and 10 
Monsanto Company 09-SY-201U Page 73 of 471

(Figure IV-2) showed no detectable hybridization bands, as expected for the negative 

control. 

Probe template spikes (Probes 8 and 10, Figure IV-2) generated from plasmid 

PV-GMPQ/HT4404 and mixed with conventional soybean DNA predigested with Spe I 

produced two expected bands that migrated at ~0.7 and ~2.1 kb (Figure V-6, lanes 5 

and 6). Plasmid PV-GMPQ/HT4404 digested with Xho I/Nco I and mixed with 

conventional soybean DNA digested with Spe I (Figure V-6, lane 7) produced an 

expected band that migrated at ~9.4 kb, which is consistent with the expected band at 

~9.9 kb. The migration of the positive hybridization controls (Figure V-6, lanes 5-7) is 

slightly different than indicated by the molecular weight marker, most likely due to 

differences in salt concentrations between samples and markers. Overall, these results 

indicate that these probes are hybridizing to their control sequences. 

MON 87705 DNA digested with either Nco I (Figure V-6, lanes 2 and 9) or Spe I 

(Figure V-6, lanes 4 and 11) showed no detectable hybridization signal. These results in 

combination with Figure V-5 indicate that MON 87705 does not contain any detectable 

backbone sequence from the transformation vector PV-GMPQ/HT4404.

 
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Figure V-5. Southern Blot Analysis of MON 87705:PV-GMPQ/HT4404 Backbone 

Probes 7 and 9 

The blot was hybridized with 32P-labeled probes that span a portion of backbone sequences (probes 7 and 9, 

Figure IV-2) of plasmid PV-GMPQ/HT4404. Each lane contains ~10 μg of digested genomic DNA 

isolated from leaf tissue. Lane designations are as follows:

Lane 1: Conventional soybean (Nco I) 

 2: MON 87705 (Nco I)

 3: Conventional soybean (Spe I)

 4: MON 87705 (Spe I)

 5: Conventional soybean (Spe I) spiked with probe templates [~0.1 genomic equivalent]

 6: Conventional soybean (Spe I) spiked with probe templates [~1 genomic equivalent]

 7: Conventional soybean (Spe I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~1 genomic equivalent]

 8: Conventional soybean (Nco I) 

 9: MON 87705 (Nco I)

 10: Conventional soybean (Spe I)

 11: MON 87705 (Spe I) 

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.
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Figure V-6. Southern Blot Analysis of MON 87705: PV-GMPQ/HT4404 

Backbone Probes 8 and 10 

The blot was hybridized with 32P-labeled probes that span a portion of backbone sequences (probes 8 and 

10, Figure IV-2) of plasmid PV-GMPQ/HT4404. Each lane contains ~10 μg of digested genomic DNA 

isolated from leaf tissue. Lane designations are as follows:

Lane 1: Conventional soybean (Nco I) 

 2: MON 87705 (Nco I)

 3: Conventional soybean (Spe I)

 4: MON 87705 (Spe I)

 5: Conventional soybean (Spe I) spiked with probe templates [~0.1 genomic equivalent]

 6: Conventional soybean (Spe I) spiked with probe templates [~1 genomic equivalent]

 7: Conventional soybean (Spe I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~1 genomic equivalent]

 8: Conventional soybean (Nco I) 

 9: MON 87705 (Nco I)

 10: Conventional soybean (Spe I)

 11: MON 87705 (Spe I) 

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel.
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V.C. Organization and Sequence of the Insert and Adjacent Genomic DNA in 

MON 87705 

The organization of the elements within the MON 87705 insert was confirmed by DNA 

sequence analyses. Several PCR primers were designed with the intent to amplify six 

overlapping regions of DNA that span the entire length of the insert (see Appendix B). 

The amplified DNA segments were subjected to DNA sequencing analyses. The insert in 

MON 87705 is 7251 base pairs and matches the sequence of PV-GMPQ/HT4404 as 

described in Tables IV-3 and V-2. 

A sequence comparison between the PCR product generated from the conventional 

soybean (A3525) and the sequence generated from the 5' and 3' flanking sequences of 

MON 87705 indicates there was a 36 bp deletion (bases 896-931) and a 2374 bp insertion 

just 5' to the MON 87705 insertion site. Given the very high homology between the 

2374 bases flanking the 5’ end of the insert and the genomic DNA flanking the 3’ end of 

the insert, the 2374 bases are most likely from the 3' end of the flanking genomic DNA 

and were duplicated at the 5' end of the insertion site when T-DNA I and T-DNA II 

integrated into the genome. This duplication has the following characteristics: 1) there is 

a single nucleotide change detected in the duplicated 2374 base pairs at the 5' flanking 

sequence; and 2) there are four unique bases located at the junction of the insert DNA and 

the 2374 bases in the 3' flanking sequence. This analysis confirms that the genomic 

sequences flanking the insert in MON 87705 are native to the soybean genome and that a 

36 base-pair deletion and a 2374 base pair duplication that contains a single base change 

occurred at the insertion site during integration of the T-DNA sequences. These 

molecular rearrangements presumably resulted from double-stranded break repair 

mechanisms in the plant during the Agrobacterium-mediated transformation process 

(Salomon and Puchta, 1998). 

V.D. Southern Blot Analysis to Examine Insert Stability in Multiple Generations 

of MON 87705 

In order to demonstrate the stability of the T-DNA I and T-DNA II insert in MON 87705 

in multiple generations, Southern blot analyses were performed using DNA obtained 

from multiple generations of the MON 87705 breeding history. For reference, the 

breeding history of MON 87705 is presented in Figure V-7. The specific generations 

tested are indicated in the legends of Figure V-8. DNA samples from R3, R4, R5, and R6 

generations of MON 87705 (refer to Figure V-7) were digested with Nco I and were 

expected to release two border segments with the expected sizes of 4.0 and 5.7 kb (Figure 

V-1). The detected hybridization bands in R4, R5, and R6 generations are compared to 

the fully characterized MON 87705 R3 generation to evaluate stability. Any instability 

associated with the insert would be detected as faint novel bands within the fingerprint on 

the Southern blot. The blot was hybridized simultaneously with two radiolabeled probes 

that cover both border segments generated by the digest (probes 1 and 6, Figure IV-1). 

This blot has two of the same positive hybridization controls (probes 1 and 6, Figure IV1) as described in Section V.A.1. The result of this analysis is shown in Figure V-8. 
Monsanto Company 09-SY-201U Page 77 of 471

Conventional soybean DNA digested with Nco I and hybridized with probes 1 and 6 

(Figure V-8, lane 4) showed hybridization bands. These hybridization signals result from 

the probes hybridizing to endogenous targets residing in the soybean genome and are not 

specific to the inserted DNA. 

Probe templates spikes (Probes 1 and 6, Figure IV-1) generated from plasmid 

PV-GMPQ/HT4404 and mixed with conventional soybean DNA predigested with Nco I 

(Figure V-8, lanes 1 and 2) produced the expected size bands at ~1.8 and ~1.1 kb. The 

detection of the probe template positive hybridization controls demonstrates that both 

probes were hybridizing to the target DNA. Plasmid PV-GMPQ/HT4404 digested with a 

combination of Xho I and Nco I and mixed with conventional soybean DNA predigested 

with Nco I (Figure V-8, lane 3) produced the expected size band at ~9.9 kb, which 

indicates that the probes are hybridizing to their corresponding sequence in the 

transformation vector. This expected band at ~9.9 kb migrated together with an 

endogenous hybridization signal observed in Figure V-8, lane 4. 

Digestion of MON 87705 from multiple generations (refer to Breeding History of 

MON 87705, Figure V-7) with restriction enzyme Nco I produced two bands at ~4.0 and 

~5.7 kb (Figure V-8, lanes 5-8) in addition to the endogenous background hybridization 

observed in the conventional soybean DNA (Figure V-8, lane 4). The ~4.0 kb band is the 

expected size for the border segment containing the 5' end of the inserted DNA (T-DNA 

I) along with the adjacent genomic DNA flanking the 5' end of the insert (Figure V-1). 

The ~5.7 kb band is the expected size for the border segment containing the 3' end of the 

inserted DNA (T-DNA I and T-DNA II) along with the adjacent genomic DNA flanking 

the 3' end of the insert (Figure V-1). However, the migration of this segment appears 

slightly lower than indicated by the molecular weight marker most likely due to 

differences in salt concentrations between the samples and marker. This restriction 

pattern is the same as the restriction pattern observed in the Southern blot analysis of the 

R3 generation shown in Figure V-2 (lanes 2 and 9). 

There were no additional unexpected bands detected, indicating that the single copy of 

T-DNA I and T-DNA II in MON 87705 is stably maintained in the selected generations. 

 
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Figure V-7. Breeding History of MON 87705 

All generations were self pollinated (⊗). The R3 generation was used for the molecular 

analyses of MON 87705 reported in Figures V-2 through V-6 and is referred to as 

MON 87705 in all Southern blot figures. The R3, R4, R5, and R6 generations were used 

for analyzing the stability of the insert in multiple generations. 

⊗
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Figure V-8. Generational Stability of MON 87705: Probes 1 and 6 

The blot was hybridized with 32P-labeled probes that spanned a portion of T-DNA I and T-DNA II 

sequences (probes 1, and 6, Figure IV-1). Each lane contains ~ 10 μg of digested genomic DNA isolated 

from leaf tissue. The breeding history of MON 87705 is illustrated in Figure V-7. Lane designations are as 

follows:

Lane 1: Conventional soybean (Nco I) spiked with probe templates [~0.1 genomic equivalent] 

 2: Conventional soybean (Nco I) spiked with probe templates [~1 genomic equivalent]

 3: Conventional soybean (Nco I) spiked with PV-GMPQ/HT4404 (Xho I/Nco I) [~1 genomic equivalent]

 4: Conventional soybean (Nco I) 

 5: MON 87705 [R3, (Nco I)]

 6: MON 87705 [R4, (Nco I)]

 7: MON 87705 [R5, (Nco I)]

 8: MON 87705 [R6, (Nco I)]

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 
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V.E. Inheritance of the Genetic Insert in MON 87705 

During development of MON 87705 segregation data were recorded to assess the 

heritability and stability of the coding sequences present in MON 87705. Chi-square 

analysis was performed over several generations to confirm the segregation and stability 

of the MON 87705 insertion. The Chi-square analysis is based on testing the observed 

segregation ratio to the expected segregation ratio according to Mendelian principles. 

The MON 87705 breeding path from which segregation data were generated is described 

in Figure V-9. The transformed R0 plant was self-pollinated to produce R1 seed. From 

the R1 segregating population, an individual plant (#90, designated MON 87705) 

homozygous for a single copy of the H6 3’UTR was identified via Invader (Wave 

Technologies, Inc.) and Southern blot analysis. 

The selected R1 MON 87705 plant was self-pollinated to give rise to a population of R2 

plants that were repeatedly self-pollinated through to the R4 generation. At each 

generation, the fixed homozygous plants were tested for the expected segregation pattern 

of 1:0 (positive: negative) for the H6 3’UTR using Invader analysis. 

At the R4 generation, homozygous MON 87705 plants were bred via traditional breeding 

with a soybean variety that did not contain the H6 3’UTR to produce F1 hemizygous 

seed. The resulting F1 plants were then self-pollinated to produce F2 seed. The 

heritability and stability of the coding sequences present in MON 87705 were assessed 

from plants of the F2, F3, F4, and F5 generations. At each of these generations, the 

plants were tested for the presence of the H6 3’UTR by Invader analysis, and hemizygous 

positive plants were then selected and self-pollinated to produce seed of the next 

generation. 

A Chi-square (χ

2

) analysis was used to compare the observed segregation ratios to the 

expected ratios according to Mendelian principles. The Chi-square value was calculated 

as: 

 χ 2

 = ∑ [( | o – e | )2 / e] 

where o = observed frequency of the genotype and e = expected frequency of the 

genotype. The level of statistical significance was predetermined to be 5% (p≤0.05). 

The results of the χ

2 analysis of the segregating progeny of MON 87705 are presented in 

Table V-3. The χ

2 values in the F2 generation indicated no statistically significant 

difference between the observed and expected 1:2:1 (homozygous positive:hemizygous 

positive:homozygous negative) segregation ratio. The χ

2 value in the F3 generation 

indicated a statistically significant difference between the observed and expected 1:2:1 

segregation ratio. However, there were ten plants out of 91 total plants tested in the assay 

in which the zygosity could not be determined by Invader analysis. These missing data 

combined with a relatively small sample size (n=81) of tested plants may have skewed 

the segregation ratio. This caused the results of the analysis to be inconclusive and the 

data from the F3 generation could not be used to accurately assess the heritability and 

stability of the coding sequences present in MON 87705. Therefore, the F4 and F5 
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generations were tested using larger sample sizes to further assess the heritability and 

stability of the inserted coding sequences. The χ

2 values in the F4 and F5 generations 

indicated no statistically significant differences between the observed and expected 1:2:1 

segregation ratios. Considering the data from three generations (F2, F4, and F5), the 

results support the conclusion that the coding sequences present in MON 87705 reside at 

a single locus within the soybean genome and are inherited according to Mendelian 

inheritance principles. These results are also consistent with the molecular 

characterization data that indicate a single genomic insertion site for the coding 

sequences present in MON 87705 that encode for the improved fatty acid profile and 

glyphosate tolerance trait (Table VI-1). 
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Transformed and regenerated R0 plant  R1  R2 (homozygous positive) 

 R3 (homozygous positive)  R4 (homozygous positive) F1 (hemizygous positive)  Breeding path continued F2 (expected segregation of 1:2:1)1

 homozygous positive : hemizygous positive : homozygous negative  F3 (expected segregation of 1:2:1)1

 homozygous positive : hemizygous positive : homozygous negative 

 F4 (expected segregation of 1:2:1)1

 homozygous positive : hemizygous positive : homozygous 

 F5 (expected segregation of 1:2:

 homozygous positive : hemizygous positive : homo

⊗ = Self pollinated Figure V-9. Breeding Path for Generating Segregation Data MON 87705 1 Chi-square analysis conducted on segregation data from the F2, F3, F4 and F5 generations

⊗Plant #90 (MON 87705) selected and self pollinated ⊗⊗

⊗

cross with variety that did not contain the H6 3’UTR 

⊗

⊗

⊗
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Table V-3. Segregation of the H6 3’UTR Gene During the Development of MON 87705 

1:2:1 Segregation

Generation1

Total 

Plants 

Tested2

Observed # 

Plants 

Homozygous 

Positive

Observed # 

Plants 

Hemizygous 

Positive

Observed # 

Plants 

Homozygous 

Negative

Expected # 

Plants 

Homozygous 

Positive

Expected # 

Plants 

Hemizygous 

Positive

Expected # 

Plants 

Homozygou

Negative 

F2 4197 1009 2091 1097 1049.25 2098.5 1049.25 F3 81 30 35 16 20.25 40.5 20.25 F4 266 68 126 72 66.5 133 66.5 F5 175 44 88 43 43.75 87.5 43.75 

1 F2 progeny were from the cross of MON 87705 homozygous positive for the H6 3’UTR with a soybean variety that did n

3’UTR. F3, F4, and F5 progeny were from self-pollinated plants of the previous generation that were hemizygous positive 

2 Plants were tested for the presence of the H6 3’UTR by Invader analysis. “Total plants” refers to the total number of plan

could be determined using the assay.
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V.F. Conclusion of Molecular Characterization 

Molecular characterization of MON 87705 by Southern blot analyses demonstrated that a 

single copy of the T-DNA I and T- DNA II sequences from the transformation vector 

PV-GMPQ/HT4404 was integrated into the soybean genome at a single locus. There 

were no additional genetic elements, including backbone sequences, from the 

transformation vector PV-GMPQ/HT4404 detected, linked or unlinked to the intact DNA 

insert, in MON 87705. 

PCR and DNA sequence analyses were performed on MON 87705, which confirmed the 

organization of the elements within the insert, determined the 5' and 3' insert-to-plant 

junctions, determined the complete DNA sequence of the insert and adjacent soybean 

genomic DNA sequence in MON 87705, and confirmed that the genomic DNA sequences 

flanking the 5' and 3' ends of the insert in MON 87705 are native to the soybean genome. 

The PCR and DNA sequence analysis identified 36 bp of conventional soybean DNA 

sequence deleted at the insertion site in MON 87705. Additionally, a 2374 bp duplication 

was identified in the 5' genomic flanking sequence of MON 87705. This duplicated 

sequence is likely from the 3′ flanking sequence of MON 87705 and it contains a single 

nucleotide change. 

Generational stability analysis demonstrated that an expected Southern blot fingerprint of 

MON 87705 has been maintained through four generations of the breeding history, 

thereby confirming the stability of the insert. Results from segregation analyses show 

heritability and stability of the insert occurred as expected across multiple generations, 

which corroborates the molecular insert stability analysis and establishes the genetic 

behavior of the DNA insert at a single chromosomal locus. 

 
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VI. CHARACTERIZATION OF THE INTRODUCED CP4 EPSPS PROTEIN 

IN MON 87705 

As described in Section V, the MON 87705 insert contains a cp4 epsps expression 

cassette and a FATB/FAD2 supression cassette. As the FATB/FAD2 cassette does not 

encode for any proteins, this section focuses on the characterization and safety of the CP4 

EPSPS protein produced by MON 87705. 

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is 

mediated by dsRNA molecules. Double stranded RNAs are commonly found in 

eukaryotes, including plants, for endogenous gene suppression and are composed of 

nucleic acids. Nucleic acids have a long history of safe consumption and are considered 

GRAS by the U.S. Food and Drug Administration. There is no evidence to suggest 

dietary consumption of RNA is associated with toxicity or allergenicity. Moreover, 

analysis of the DNA segments encoding this dsRNA showed that they lack the sequences 

required for translation initiation and protein synthesis. The production of a protein from 

the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several 

biotechnology-derived plant products previously deregulated by APHIS were developed 

using RNA-based suppression mechanisms, including virus-resistant papaya and squash, 

high oleic soybean, FLAVR SAVR tomatoes, and plum trees resistant to Plum pox virus. 

Based on this information, it is concluded that the inserted DNA and resulting dsRNA are 

safe and unlikely to produce a protein or polypeptide. As a result, the RNA-based 

suppression technology used in MON 87705 poses no novel risks from a food, feed or 

environmental perspective (FDA, 1994; USDA-APHIS, 1994; USDA-APHIS, 1997; 

USDA-APHIS, 2006; USDA-APHIS, 2007). 

The remainder of this section summarizes the assessment of the CP4 EPSPS protein 

produced in MON 87705 including: 1) the identity of the CP4 EPSPS protein from 

MON 87705; 2) demonstration of the equivalence of the plant-produced and E. coliproduced CP4 EPSPS proteins used in laboratory and regulatory safety evaluations; 3) 

the CP4 EPSPS protein expression levels in MON 87705 soybean tissues; and 4) an 

allergenicity assessment for the CP4 EPSPS protein. Results indicate that the 

MON 87705-produced CP4 EPSPS protein is equivalent to E. coli-produced protein. 

Data also support a conclusion of safe consumption based on several lines of evidence, 

all of which will be submitted to FDA as part of the pre-market consultation. 

VI.A. Identity and Function of the CP4 EPSPS Protein from MON 87705 

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS: EC2.5.1.19) family of 

enzymes is ubiquitous to plants and microorganisms. EPSPS proteins have been isolated 

from both sources, and their properties have been extensively studied (Harrison et al., 

1996; Haslam, 1993; Klee et al., 1987; Schonbrunn et al., 2001; Steinrucken and 

Amrhein, 1984). The shikimate pathway and the EPSPS protein are absent in mammals, 

fish, birds, reptiles, and insects (Alibhai and Stallings, 2001). The bacterial and plant 

enzymes are mono-functional with a molecular weight of 44-48 kDa (Kishore et al., 

1988). EPSPS proteins catalyze the transfer of the enolpyruvyl group from 

phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P), thereby 

yielding inorganic phosphate and 5-enolpyruvylshikimate-3-phosphate (EPSP) (Alibhai 
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and Stallings, 2001). Due to the specificity of EPSPS for its substrates, the only known 

catalytic product generated is EPSP, which is the penultimate product of the shikimic 

acid pathway. Shikimic acid is a substrate for the biosynthesis of the aromatic amino 

acids (phenylalanine, tryptophan and tyrosine) and other aromatic molecules. It has been 

estimated that aromatic molecules, all of which are derived from shikimic acid, represent 

35% or more of the dry weight of a plant (Franz et al., 1997). 

The EPSPS transgene in MON 87705 is derived from Agrobacterium sp. strain CP4 (cp4 

epsps). The cp4 epsps coding sequence encodes a 47.6 kDa EPSPS protein consisting of 

a single polypeptide of 455 amino acids (Padgette et al., 1996b). The CP4 EPSPS protein 

is similar and functionally identical to endogenous plant EPSPS enzymes, but has a much 

reduced affinity for glyphosate, the active ingredient in the Roundup family of 

agricultural herbicides, relative to endogenous plant EPSPS (Padgette et al., 1996b). In 

conventional plants, glyphosate binds to the endogenous plant EPSPS enzyme and blocks 

the biosynthesis of shikimate-3-phosphate, thereby depriving plants of essential amino 

acids (Haslam, 1993; Steinrücken and Amrhein, 1980). In Roundup Ready plants, which 

are tolerant to the Roundup family of agricultural herbicides, requirements for aromatic 

amino acids and other metabolites are met by the continued action of the CP4 EPSPS 

enzyme in the presence of glyphosate (Padgette et al., 1996b). The CP4 EPSPS protein 

expressed in MON 87705 is identical to the CP4 EPSPS protein in other Roundup Ready 

crops including Roundup Ready soybean (404-3-2), Roundup Ready 2 Yield soybean 

(MON 89788), Roundup Ready Corn 2, Roundup Ready canola, Roundup Ready sugar 

beet, and Roundup Ready cotton. 

VI.B. Characterization of the Full Length CP4 EPSPS Protein from MON 87705 

The safety assessment of crops derived through biotechnology includes characterization 

of the introduced protein produced from the inserted DNA, confirmation of its functional 

and physicochemical properties, and confirmation of the safety of the protein. The level 

of CP4 EPSPS protein produced in MON 87705 is too low to allow purification of 

sufficient quantities for use in subsequent safety assessment studies. Therefore, it is 

necessary to produce the protein in high-expressing recombinant host systems (such as 

bacteria) in order to obtain sufficient quantities of the CP4 EPSPS protein. CP4 EPSPS 

protein was produced in E. coli, and subsequently purified and characterized. A small 

quantity of the CP4 EPSPS protein was also purified from harvested MON 87705 seed. 

The equivalence of the physicochemical characteristics and functional activity between 

the MON 87705-produced and E. coli-produced CP4 EPSPS proteins was confirmed by a 

panel of analytical techniques, including: (1) sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE) to establish equivalence of the apparent molecular weight 

between MON 87705-produced and the E. coli-produced reference standard protein, (2) 

western blot analysis to establish immunoreactive equivalence between MON 87705-

produced and the E. coli-produced reference standard protein using an anti-CP4 EPSPS 

antibody, (3) N-terminal sequence analysis, (4) matrix-assisted laser 

desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to generate a 

tryptic peptide map of MON 87705-produced CP4 EPSPS, (5) CP4 EPSPS enzymatic 

activity analysis to demonstrate functional equivalence between MON 87705-produced 

and the E. coli-produced reference standard protein, and (6) glycosylation analysis to 
Monsanto Company 09-SY-201U Page 87 of 471

establish equivalent glycosylation status between MON 87705-produced and E. coliproduced reference standard protein. The details of the materials, methods, and results 

are described in Appendix C while the conclusions are summarized below. 

A comparison of the MON 87705-produced CP4 EPSPS to the E. coli-produced 

CP4 EPSPS reference standard protein confirmed the identity of the MON 87705-

produced CP4 EPSPS protein and established the equivalence of the plant produced 

protein to the E. coli-produced CP4 EPSPS reference standard protein. The molecular 

weight of the MON 87705-produced and E. coli-produced CP4 EPSPS protein was 

estimated by SDS-PAGE. The SDS-PAGE demonstrated that the proteins migrated 

identically, indicating that the CP4 EPSPS proteins from both sources are equivalent in 

their molecular weight. The electrophoretic mobility and immunoreactive properties of 

the MON 87705-produced CP4 EPSPS protein were shown to be equivalent to those of 

the E. coli-produced CP4 EPSPS protein reference standard by immunoblot. The Nterminus of the MON 87705 produced CP4 EPSPS protein was consistent with the 

predicted amino acid sequence translated from the CP4 EPSPS coding sequence, and the 

MALDI-TOF MS analysis yielded peptide masses consistent with the expected peptide 

masses from the translated CP4 EPSPS coding sequence. The MON 87705- produced 

and the E. coli-produced CP4 EPSPS protein reference standard were also found to be 

equivalent based on the functional activities and the lack of glycosylation. Taken 

together, these data provide a detailed characterization of the CP4 EPSPS protein isolated 

from MON 87705 and established its equivalence to the E. coli-produced CP4 EPSPS 

reference standard protein. Furthermore, since CP4 EPSPS proteins isolated from other 

Roundup Ready crops (Roundup Ready soybean, Roundup Ready 2 Yield soybean, 

Roundup Ready canola, Roundup Ready cotton and Roundup Ready sugar beet) have 

established equivalence to the E. coli-produced protein standard, by inference, the 

MON 87705-derived CP4 EPSPS protein is likely to possess equivalent biochemical and 

physiological characteristics with the CP4 EPSPS proteins expressed in other Roundup 

Ready crops, all of which have been deregulated by USDA-APHIS. 

VI.C. Expression Levels of CP4 EPSPS Protein in MON 87705 

CP4 EPSPS protein levels in various tissues of MON 87705 that are relevant to the risk 

assessment were assessed by a validated enzyme-linked immunosorbent assay (ELISA). 

Tissues of MON 87705 and conventional control were collected during the 2007/2008 

growing season from five field sites in Chile (City, Province): Quilapilum, Chacabuco; 

Melipilla, Melipilla; Calera de Tango, Maipo; Rancagua, Cachapoal; and San Fernando, 

Colchagua. These field sites were representative of soybean producing regions suitable 

for commercial production. At each site, three replicated plots of plants containing 

MON 87705, as well as a conventional soybean control, were planted using a randomized 

complete block field design. Over-season leaf (OSL 1-4), root, forage, and mature seed 

tissues were collected from each replicated plot at all field sites. A description of tissues 

collected is provided below. 
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Table VI-1. Tissues collected for MON 87705 

Tissue Soybean development stage Days after planting 

(DAP) 

OSL-1 V2-V3 31-35 

OSL-2 V7 46-50 

OSL-3 V10 62-66 

OSL-4 V14 84-88 

Forage R5-R6 101-106 

Root R5-R6 101-106 

Mature Seed R8 154-158 

The CP4 EPSPS protein levels were determined in all seven tissue types described above. 

The results obtained from ELISA analysis are summarized in Table VI-2 and the details 

of the materials and methods are described in Appendix D. In summary, the 2007/2008 

Chilé expression study showed the CP4 EPSPS protein in MON 87705 was detected in 

all tissue types across all five sites with a range from 40 – 1000 μg/g dwt. The levels of 

the CP4 EPSPS protein from the conventional control (A3525) were less than the assay 

limits of detection (LOD) or limit of quantitation LOQ in all tissue types. The mean CP4 

EPSPS protein levels across the five sites were highest in leaf (ranging from OSL-1 200 

μg/g dwt to OSL-2 530 μg/g dwt), followed by root (120 μg/g dwt), seed (110 μg/g dwt) 

and forage (77 μg/g dwt). 

 
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Table VI-2. Summary of CP4 EPSPS Protein Levels in Leaf, Seed, Root, and Forage 

Tissues from MON 87705 Grown in 2007/2008 Chile Field Trials 

Tissue 

Type1

CP4 EPSPS 

protein

μg/g fwt2

(SD)3

Range4

μg/g fwt2

CP4 EPSPS 

protein

μg/g dwt5

(SD)2

Range4

μg/g dwt5

LOQ/LOD

μg/g fwt2

 

OSL-1 36 (14) 16-65 200 (72) 84-340 0.57/0.26 

OSL-2 110 (51) 60-230 530 (230) 290-1000 0.57/0.26 

 

OSL-3 51 (21) 11-84 220 (94) 47-350 0.57/0.26 

 

OSL-4 51 (21) 27-94 210 (92) 110-410 0.57/0.26 

 

Root 32 (5.3) 22-40 120 (24) 77-160 0.57/0.10 

 

Forage 24 (6.4) 14-34 77 (24) 41-120 0.57/0.11 

Mature 

Seed

100 (39) 35-190 110 (44) 40-210 0.34/0.26 

 

1

The OSL-1, OSL-2, OSL-3, OSL-4 samples were collected approximately at V2 – V3, V7, V10; and V14 

stages, respectively. The forage and root were collected approximately at R5-R6 stage, and the mature 

seed was collected at R8 stage. 

2

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a fresh weight (fwt) 

basis. 

3

The means and standard deviations were calculated for each tissue type across all sites (n=15 for all tissues, 

except OSL-2 where n=12 and OSL-3 where n=19). 

4

Minimum and maximum values were determined for each tissue type across all sites. 

5

Protein levels are expressed as microgram (μg) of protein per gram (g) of tissue on a dry weight (dwt) 

basis. The dry weight values were calculated by dividing the μg/g fwt by the dry weight conversion 

factors obtained from moisture analysis data. 

 
Monsanto Company 09-SY-201U Page 90 of 471

VI.D. Assessment of Potential Allergenicity of the CP4 EPSPS Protein from 

MON 87705 

According to guidelines adopted by the Codex Alimentarius Commission (Codex, 2003) 

for the allergy safety evaluation of novel proteins, the allergenic potential of a novel 

protein is assessed by comparing the biochemical characteristics of the novel protein to 

characteristics of known allergens (Codex, 2003). A protein is not likely to be associated 

with allergenicity if: 1) the protein is from a nonallergenic source; 2) the protein 

represents only a very small portion of the total plant protein; 3) the protein does not 

share structural similarities to known allergens based on the amino acid sequence; and 4) 

the protein is rapidly digested in mammalian gastrointestinal systems. The CP4 EPSPS 

protein in MON 87705 has been assessed for its potential allergenicity according to these 

safety assessment guidelines. 

The CP4 EPSPS protein is from Agrobacterium sp., strain CP4, an organism that is not a 

source of known allergens. Bioinformatics analyses demonstrated that the CP4 EPSPS 

protein does not share immunologically relevant amino acid sequence similarities with 

known allergens and, therefore, is highly unlikely to contain immunologically crossreactive allergenic epitopes. Digestive fate experiments conducted with the CP4 EPSPS 

protein demonstrated that the full-length protein is rapidly digested in simulated gastric 

fluid (SGF) and in simulated intestinal fluid (SIF) in vitro assays (Harrison et al., 1996). 

Finally, the CP4 EPSPS protein represents no more than 0.031% of the total protein in 

the seed of MON 87705, a relatively low abundance for this protein compared to the rest 

of the seed protein content. Taken together, these data support the conclusion that the 

CP4 EPSPS protein present in MON 87705 is not similar to known allergens and does 

not pose a significant allergenic risk to humans or animals. 

VI.E. Safety Assessment Summary of CP4 EPSPS Protein 

The U.S. Environmental Protection Agency (EPA) has established an exemption from the 

requirement of a tolerance for residues of CP4 EPSPS protein and the genetic material 

necessary for its production in all plants (EPA, 1996). This exemption was based on a 

safety assessment that included rapid digestion in simulated mammalian gastrointestinal 

fluids, lack of homology to toxins and allergens, and lack of toxicity in an acute oral 

mouse gavage study. Since the MON 87705-produced CP4 EPSPS protein is equivalent 

to the exempted CP4 EPSPS protein, a similar conclusion can be reached that the 

MON 87705-produced CP4 EPSPS is safe for human and animal consumption. The 

comprehensive food and feed safety and nutritional assessment of MON 87705 also is 

scheduled to be submitted to the FDA, which will include the following conclusions: 

a) The donor organism, Agrobacterium species strain CP4 is not known for human 

or animal pathogenicity, and is not commonly allergenic. Agrobacterium sp. 

strain CP4 has been previously reviewed as a part of the safety assessment of the 

donor organism during Monsanto consultations with the FDA regarding Roundup 

Ready soybean (1994), Roundup Ready canola (1995), Roundup Ready cotton 

(1995), Roundup Ready Corn 2 (1996), Roundup Ready sugar beet (1998), 

Roundup Ready Flex cotton (2005), and Roundup Ready 2 Yield soybean (2007). 
Monsanto Company 09-SY-201U Page 91 of 471

b) EPSPS exerts its function in the shikimate pathway that is integral to aromatic 

amino acid biosynthesis in plants and microorganisms (Levin and Sprinson, 1964; 

Steinrücken and Amrhein, 1980). Therefore, this enzyme and its activity are 

found widely in food and feed derived from plant and microbial sources. Genes 

for numerous EPSPSs have been cloned (Padgette et al., 1996b), and the catalytic 

domains of this group of proteins are conserved. Bacterial EPSPSs have been 

well characterized with respect to their three dimensional X-ray crystal structures 

(Stallings et al., 1991) and detailed kinetic and chemical mechanisms (Anderson 

and Johnson, 1990). 

c) The EPSPS from Agrobacterium sp. strain CP4 is highly tolerant to inhibition by 

glyphosate and has high catalytic efficiency, compared to most glyphosatetolerant EPSPSs (Barry et al., 1992; Padgette et al., 1996b). The CP4 EPSPS 

protein thus represents one of many different EPSPSs found in nature, and the 

CP4 and native plant EPSPS enzymes are functionally equivalent except for their 

affinity to glyphosate. The CP4 EPSPS protein present in MON 87705 is similar 

to EPSPSs consumed in a variety of food and feed sources. CP4 EPSPS protein is 

homologous to EPSPSs naturally present in plants, including food crops (e.g., 

soybean and corn) and fungal and microbial food sources such as baker’s yeast 

(Saccharomyces cerevisiae), all of which have a history of safe human 

consumption (Harrison et al., 1996; Padgette et al., 1996b). The similarity of the 

CP4 EPSPS protein to EPSPSs in a variety of foods supports extensive human 

consumption of the family of EPSPS proteins and the lack of health concerns. 

Furthermore, the ubiquitous presence of homologous EPSPS enzymes in food 

crops and common microorganisms establishes that EPSPS proteins, and their 

enzyme activity, pose no hazards for human and animal consumption. 

d) The CP4 EPSPS protein does not share amino acid sequence similarities with 

known allergens, gliadins, glutenins, or protein toxins which have adverse effects 

to mammals. This has been demonstrated by extensive assessments with 

bioinformatic tools, such as the FASTA sequence alignment tool and eight-amino 

acid sliding window search. An amino acid sequence may be considered to have 

allergenic potential if it has an exact sequence identity of at least eight linearly 

contiguous amino acids with a potential allergen epitope (Metcalfe, 1996 

Hileman et al., 2002). Using a sliding window of less than eight amino acids can 

produce matches containing significant uncertainty depending on the length of the 

query sequence (Silvanovich et al., 2006) and are not useful to the allergy 

assessment process (Thomas et al., 2004). 

e) The CP4 EPSPS protein is readily digestible in simulated gastric and simulated 

intestinal fluids (Harrison et al., 1996). Rapid degradation of the full-length CP4 

EPSPS protein in SGF and SIF makes it highly unlikely for CP4 EPSPS protein to 

be absorbed by epithelial cells of the small intestine in a biologically active form. 

f) An acute toxicology study was conducted with a CP4 EPSPS protein (Harrison et 

al., 1996) that was shown to be physicochemically and functionally equivalent to 
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the CP4 EPSPS protein produced in MON 87705. Results indicate that the CP4 

EPSPS protein did not cause any adverse effects in mice, with a No Observable 

Adverse Effect Level (NOAEL) of 572 mg/kg, the highest dose level tested. 

 

g) Potential human health risks from consumption of the CP4 EPSPS protein in 

foods derived from MON 87705 were evaluated by calculating a Margin of 

Exposure (MOE) between the acute mouse NOAEL for CP4 EPSPS protein and 

95th percentile “eater-only” estimates of acute dietary exposure determined using 

the Dietary Exposure Evaluation Model (DEEM-FCID version 2.03, Exponent 

Inc.) and food consumption data from the 1994-1996 and 1998 USDA Continuing 

Survey of Food Intakes by Individuals (CSFII). The MOEs for acute dietary 

intake of the CP4 EPSPS protein were estimated to be 60,000 and 1,600 for the 

general population and non-nursing infants, respectively. These very large MOEs 

indicate that there is no meaningful risk to human health from dietary exposure to 

the CP4 EPSPS protein produced by MON 87705. 

h) Potential health risks to animals from the presence of CP4 EPSPS protein in feed 

were evaluated by calculating an estimate of daily dietary intake (DDI). In the 

worst case scenario, the percentage of the CP4 EPSPS protein consumed from 

MON 87705 as part of the daily protein intake for a dairy cow is 0.0907% and for 

both the broiler and pig is less than 0.0325%. 

Using the guidance provided by the FDA, a conclusion of “no concern” is reached for the 

donor organism and the CP4 EPSPS protein. The food and feed products containing 

MON 87705 or derived from MON 87705 are as safe as soybean currently on the market 

for human and animal consumption. 
Monsanto Company 09-SY-201U Page 93 of 471

VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENT OF 

MON 87705 

Compositional comparisons between biotechnology-derived and conventional crops 

represent an integral part of a nutritional and safety assessment. Compositional 

assessments are performed using the principles and analytes outlined in the OECD 

consensus documents for soybean composition (OECD, 2001). These principles are 

accepted globally and have been employed previously in assessments of soybean 

products derived through biotechnology. 

Compositional equivalence between biotechnology-derived and conventional crops 

provides an “equal or increased assurance of the safety of foods derived from genetically 

modified plants” (OECD, 1998). The OECD consensus documents emphasize 

quantitative measurements of essential nutrients, and known antinutrients and toxicants. 

This is based on the premise that such comprehensive and detailed analyses will most 

effectively discern any compositional changes that imply potential safety and 

antinutritional concerns. Levels of the components in seed and forage of the 

biotechnology-derived crop product are compared to: 1) corresponding levels in a nonmodified comparator, typically the nontransgenic parental line grown under identical 

conditions, and 2) natural ranges generated from an in-study evaluation of commercial 

varieties or from data published in the scientific literature. 

MON 87705 was developed to generate soybean oil with lower levels of saturated fats 

(16:0 palmitic acid and 18:0 stearic acid) and higher levels of 18:1 oleic acid, with an 

associated decrease in 18:2 linoleic acid, through suppression of FAD2 and FATB RNAs 

(Figure VII-1). MON 87705 contains the same major fatty acids that are found in 

conventional soybean, including 16:0 palmitic, 18:0 stearic, 18:1 oleic, 18:2 linoleic and 

18:3 linolenic fatty acids. MON 87705 has a fatty acid profile that is comparable to other 

commercial high oleic vegetable oils (high oleic canola, high oleic safflower, high oleic 

sunflower), traditional oils, such as olive oil, that have a long-history of consumption in 

the diet, and canola oil that was granted GRAS status by the U.S. FDA. MON 87705 

also contains the cp4 epsps gene encoding the CP4 EPSPS protein that is expressed 

throughout the plant conferring tolerance to glyphosate, the active ingredient in the 

Roundup family of agricultural herbicides. 

Compositional analyses were conducted to assess whether the nutrient and antinutrient 

levels in the seed and forage derived from MON 87705 are comparable to those in the 

conventional soybean control, A3525, which has background genetics similar to 

MON 87705, but lacks the introduced traits. In addition, commercial conventional 

soybean varieties were included in the seed and forage composition analyses to establish 

a range of natural variability for each analyte, defined by a 99% tolerance interval. 

Statistically significant differences were determined at the 5% level of significance 

(p<0.05) using established statistical methods. 

Seed and forage of MON 87705 and the conventional soybean control were harvested 

from soybean grown in three replicated plots, planted in a randomized complete block 

design, at each of five sites across Chile during the 2007-2008 growing season: 

Quilapilun, Chacabuco Province (QUI); Melipilla, Melipilla Province (MEL); Calera de 

Tango, Maipo Province (CdT); Rancagua, Cachapoal Province (RAN); and San 
Monsanto Company 09-SY-201U Page 94 of 471

Fernando, Colchagua Province (SFR). Samples from all three replicates of MON 87705 

and the control were collected from all three plots and analyzed. Four different 

commercial reference soybean varieties also were grown at each site for a total of 20 

varieties. Samples from the commercial reference varieties grown at each site were 

collected from all three plots. All replicates from 19 of 20 commercial conventional 

reference soybean varieties were analyzed; however, one reference variety had all 

replicates damaged by an early frost and was excluded from the study. All MON 87705, 

control and reference soybean varieties were grown under normal agronomic field 

conditions for their respective geographic regions. Forage was collected at the R6 plant 

growth stage, and harvested soybean seed was collected at physiological maturity. The 

seed and forage collected from MON 87705, the conventional control, and the reference 

varieties were analyzed for compositional components. 

In all, 67 analytical components were measured, 60 in seed and seven in forage. The 

analytes in forage included proximates (ash, fat, moisture, protein, and carbohydrates by 

calculation), acid detergent fiber (ADF), and neutral detergent fiber (NDF). Seed 

samples were analyzed for proximates, ADF, NDF, amino acids (18), fatty acids (26; C8-

C24), trypsin inhibitors, phytic acid, lectin, isoflavones (daidzein, glycitein, and 

genistein), vitamin E, raffinose, and stachyose. Materials and methods used for 

compositional analysis of the seed and forage of MON 87705, the conventional soybean 

control, and commercial conventional reference soybean varieties are presented in 

Appendix E. 

The composition data then were statistically compared to that of the conventional 

soybean control to establish substantial equivalence. Of the measured components, 17 

fatty acids in seed had more than 50% of the observations below the assay limit of 

quantitation (LOQ) and could not be statistically analyzed. Thus, statistical analyses 

were conducted for 50 components (43 in seed and seven in forage). The data set was 

examined for evidence of biologically relevant changes using a mixed model of variance. 

Six sets of statistical analyses were conducted, five based on the data from each of the 

replicated field sites (individual-site) and the sixth analysis based on data from a 

combination of all five field sites (combined-site). The statistical summaries of the 

combined-site analysis and the individual-site analyses, reported literature and the 

International Life Sciences Institute-Crop Composition Database (ILSI-CCD at 

http://www.cropcomposition.org) ranges for the analytical components present in seed 

are provided in Appendix E. The compositional data set was examined for evidence of 

statistically significant differences (p≤0.05) between MON 87705 and the conventional 

soybean control. A summary of the significant differences observed between 

MON 87705 and the control are presented in Table VII-2. 

Results of the comparisons indicate that except for the intended fatty acid changes, the 

composition of the seed and forage of MON 87705 is equivalent to that of the 

conventional soybean control A3525, in accordance with OECD guidelines. Moreover, 

no new fatty acids beyond those presently found in soybean were detected in MON 

87705. Therefore, MON 87705 is regarded as safe and nutritious as conventional 

soybean for food and feed use. Further details of this assessment are provided in Section 

VII-A. 
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Figure VII-1. Schematic of the Soybean Fatty Acid Biosynthetic Pathway and 

Summary of Modified Fatty Acids in MON 87705 

Panel A: Schematic of the soybean fatty acid biosynthetic pathway, 

Panel B: MON 87705 soybean oil compared to commodity soybean oil and other vegetable oils 

Indicates suppression of endogenous FATB and FAD2 in MON 87705 seeds 

VII.A. Overall Assessment of the Composition of Forage and Seed from MON 87705 

Compared to the Conventional Soybean Control 

Based on the comprehensive assessment procedures discussed above, MON 87705 is 

compositionally equivalent to conventional soybean except for the intended changes in 

fatty acid levels. Combined-site analysis of both forage and seed samples showed no 

statistically significant difference (p>0.05) between MON 87705 and the control for 39 of 

50 comparisons. Significant differences (p<0.05) between MON 87705 and the 

conventional soybean control were detected for 10 analytes in seed (arginine, cystine, fat, 

16:0 palmitic acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic 

acid, 20:0 arachidic acid, and 20:1 eicosenoic acid) and one analyte (ash) in forage. 

The compositional analyses confirmed that MON 87705 had the intended changes in the 

levels of four major soybean oil fatty acids (16:0 palmitic, 18:0 stearic, 18:1 oleic and 

18:2 linoleic). For the remaining seven comparisons where a significant difference 

(p<0.05) was detected, an analysis, including magnitude of the differences, 
Monsanto Company 09-SY-201U Page 96 of 471

reproducibility across individual sites, and comparisons of mean analyte values to the 

99% tolerance interval and literature values, indicated they were not materially different 

and were not biologically meaningfully different from a food and feed safety perspective. 

Further assessment of the statistically significant differences observed between 

MON 87705 and the conventional soybean control is provided in the following sections. 

Therefore, the compositional assessment of MON 87705 supports the conclusion that, 

except for intended changes in seed fatty acid composition, seed and forage produced 

from MON 87705 are compositionally equivalent to those of conventional soybean. 

VII.A.1 Intended Changes to Fatty Acid Levels in MON 87705 Seed 

As described previously, MON 87705 was developed to generate soybean oil with lower 

levels of saturated fats (16:0 palmitic acid and 18:0 stearic acid) and higher levels of 18:1 

oleic acid, with an associated decrease in 18:2 linoleic acid, through suppression of FATB

and FAD2 RNAs (Figure VII-1). As expected, all of the intended changes in fatty acid 

levels were statistically significant in the combined-site analysis and are summarized in 

the Table VII-1 below. 
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Table VII-1. Summary of Intended Changes in Fatty Acid Levels for MON 87705 

vs. the Conventional Soybean Control (A3525) in the Combined-Site Analysis 

Fatty Acid (% total) 

MON 87705

Mean 

[Range] 

A3525 

Mean 

[Range] 

Commercial 

Tolerance 

Interval2

16:0 Palmitic1 2.36 

[2.25 - 2.44] 

10.83 

[10.51 – 11.08]

[7.62, 12.55] 

18:0 Stearic1 3.31 

[3.07 - 3.82] 

4.50 

[4.24 – 4.85] 

[2.87, 7.15] 

18:1 Oleic1 76.47 

[73.13 - 79.17]

22.81 

[21.41 – 25.08]

[18.40, 30.22] 

18:2 Linoleic1 10.10 

[7.85 - 12.42] 

52.86 

[51.68 – 53.89]

[47.75, 56.46] 

1

Significance level = <0.001

2

With 95% confidence, interval contains 99% of the values expressed in the population of 

commercial soybean varieties. Negative limits were set to zero.

The results show that the level of the saturated 16:0 palmitic acid decreased from 10.83% 

total FA in the conventional control to 2.36% total FA in MON 87705, and the level of 

the saturated 18:0 stearic acid decreased from 4.50% total FA in the conventional control 

to 3.31% total FA in MON 87705. Thus, total saturated fat was decreased from 

approximately 15.3% total FA in the conventional control to 5.7% total FA in 

MON 87705. In addition, the mean level of 18:1 oleic acid increased from 22.81% total 

FA in the conventional control to 76.47% total FA in MON 87705. These changes were 

associated with a decrease in the mean level of 18:2 linoleic acid from 52.86% total FA 

in the conventional control to 10.10% total FA in MON 87705. As expected, all intended 

changes in fatty acid levels in the seed of MON 87705 were statistically significant in the 

combined-site analysis and consistently observed at all five of the individual sites. Thus, 

compositional analysis confirmed MON 87705 had the intended fatty acid profile 

required for improved nutrition and soybean oil stability. 

VII.A.2 Fatty Acid Levels in Soybean Seed 

Of the 26 fatty acids analyzed in seed, 17 fatty acids had more than 50% of the 

observations below the assay limit of quantitation and, as a result, were excluded from 

the statistical analysis. Of the nine fatty acids that could be statistically analyzed, 

significant differences (p<0.05) were observed for seven fatty acids in the combined-site 

analysis (Table VII-2). Four of these differences were due to the intended changes in 

fatty acids, as described in Section VII.A.1. The three remaining significant differences 
Monsanto Company 09-SY-201U Page 98 of 471

in the combined-site analysis were for 18:3 linolenic, 20:0 arachidic and 20:1 eicosenoic 

acids. Given the intended changes in fatty acid metabolism, these additional differences 

in fatty acid levels were not unexpected. The biological relevance of these differences 

was assessed based on the magnitude of the difference, reproducibility across sites, and 

comparison of mean analyte values to the 99% tolerance interval for the population of 

commercial conventional soybean varieties grown concurrently at the same field sites. 

A combined-site statistical difference (p<0.05) between MON 87705 and the 

conventional control was observed in the levels of 18:3 linolenic acid. The decrease in 

linolenic acid is expected given that it is produced from 18:2 linoleic acid which was 

reduced by the suppression of the FAD2 gene. Examination of the reproducibility within 

sites shows the levels of 18:3 linolenic acid were significantly lower than the soybean 

control in four of five individual-site analyses, with the absolute magnitude of the 

differences being small (<1.5% total FA; Appendix E). In addition, all the mean levels of 

18:3 linolenic acid in MON 87705 seed from the combined-site and individual-site 

analyses were well within the 99% tolerance interval, and therefore these differences are 

not considered biologically relevant compositional changes. 

Combined-site statistical differences between MON 87705 and the conventional control 

also were observed in levels of two minor fatty acids, 20:0 arachidic acid, and 20:1 

eicosenoic acid. The mean level of 20:0 arachidic acid in MON 87705 was significantly 

lower than in the conventional soybean control in the combined-site analysis. An 

examination of the reproducibility within sites showed that the levels of 20:0 arachidic 

acid were consistently lower than the soybean control in all five individual-site analyses. 

However, the absolute magnitude of the differences was small (<0.063% total FA; 

Appendix E), all combined-site and individual-site means were well within the 99% 

tolerance interval, and therefore these differences are not considered biologically relevant 

compositional changes. 

The mean level of 20:1 eicosenoic acid in MON 87705 was significantly higher than in 

the conventional soybean control in the combine-site analysis. An examination of the 

reproducibility within sites showed that the levels of 20:1 eicosenoic acid were 

consistently higher than the soybean control in all five individual-site analyses. 

However, the absolute magnitude of these differences was small (<0.19% total FA; 

Appendix E). The combined-site mean for 20:1 eicosenoic (0.18% total FA) was slightly 

(0.09% total FA) outside the upper end (0.25% total FA) of the 99% tolerance interval 

but within the values reported in ILSI-CCD. In addition, 20:1 eicosenoic acid has a 

history of consumption in other commonly consumed vegetable oils, such as canola 

(4.3% total FA), corn (0.6% total FA), mustard seed (13.0% total FA), peanut (1.7% total 

FA), high oleic safflower (0.5% total FA, and high oleic sunflower (0.5% total FA) 

(Codex, 2005). Therefore, the small change in the mean level of 20:1 eicosenoic acid in 

MON 87705 is not considered a biologically relevant compositional change. 

These results lead to the conclusion that apart from the intended changes in the levels of 

four fatty acids (16:0 palmitic, 18:0 stearic, 18:1 oleic and 18:2 linoleic), the seed from 

MON 87705 is compositionally equivalent to conventional soybean with regard to the 

levels of other fatty acids. The differences observed for 18:3 linolenic, 20:0 arachidic, 

and 20:1 eicosenoic fatty acids were not unexpected, given the intended shift made in 

fatty acid metabolism (see Figure VII-1). Furthermore, the mean levels of these fatty 
Monsanto Company 09-SY-201U Page 99 of 471

acids were within the 99% tolerance interval and/or the ILSI-CCD and literature values. 

Therefore, these differences are not considered biologically meaningful from a food and 

feed safety or nutritional perspective. 

VII.A.3. Levels of Non-Fatty Acid Nutrients in Soybean Seed 

In addition to fatty acids, soybean seed also was analyzed for the following 26 nutrients: 

proximates (5), ADF, NDF, amino acids (18), and vitamin E. No statistically significant 

differences (p<0.05) were observed for 23 nutrient analytes. Three analytes were 

statistically different (p<0.05) between MON 87705 and the conventional control in the 

combined-site analysis: total fat, arginine and cystine (Table VII-2). The biological 

relevance of these differences was assessed based on the magnitude of the difference, 

reproducibility across sites, and comparison of mean analyte values to the 99% tolerance 

interval for the population of commercial conventional soybean varieties grown 

concurrently at the same field sites. Mean analyte values were further compared to ILSICCD and literature ranges. 

The mean level of total fat was significantly lower (p<0.05) in MON 87705 than the 

conventional soybean control in the combined-site analysis; however, the absolute 

magnitude of the mean difference was small (1.04% dw; Appendix E). There were no 

differences in total fat in any of the individual-site analyses. Furthermore, the mean level 

of total fat in MON 87705 was well within the 99% tolerance interval. Therefore, the 

difference in total fat in MON 87705 compared to the control is not considered 

biologically meaningful. 

The mean level of cystine was significantly higher (p<0.05) in MON 87705 than the 

conventional soybean control in the combined-site analysis; however, the absolute 

magnitude of the mean difference was small (0.022% dw; Appendix E). There were no 

differences in cystine levels in any of the individual-site analyses. Furthermore, the mean 

level of cystine in MON 87705 was within the 99% tolerance interval. Therefore, the 

difference in cystine in MON 87705 compared to the control is not considered 

biologically meaningful. 

The mean level of arginine was significantly higher (p<0.05) in MON 87705 than the 

conventional soybean control in the combined-site analysis; however, the absolute 

magnitude of the mean difference was small (0.1% dw). Examination of the 

reproducibility within sites shows that the mean level of arginine was significantly higher 

in only one of five individual-site analyses; however, the absolute magnitude of the mean 

difference was small (0.18% dw; Appendix E) These differences are not biologically 

relevant changes in composition, given that the mean levels of arginine in MON 87705 in 

the combined-site and individual-site analyses were all well within the 99% tolerance 

interval. 

These results lead to the conclusion that the seed from MON 87705 is compositionally 

equivalent to conventional soybean with regard to the levels of nutrients. The differences 

observed for nutrients were limited in number, not consistently observed across sites, and 

reflect the natural variation of conventional soybean. Furthermore, the mean levels of 

nutrient analytes were within the 99% tolerance interval and ILSI-CCD values. 
Monsanto Company 09-SY-201U Page 100 of 471

Therefore, these differences are not considered biologically meaningful from a food and 

feed safety or nutritional perspective. 

VII.A.4. Naturally Occurring Anti-Nutrient Levels in Soybean Seed 

Soybean seed contains several well-described antinutritional factors according to OECD 

(2001), which include: trypsin inhibitors, lectins, isoflavones (genistein, daidzein and 

glycitein), stachyose, raffinose, and phytic acid. Combined-site analysis of antinutrients 

showed no significant differences (p>0.05) between MON 87705 and the conventional 

soybean control. Additional information is provided below to complete the discussion 

for the group of antinutrients. 

Trypsin inhibitors are heat-labile antinutrients that interfere with the digestion of proteins 

and result in decreased animal growth (Liener, 1994). Lectins are also heat labile, and 

can inhibit growth and cause death in animals if raw soybean is consumed (Liener, 1994). 

Both trypsin inhibitors and lectins are inactivated during processing of soybean protein 

products or soybean meal and, when processed appropriately, the final edible soybean 

fractions should contain minimal levels of these antinutrients. No significant differences 

(p≥0.05) were observed in trypsin inhibitor levels between MON 87705 and the 

conventional soybean control in the combined-site or individual-site analyses. 

There are three principle isoflavones in soybean seed, namely daidzein, genistein, and 

glycitein. Although they have been reported to possess biochemical activities, including 

estrogenic and anti-estrogenic effects, it is not universally accepted that the isoflavones 

are antinutrients because they have also been reported to have beneficial antioxidant, 

anticarcinogenic and heart-healthy hypocholesterolemic effects (OECD, 2001). It is well 

documented that isoflavone levels in soybean seed are highly variable and are greatly 

influenced by many factors (OECD, 2001; Messina, 2001; Nelson et al., 2001). No 

significant differences (p≥0.05) in isoflavone levels were observed between MON 87705 

and the conventional soybean control for the combined-site or individual-site analyses. 

Stachyose and raffinose are low molecular weight carbohydrates present in soybean seed 

that are considered to be antinutrients due to their consumption, which causes flatulence. 

No significant differences (p≥0.05) in raffinose levels were observed between 

MON 87705 and the conventional soybean control in the combined-site or individual-site 

analyses. Stachyose levels showed no differences between MON 87705 and the 

conventional soybean control in the combined-site analysis, but were different at one site 

(Table VII-2). This difference is not considered biologically relevant because it was 

observed only at one site and was not observed consistently across all sites. 

Phytic acid present in soybean seed chelates mineral nutrients, including calcium, 

magnesium, potassium, iron and zinc, rendering them biologically unavailable to 

monogastric animals consuming the seed (Liener, 2000). Unlike trypsin inhibitors, 

phytic acid is not heat labile, and remains stable through most soybean processing steps. 

No significant differences (p≥0.05) in phytic acid levels were observed between 

MON 87705 and the conventional soybean control for the combined-site or individualsite analyses. 
Monsanto Company 09-SY-201U Page 101 of 471

Based on the data and information presented above, it is concluded that the seed from 

MON 87705 is compositionally equivalent to conventional soybean with regard to the 

levels of antinutrients. 

VII.A.5. Proximate and Fiber Levels in Forage 

Combined-site analysis of forage showed one significant difference (p<0.05) between 

MON 87705 and the conventional soybean control for ash. The biological relevance of 

this difference was assessed based on the magnitude of the difference, reproducibility 

across sites, and comparison of mean analyte values to the 99% tolerance interval for the 

population of commercial conventional soybean varieties grown concurrently at the same 

field sites. 

The mean level of ash was significantly higher (p<0.05) in MON 87705 than the 

conventional soybean control; however, the absolute magnitude of this difference was 

small (0.57% dw). There were no differences in ash in any of the individual-site 

analyses. Furthermore, the mean level of ash in MON 87705 was well within the 99% 

tolerance interval. Therefore, the difference in ash in MON 87705 compared to the 

control is not considered biologically meaningful. These results lead to the conclusion 

that the forage from MON 87705 is compositionally equivalent to that from conventional 

soybean. 

VII.B. Compositional Equivalence of MON 87705 Seed and Forage to Conventional 

Soybean 

Consistent with OECD guidelines for soybean composition (OECD, 2001) compositional 

analyses were conducted to assess whether levels of nutrients, antinutrients, and key 

secondary metabolites in seed and forage derived from MON 87705 are comparable to 

those in the conventional soybean control, A3525, which has background genetics similar 

to MON 87705 but lacks the introduced improved fatty acid profile and glyphosate 

tolerance traits. Intended changes in the levels of the seed fatty acids 16:0 palmitic, 18:0 

stearic, 18:1 oleic, and 18:2 linoleic comprised four of the 11 significant differences in 

the combined-site analyses. For the remaining seven comparisons where a significant 

difference (p<0.05) was detected, an analysis, including magnitude of differences, 

reproducibility across individual sites, and comparisons of mean test analyte values to the 

99% tolerance interval and published values, indicates the differences are not materially 

different and/or not biologically meaningful from a food and feed safety or nutritional 

perspective. Therefore, the compositional and nutritional assessment of MON 87705 

supports the conclusion that, except for intended changes in seed fatty acid composition, 

forage and seed produced from MON 87705 are compositionally equivalent to those of 

conventional soybean. 
Monsanto Company 09-SY-201U Page 102 of 471

Table VII-2. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MOConventional Control (A3525) and Commercial Reference Varieties 

Mean Difference 

(MON 87705 minus Control)

Component (Units)1 

MON 87705

Mean 

Control 

Mean 

Mean 

Difference 

(% of A3525) 

Significance

(p-Value) 

MON 8770

Range 

Statistical Differences Observed in Combined-Site Analysis Forage Proximate (% DW) Ash 8.75 8.18 6.99 0.020 [7.39 - 10.1Seed Amino Acid (% DW) Arginine 2.78 2.68 3.74 0.048 [2.43 - 3.16Cystine 0.61 0.59 3.66 0.043 [0.57 - 0.64Seed Fatty Acid (% Total FA) 16:0 Palmitic 2.36 10.83 -78.18 <0.001 [2.25 - 2.4418:0 Stearic 3.31 4.50 -26.39 <0.001 [3.07 - 3.8218:1 Oleic 76.47 22.81 235.20 <0.001 [73.13 - 79.118:2 Linoleic 10.10 52.86 -80.90 <0.001 [7.85 - 12.42
Monsanto Company 09-SY-201U Page 103 of 471

Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in Combined-Site AnalysisSeed Fatty Acid (% Total FA)18:3 Linolenic 6.69 8.02 -16.59 <0.001 [5.55 - 7.81] 20:0 Arachidic 0.30 0.34 -11.72 0.005 [0.28 - 0.36] 20:1 Eicosenoic 0.34 0.19 79.85 <0.001 [0.27 - 0.40] Seed Proximate (% DW)Total Fat 18.29 19.33 -5.38 <0.001 [16.55 - 19.50Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)16:0 Palmitic Site CdT 2.31 10.80 -78.62 <0.001 [2.29 - 2.32] 16:0 Palmitic Site MEL 2.39 10.83 -77.92 <0.001 [2.35 - 2.42]16:0 Palmitic Site QUI 2.30 10.56 -78.24 0.005 [2.25 - 2.37]
Monsanto Company 09-SY-201U Page 104 of 471

Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)16:0 Palmitic Site RAN 2.40 10.96 -78.12 <0.001 [2.39 - 2.4016:0 Palmitic Site SFR 2.42 11.00 -78.00 <0.001 [2.40 - 2.4418:0 Stearic Site CdT 3.17 4.58 -30.88 <0.001 [3.09 - 3.2318:0 Stearic Site MEL 3.33 4.39 -24.06 0.018 [3.20 - 3.4718:0 Stearic Site QUI 3.51 4.82 -27.20 0.004 [3.15 - 3.8218:0 Stearic Site RAN 3.34 4.50 -25.73 0.001 [3.28 - 3.4118:0 Stearic Site SFR 3.22 4.31 -25.26 0.001 [3.07 - 3.41
Monsanto Company 09-SY-201U Page 105 of 471

Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:1 Oleic Site CdT 76.44 23.02 232.08 <0.001 [76.35 - 76.618:1 Oleic Site MEL 76.10 22.31 241.09 <0.001 [75.68 - 76.318:1 Oleic Site QUI 78.61 24.95 215.05 0.003 [77.70 - 79.118:1 Oleic Site RAN 74.69 21.53 246.87 <0.001 [73.13 - 75.918:1 Oleic Site SFR 76.49 22.42 241.12 <0.001 [75.33 - 77.218:2 Linoleic Site CdT 10.09 52.43 -80.75 <0.001 [9.94 - 10.2218:2 Linoleic Site MEL 10.50 53.48 -80.38 <0.001 [10.16 - 10.9
Monsanto Company 09-SY-201U Page 106 of 471

Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:2 Linoleic Site QUI 8.75 51.70 -83.07 0.006 [7.85 - 10.0218:2 Linoleic Site RAN 11.32 53.73 -78.92 <0.001 [10.37 - 12.418:2 Linoleic Site SFR 9.82 52.84 -81.42 <0.001 [9.33 - 10.5518:3 Linolenic Site CdT 6.90 8.15 -15.32 0.001 [6.85 - 6.9418:3 Linolenic Site MEL 6.58 8.00 -17.72 0.002 [6.53 - 6.6518:3 Linolenic Site QUI 5.64 7.02 -19.69 0.029 [5.55 - 5.7118:3 Linolenic Site SFR 6.98 8.49 -17.72 0.009 [6.79 - 7.26
Monsanto Company 09-SY-201U Page 107 of 471

Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)20:0 Arachidic Site CdT 0.29 0.35 -18.10 0.016 [0.28 - 0.2920:0 Arachidic Site MEL 0.30 0.34 -11.86 0.026 [0.29 - 0.3020:0 Arachidic Site QUI 0.33 0.36 -8.84 0.041 [0.30 - 0.3620:0 Arachidic Site RAN 0.28 0.33 -13.08 0.014 [0.28 - 0.2920:0 Arachidic Site SFR 0.29 0.32 -8.18 0.006 [0.29 - 0.2920:1 Eicosenoic Site CdT 0.36 0.21 76.81 <0.001 [0.36 - 0.3820:1 Eicosenoic Site MEL 0.35 0.20 70.85 0.001 [0.34 - 0.36
Monsanto Company 09-SY-201U Page 108 of 471

Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA) 20:1 Eicosenoic Site QUI 0.38 0.20 89.53 0.049 [0.37 - 0.4020:1 Eicosenoic Site RAN 0.29 0.16 82.18 0.003 [0.27 - 0.3120:1 Eicosenoic Site SFR 0.33 0.18 80.72 0.005 [0.32 - 0.35Seed Fiber (% DW)Acid Detergent Fiber Site CdT 18.23 16.27 12.10 0.049 [17.57 - 18.5Acid Detergent Fiber Site RAN 16.32 13.94 17.07 0.002 [15.71 - 16.7Statistical Differences Observed in One SiteForage Proximate (% DW)Carbohydrates Site RAN 69.77 72.09 -3.22 0.027 [68.94 - 71.0Total Fat Site MEL 5.79 6.76 -14.29 0.030 [5.37 - 6.57
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Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in One SiteSeed Amino Acid (% DW)Alanine Site SFR 1.51 1.44 4.62 0.024 [1.49 - 1.54Arginine Site SFR 2.52 2.34 7.56 0.047 [2.43 - 2.64Aspartic Acid Site SFR 3.76 3.56 5.48 0.009 [3.67 - 3.88Glutamic Acid Site SFR 5.90 5.53 6.62 0.008 [5.72 - 6.12Histidine Site SFR 0.90 0.85 5.90 0.018 [0.88 - 0.94Leucine Site SFR 2.54 2.41 5.12 0.014 [2.47 - 2.61Lysine Site SFR 2.25 2.13 5.32 0.007 [2.19 - 2.30
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Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in One SiteSeed Amino Acid (% DW)Phenylalanine Site SFR 1.68 1.60 4.83 0.019 [1.64 - 1.73Proline Site SFR 1.62 1.55 5.02 0.021 [1.59 - 1.66Tyrosine Site SFR 1.18 1.12 5.72 0.042 [1.17 - 1.20Seed Fatty Acid (% Total FA) 24:0 Lignoceric Site CdT 0.15 0.15 -3.24 0.008 [0.14 - 0.15Seed Fiber (% DW)Neutral Detergent Fiber Site CdT 21.04 17.99 16.97 0.009 [20.47 - 22.1Seed Proximate (% DW)Carbohydrates Site CdT 41.82 40.05 4.40 0.016 [41.62 - 42.0Seed Vitamin (mg/100g DW)Vitamin E Site MEL 3.26 3.83 -15.05 0.005 [3.15 - 3.45
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Table VII-2 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Component Le

vs. the Conventional Control (A3525) and Commercial Reference Varieties 

 Mean Difference 

(MON 87705 minus Control)

Component (Units)1

MON 87705

Mean

 Control 

Mean

Mean 

Difference 

(% of A3525)

Significance

(p-Value)

MON 87705 

Range

Statistical Differences Observed in One SiteSeed Antinutrient (% DW)Stachyose Site CdT 3.76 3.10 21.27 0.046 [3.55 - 4.161DW = dry weight; FA = fatty acid. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Nto zero.
Monsanto Company 09-SY-201U Page 112 of 471

VII.C. Compositional Comparison of Processed Fractions from Soybean Seed of 

MON 87705 and the Conventional Control 

To prepare soybean processed fractions, seed samples were collected from field trials 

conducted with MON 87705 and the conventional soybean control at two field sites 

(Jefferson County, IA and Clinton County, IL) in the U.S. during the 2007 growing 

season. In addition, 12 commercial conventional soybean varieties were grown at three 

field sites in the U.S. and processed to determine a 99% tolerance interval for each 

component analyzed. The seed samples were processed into defatted toasted soybean 

meal (TD soybean meal); refined, bleached, and deodorized soybean oil (RBD oil); 

protein isolate; and crude lecithin fractions. The processed fractions were analyzed 

according to the principles outlined in the OECD consensus document for soybean 

composition (OECD, 2001). The TD soybean meal was analyzed for proximates 

(moisture, protein, fat, ash, and carbohydrates by calculation), ADF, NDF, amino acids, 

trypsin inhibitors and phytic acid. The RBD oil was analyzed for fatty acids and vitamin 

E (α-tocopherol). The protein isolate fraction was analyzed for amino acids and 

moisture. The crude lecithin fraction was analyzed for phosphatides (α-phosphatidic 

acid, α-phosphatidylcholine, α-phosphatidylethanolamine, and α-phosphatidylinositiol). 

Compositional analyses were conducted to assess whether the processed fractions from 

MON 87705 are comparable to those of the conventional soybean control, A3525, which 

has background genetics similar to MON 87705, but lacks the introduced improved fatty 

acid profile and glyphosate tolerance traits. Statistically significant differences were 

determined at the 5% level of significance (p<0.05) using established statistical methods. 

The statistical analysis compared MON 87705 and the conventional control across the 

two sites (combined-site). Statistical summary of the composition of each processed 

fraction and summary of the significant differences observed between the processed 

fractions prepared from the seed of MON 87705 and the conventional control are 

included in Appendix E. 

Results show that there were no statistically significant differences (p>0.05) between 

MON 87705 and the conventional control for components measured in the protein isolate 

fraction or phosphatides of crude lecithin. Comparison of the composition of TD 

soybean meal processed from MON 87705 and the conventional soybean control showed 

no differences (p>0.05) for 21 of the 27 components analyzed. Significant differences 

(p<0.05) were observed for six components of the TD soybean meal: alanine, glycine, 

isoleucine, lysine, valine, and NDF. The absolute magnitude of the differences was small 

(<1.8% dw) and the MON 87705 mean values fell within the 99% tolerance interval for 

the conventional soybean varieties and also within the range of published values for 

conventional soybean. The low levels of residual oil (0.78% dw, as total fat) present in 

the TD soybean meal from MON 87705 also are expected to reflect the intended changes 

in fatty acid levels observed in seed. 

As expected, and consistent with the results obtained for seed fatty acid levels, the 

intended fatty acid changes (16:0 palmitic, 18:0 palmitic, 18:1 oleic and 18:2 linoleic) 

also were observed in RBD oil. In addition, six fatty acids were detected in RBD oil that 

were not detected in seed: 14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric 

(heptadecanoic) acid, 17:1 9c heptadecenoic acid, 18:2 other trans isomer fatty acids 
Monsanto Company 09-SY-201U Page 113 of 471

(excluding 9t,12t linolelaidic), and 18:2 6c,9c, octadecadienoic acid. As observed in 

seed, levels of several less abundant fatty acids were significantly different (p<0.05) 

between the RBD oil from MON 87705 and the conventional control. Differences were 

observed for 14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric (heptadecanoic) 

acid, 20:0 arachidic, 20:1 eicosenoic and 22:0 behenic acids. However, the absolute 

magnitude of the differences was small (<0.15% total FA), and the MON 87705 mean 

values fell within the 99% tolerance intervals for the reference varieties and/or within 

published ranges for conventional soybean oil (Codex, 2005; Appendix E). 

A significant increase (p<0.05) in the level of the minor fatty acid 17:1 9c heptadecenoic 

acid was observed in MON 87705 compared to conventional control RBD oil. This is not 

unexpected, given the intended shift in fatty acid levels in MON 87705. The mean level 

of 17:1 9c heptadecenoic acid in MON 87705 (0.12% total FA) was outside the range of 

values obtained for the RBD oil from commercial references. However, 17:1 9c 

heptadecenoic acid is present at similar or higher levels in a variety of oils (canola, corn, 

peanut, high oleic safflower, and high oleic sunflower; Codex, 2005) and foods (tofu, 

ground beef, and soft-spread margarine; USDA-ARS, 2007). Therefore, there are no 

adverse food and feed safety or nutrition effects associated with the levels of 17:1 9c 

heptadecenoic acid observed in MON 87705 soybean oil. The remaining minor fatty 

acids 18:2 other trans (excluding linolelaidic) and 18:2 6c,9c, octadecadienoic acid, not 

detected in seed and are believed to arise from the spontaneous isomerization of 

unsaturated fatty acids during the oil refining process. Levels of these fatty acids were 

significantly lower (p<0.05) in MON 87705 compared to control RBD oil and thus, these 

differences were not considered biologically relevant from a food and feed safety or 

nutritional perspective (Chardigny et al., 1996). 

Therefore, this supports the conclusion that, except for the intended changes in fatty acid 

composition, minor differences in the levels of less abundant fatty acids and occurrence 

of low levels of minor fatty acids due to spontaneous isomerisation during the oil refining 

process, the processed fractions produced from MON 87705 are compositionally 

equivalent to those of conventional soybean. 

VII.D Safety and Nutritional Assessment of the Intended Changes in MON 87705 

MON 87705 was developed to generate soybean oil with decreased levels of saturated 

fats (16:0 palmitic acid and 18:0 stearic acid) and increased levels of 18:1 oleic acid, with 

an associated decrease in 18:2 linoleic acid. Replacement of conventional soybean oil 

with MON 87705 soybean oil under the proposed food uses results in changes in the fatty 

acid composition in the U.S. diet that lead to higher oleic acid intake,and lower 

consumption of saturated fats (i.e., 16:0 palmitic and 18:0 linoleic acid) with no impact 

on total fat intake. This assessment assumes all of the targeted oil components of the 

foods proposed for replacement that are consumed in the U.S. are replaced with 

MON 87705 soybean oil. Therefore, the results presented in this petition represent a 

theoretical maximal effect of MON 87705 soybean oil on fatty acid composition of the 

diet. The nutritional impact from the use of MON 87705 soybean oil in targeted foods 

under the intended conditions of use is estimated to result in changes in fatty acid 

consumption that are within current dietary guidelines for fatty acid intake (Lichtenstein 

et al., 2006; USDA-ERS 2005; WHO/FAO, 2003). A discussion of the safety and 
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nutritional impact resulting from the intended changes in MON 87705 is included in 

Appendix M. 

VII.E Safety and Nutrition Assessment Conclusion 

In conclusion, except for the intended changes in fatty acid levels, the compositional 

equivalence of MON 87705 seed, forage, and processed fractions to conventional 

soybean has been demonstrated in accordance with OECD guidelines. In addition, the 

nutritional impact from the use of MON 87705 soybean oil in targeted foods under the 

intended conditions of use is estimated to result in changes in fatty acid consumption that 

are within current dietary guidelines for fatty acid intake. Therefore, MON 87705 is 

regarded to be as safe and nutritious as conventional soybean for food and feed use. 
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VIII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT 

This section provides an evaluation of the phenotypic and agronomic characteristics and 

environmental interactions of MON 87705 compared to the conventional A3525 control, 

a conventional soybean variety that has background genetics similar to MON 87705 but 

does not possess the improved fatty acid profile and glyphosate tolerance trait. 

Phenotypic and agronomic characteristics of MON 87705 were evaluated in a 

comparative manner to assess plant pest potential (OECD, 1993). In the phenotypic, 

agronomic, and environmental interactions assessment of MON 87705, data were 

collected to evaluate specific aspects of altered plant pest potential based on requirements 

of USDA-APHIS set forth at 7 CFR § 340.6. The MON 87705 plant characterization and 

environmental interactions data cover six general categories: 1) germination, dormancy, 

and emergence; 2) vegetative growth; 3) reproductive growth (including pollen 

characteristics); 4) seed retention on the plant and lodging; 5) plant-symbiont 

associations; and 6) plant interactions with insect, disease, and abiotic stressors. An 

overview of the characteristics assessed is presented in Table VIII-1. 

Results from the phenotypic and agronomic assessments indicate that MON 87705 does 

not possess characteristics that would confer a plant pest risk or significant environmental 

impact compared to conventional soybean. Data on environmental interactions also 

indicate that MON 87705 does not confer any increased susceptibility or tolerance to 

specific diseases, insects, or abiotic stressors. 

VIII.A. Characteristics Measured for Assessment 

The phenotypic, agronomic, and environmental interactions data were evaluated from a 

basis of familiarity (OECD, 1993) and were comprised of a combination of field, 

greenhouse, and laboratory studies conducted by scientists who are familiar with the 

production and evaluation of soybean. In each of these assessments, MON 87705 was 

compared to an appropriate conventional control that had a genetic background similar to 

MON 87705 but did not possess the improved fatty acid profile and glyphosate tolerance 

trait. In addition, multiple commercial soybean varieties (see Appendix F and Tables F1, G-1, and I-1) were included to provide a range of comparative values that are 

representative of existing commercial soybean varieties for each measured phenotypic, 

agronomic, and environmental interaction characteristic. Data collected from the 

commercial reference varieties reflect a range of selection and breeding for desirable 

characteristics and therefore can provide context for interpreting experimental results. 
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Table VIII-1. Phenotypic, Agronomic and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials or Greenhouse Studies 

Data 

Category 

Characteristics 

measured 

Evaluation timing1 Evaluation description 

(measurement endpoints) 

Germination

, dormancy, 

and 

emergence 

Normal germinated Day 5 and 8 (20/30°C) % of seed producing seedlings exhibiting 

normal developmental characteristics 

Abnormal germinated Day 8 (20/30°C) % of seed that could not be classified as 

normal germinated 

Germinated Day 5, 8, and 13 (10, 20, 30, 

10/20 and 10/30°C) 

% of seed that had germinated normally 

and abnormally 

Dead Day 5 and 8 (10, 20, 30, 

10/20, 10/30, and 20/30°C); 

Day 13 (10, 20, 30, 10/20 and 

10/30°C) 

% of seeds that had visibly deteriorated 

and had become soft to the touch 

Hard viable and 

nonviable 

Day 8 (20/30°C); Day 13 (10, 

20, 30, 10/20 and 10/30°C) 

% of seeds that did not imbibe water and 

remained hard to the touch 

Firm swollen viable 

and nonviable 

Day 8 (20/30°C); Day 13 (10, 

20, 30, 10/20 and 10/30°C) 

% of seeds that imbibed water 

Early stand count V2 - V4 Number of emerged plants in two rows, 

standardized to 20 ft rows 

Final stand count Maturity, R8 Number of plants in two rows, 

standardized to 20 ft rows 

Vegetative 

growth 

Seedling vigor V2 - V4 Rated on a 1-9 scale, where 1-3 = 

excellent, 4-6 = average, and 7-9 = poor 

vigor 

Growth stage 

assessment 

Every two-three weeks, V2-

R8 

Average soybean plant growth stage per 

plot 

Flower color Flowering, R1-R2 Color of flowers: purple, white, or mixed 

Plant pubescence Maturity, R8 Pubescence on plants in each plot 

categorized as hairy, hairless, or mixed 

Plant height Maturity, R8 Distance from the soil surface to the 

uppermost node on the main stem of five 

representative plants per plot 

Reproductiv

e growth 

Days to 50% flowering Flowering, R1-R2 Calendar day number when approximately 

50% of the plants in each plot were 

flowering 

Pollen viability Flowering, R1-R2 Viable and nonviable pollen based on 

pollen grain staining characteristics 

Pollen morphology Flowering, R1-R2 Diameter of viable pollen grains 

Seed moisture Harvest Percent moisture content of harvested seed 

100 seed weight (g) Harvest Mass of 100 harvested seeds 

Test weight (lb/bu) Harvest Mass of a bushel of harvested seed 

Yield (bu/ac) Harvest Bushels of harvested seed per acre, 

adjusted to 13% moisture 

Seed 

retention and 

lodging 

Lodging Maturity, R8 Rated on 0-9 scale, where 0 = completely 

erect and 9 = completely flat or lodged 

Pod shattering Maturity, R8 Rated on 0-9 scale, where 0 = no shattering 

and 9 = completely shattered 

Plantsymbiont 

interactions 

Biomass 6 weeks after emergence in 

greenhouse 

Nodule, root, and shoot dry weight 

Nodule number 6 weeks after emergence in 

greenhouse 

Nodule number 

Total nitrogen 6 weeks after emergence in 

greenhouse 

Shoot total nitrogen (% and g/plant) 

 
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Table VIII-1 (continued). Phenotypic, Agronomic and Environmental 

Interaction Characteristics Evaluated in U.S. Field Trials or Greenhouse Studies 

Data 

Category

Characteristics 

measured

Evaluation timing1 Evaluation description 

(measurement endpoints)

Plant 

interactions 

Plant response to 

abiotic stressors and 

disease damage 

Four times per growing 

season 

Qualitative assessment of each plot, with 

rating on a 0-9 scale, where 0 = no 

symptoms and 9 = severe symptoms 

Arthropod damage Four times during growing 

season 

Qualitative assessment of each plot, with 

rating on a 0-5 scale, where 0 = no 

symptoms and 5 = severe symptoms 

Arthropod abundance Three times during growing 

season 

Quantitative assessment of pest and 

beneficial arthropods 1

Soybean plant growth stages were determined using descriptions and guidelines outlined in Soybean 

Growth and Development (Pedersen, 2004). 

VIII.B. Interpretation of Phenotypic and Environmental Interaction Data 

Plant pest risk assessments for biotechnology-derived crops are, by standard, comparative 

assessments. Familiarity provides a basis from which the potential environmental impact 

of a biotechnology-derived plant can be evaluated. The concept of familiarity is based on 

the fact that the biotechnology-derived plant is developed from a well-characterized 

conventional plant variety. Familiarity considers the biology of the crop, the introduced 

trait, the receiving environment and the interaction of these factors, and provides a basis 

for comparative environmental risk assessment between a biotechnology-derived plant 

and its conventional counterpart. 

Expert knowledge and experience with conventionally bred soybean was the basis for 

selecting appropriate endpoints and estimating the range of responses that would be 

considered typical for soybean. As such, assessment of phenotypic and agronomic 

characteristics and environmental interactions was essential to compare the 

biotechnology-derived plant to the conventional counterpart. An overview of the 

characteristics assessed is presented in Table VIII-1. A subset of the data relating to 

well-understood weediness criteria (e.g., seed dormancy, pre-harvest seed loss 

characteristics, and lodging) was used to assess whether there was an increase in 

weediness potential, an element of APHIS’s plant pest determination. Based on all of the 

data collected, an assessment was made whether the biotechnology-derived plant is likely 

to pose an increased plant pest risk compared to the conventional counterpart. 

Experienced scientists familiar with each experimental design and evaluation criteria 

were involved in all components of data collection, summarization, and analysis. Study 

personnel assessed that measurements were taken properly, data were consistent with 

expectations based on experience with the crop, and the experiment was carefully 

monitored. Prior to analysis, the overall dataset was evaluated for evidence of 

biologically relevant changes, and for possible evidence of an unexpected plant response. 

These scientists did not identify any unexpected observations or issues in the course of 

these evaluations. Data were then subjected to statistical analysis. 
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VIII.C. Interpretation of Detected Differences Criteria 

Comparative plant characterization data between a biotechnology-derived crop and the 

control are interpreted in the context of contributions to increased plant pest potential as 

assessed by APHIS. Under the framework of familiarity, characteristics for which no 

differences are detected support a conclusion of no increased plant pest potential of the 

biotechnology-derived crop compared to the conventional crop. Characteristics for 

which differences are detected are considered in a step-wise method (Figure VIII-1 or a 

similar method). All detected differences for a characteristic are considered in the 

context of whether or not the difference would increase the plant pest potential of the 

biotechnology-derived crop. Ultimately, a weight of evidence approach considering all 

characteristics and studies was used for the overall risk assessment of differences and 

their significance. In detail, Figure VIII-1 illustrates the stepwise assessment process 

employed: 

Step 1 

Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or 

ecological change for the crop in terms of plant pest potential and subsequent steps are not considered. If 

the answer is “yes” or uncertain the subsequent step is considered. 

Figure VIII-1. Schematic Diagram of Agronomic and Phenotypic Data 

Interpretation Methods 

Hazard identification and 

risk assessment on 

difference 

Outside variation for crop? No 

Yes 

No 

Yes 

Adverse in terms of plant pest 

potential? 

Yes 

Outside variation of study 

No 

Not adverse; the direction 

or magnitude of the 

detected difference in the 

measured characteristic 

does not contribute to a 

biological or ecological 

change for the crop in 

terms of plant pest 

potential 

Yes 

Statistical differences detected 

in combined-site analysis? 

No 

Differences detected in the combined-site 

and individual site analyses are evaluated*

Step 2 

Step 3 

Step 4 

The measured

characteristic does not 

contribute to a biological 

or ecological change for 

the crop in terms of plant 

pest potential 

Step 5 

Step 6 

*See text for interpretation of differences 

 detected in the individual site analysis 
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▪ Steps 1 and 2. Evaluate Detected Statistical Differences. Combined-site and 

individual-site statistical analyses are conducted and evaluated on each measured 

characteristic. Differences detected in the individual-site analysis must be observed in 

the combined site analysis to be considered further for plant pest potential. Any 

difference detected in the combined-site analysis is further assessed. 

▪ Step 3. Evaluate Differences Relative to Reference Range. If a difference is detected 

in the combined-site analysis across multiple environments, then the test substance mean 

value is assessed relative to the reference substances. 

▪ Step 4. Evaluate Differences in the Context of the Crop. If the test substance mean is 

outside the variation of the reference substances (e.g., reference range), the test substance 

mean is considered in the context of known values common for the crop. 

▪ Step 5. Plant Pest Potential. If the test substance mean is outside the range of values 

common for the crop, the detected difference is then assessed for whether or not it is 

adverse in terms of plant pest potential. 

▪ Step 6. Conduct Risk Assessment on Identified Hazard. If an adverse effect (hazard) 

is identified, risk assessment on the difference is conducted. The risk assessment 

considers contributions to enhanced plant pest potential of the crop itself, the impact of 

differences detected in other measured characteristics, and potential for and effects of 

trait transfer to feral populations of the crop or to a sexually compatible species. 

VIII.D. Phenotypic, Agronomic and Environmental Interactions Characteristics 

As a significant part of the evaluation of MON 87705, plant phenotypic and agronomic 

characteristics including seed dormancy and germination, phenotypic, agronomic and 

environmental interactions, pollen characteristics, and symbiont interactions were 

evaluated. The phenotypic, agronomic, and environmental interaction evaluations are 

based on replicated laboratory, greenhouse, and/or multi-site field trials and experiments. 

In evaluating the phenotypic and agronomic characteristics of MON 87705, data were 

collected that address specific environmental risks regarding plant pest potential based on 

the considerations of USDA-APHIS. 

VIII.D.1. Seed Dormancy and Germination Characteristic 

APHIS considers the potential for weediness to constitute a plant pest factor (CFR § 

340.6). Seed germination and dormancy mechanisms vary with species and their genetic 

basis tends to be complex. Seed dormancy (e.g., hard seed) is an important characteristic 

that is often associated with plants that are considered as weeds (Anderson, 1996; 

Lingenfelter and Hartwig, 2003), where in soybean it is not uncommon to observe low 

levels of hard seed (Mullin and Xu, 2001; Potts et al., 1978). Standardized germination 

assays are available and routinely used to measure the germination characteristics of 

soybean seed. The Association of Official Seed Analysts, an internationally recognized 

seed testing organization, recommends a temperature range of 20/30° C as optimal for 

germination of soybean (AOSA, 2007). 

Comparative assessments of seed dormancy and germination characteristics were 

conducted on MON 87705 and A3525, where A3525 served as a comparable control 
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because it has background genetics similar to MON 87705 but does not possess the 

improved fatty acid profile and glyphosate tolerance trait. In addition, eight 

commercially available soybean varieties were included as references to provide baseline 

values common to soybean. The seed lots for MON 87705, the conventional soybean 

control and reference varieties were produced during 2007 in Iowa (IA), Indiana (IN), 

and Missouri (MO), geographic areas which represent environmentally relevant 

conditions for soybean production for this product. In addition to the AOSA 

recommended temperature range of 20/30 °C, seed was tested at five other temperature 

regimes of 10, 20, 30, 10/20, and 10/30 °C to assess seed germination properties. The 

details of the materials, experimental methods, and germination data from all individual 

production sites are presented in Appendix F. 

No statistically significant differences were detected between MON 87705 and the 

control for percent viable hard seed in any temperature regime with the exception of the 

20 °C temperature regime (Table VIII-2). At 20 °C, MON 87705 had lower percent 

viable hard seed than the control (0.0 vs. 0.3%). The mean value for percent viable hard 

seed of MON 87705 was within the reference range (0.0 – 0.3%). Thus, the statistical 

difference detected for percent hard seed is unlikely to be biologically meaningful in 

terms of increased weed potential of MON 87705 compared to conventional soybean. 

Furthermore, a decrease in hard seed would not contribute to increased weediness of 

soybean. 

No statistically significant differences were detected between MON 87705 and the 

control for percent viable firm swollen seed in any temperature regime (Table VIII-2). 

Within some temperature regimes, it was not possible to conduct an analysis of variance 

for percent viable firm swollen seed because there was no variability present in the data. 

For these data, the values for MON 87705 and the control were all zero, indicating no 

biological differences. Additionally, no statistically significant differences were detected 

between MON 87705 and the control for percent germinated, viable hard, dead, or viable 

firm swollen seed in the 10, 10/20, or 10/30 °C temperature regimes. 

Three other statistically significant differences were detected between MON 87705 and 

the control in the combined-site analysis (Table VIII-2). MON 87705 had lower percent 

germinated seed than the control at 30 °C (92.8 vs. 95.5%), and had higher percent dead 

seed than the control at 30 °C (7.3 vs. 4.5%) and 20/30 oC (2.7 vs. 1.3%). Lower percent 

germinated seed and higher percent dead seed would not contribute to increased 

weediness. Furthermore, all values were well within the recommended standards for 

certified soybean seed (AOSCA, 2009b). 

The biological characteristics evaluated in this study were used to characterize 

MON 87705 in the context of plant pest risk assessment. Based on the dormancy and 

germination characteristics assessed, the results of this study, particularly the lack of 

increased hard seed, demonstrate there were no changes indicative of increased weed 

potential of MON 87705 relative to conventional soybean. 
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Table VIII-2. Germination Characteristics of MON 87705 and A3525 

Temp. 

Regime 1 Category

Mean % (S.E.)2

MON 87705 A3525 Reference Range 3

10 °C Germinated 97.7 (0.7) 98.4 (0.4) 98.2 – 99.5

Viable Hard 0.1 (0.1) 0.3 (0.1) 0.0 – 0.8

Dead 2.1 (0.8) 1.1 (0.4) 0.3 – 1.3

Viable Firm-Swollen 0.2 (0.1) 0.3 (0.1) 0.0 – 0.5

20 °C Germinated 99.1 (0.5) 98.8 (0.3) 96.9 – 99.8

Viable Hard 0.0 (0.0) * 0.3 (0.1) 0.0 – 0.3

Dead 0.9 (0.5) 0.8 (0.3) 0.3 – 3.1

Viable Firm-Swollen 0.0 (0.0)† 0.0 (0.0) 0.0 – 0.0

30 °C Germinated 92.8 (2.4)* 95.5 (0.6) 94.4 – 99.5

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3

Dead 7.3 (2.4) * 4.5 (0.6) 0.3 – 5.4

Viable Firm-Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0

10/20 °C Germinated 99.3 (0.2) 99.6 (0.2) 98.4 – 100.0

Viable Hard 0.1 (0.1) 0.1 (0.1) 0.0 – 0.3

Dead 0.6 (0.1) 0.3 (0.2) 0.0 – 1.6

Viable Firm-Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0

10/30 °C Germinated 98.0 (0.7) 98.8 (0.4) 98.0 – 99.5

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3

Dead 2.0 (0.7) 1.3 (0.4) 0.5 – 1.8

Viable Firm-Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0

20/30 °C Normal Germinated 92.4 (1.0) 93.6 (0.7) 57.0 – 98.5

(AOSA) Abnormal Germinated 4.9 (0.7) 5.0 (0.7) 1.3 – 42.5

Viable Hard 0.0 (0.0) 0.2 (0.1) 0.0 – 0.4

Dead 2.7 (0.8) * 1.3 (0.4) 0.3 – 2.4

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0 – 0.1

Note: The data were analyzed according to three randomized complete block (RCB) designs with four 

replications; each site represented a separate RCB. 

*Indicates a statistically significant difference between MON 87705 and the control (p < 0.05). 

†No statistical comparison could be made due to lack of variability in the data. 

1

 For the alternating temperature regimes of 10/20, 10/30, or 20/30 °C, the lower temperature was 

maintained for 16 hours, and the higher temperature for eight hours. 2

 Means based on four replicates (n = 4) of approximately 100 seeds. In some instances, the total 

percentage for both MON 87705 and the control did not equal exactly 100% due to numerical rounding of 

the means. S.E. = Standard Error. 3 Minimum and maximum means determined from among the eight commercially available reference 

soybean varieties produced at the sites.

 
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VIII.D.2. Field Phenotypic, Agronomic Characteristics and Environmental 

Interactions 

Plant growth, development, and yield characteristics were assessed under field conditions 

as part of the plant characterization assessment of MON 87705. These data were 

developed to provide APHIS with a detailed characterization of MON 87705 relative to 

the conventional soybean control, A3525, and commercially available soybean. 

According to CFR § 340.6, as part of the petition to seek deregulation, a petitioner must 

submit “A detailed description of the phenotype of the regulated article.” This 

information is being provided to assess whether there are phenotypic differences between 

MON 87705 compared to the unmodified recipient organism that may impact its pest 

potential. Environmental interactions were also assessed as an indirect indicator of 

phenotypic changes to MON 87705 relative to the same comparators described above. 

The purpose of these field evaluations was to assess the phenotypic and agronomic 

characteristics and the plant-insect, plant-disease, or plant-abiotic stressor interactions of 

MON 87705 compared to the control. Certain growth, reproduction, and pre-harvest seed 

loss characteristics (such as lodging and pod shattering) can be used in the assessment of 

whether MON 87705 has enhanced plant pest potential. 

Data were collected at 17 field locations during 2007 to thoroughly evaluate phenotypic, 

agronomic, and environmental interaction characteristics. These 17 locations provided a 

diverse range of environmental and agronomic conditions representative of commercial 

soybean production areas in the U.S. (Table VIII-3). The experiments were arranged as 

randomized complete block designs. The categories and timings of phenotypic 

characteristics and environmental interactions evaluated are included in Table VIII-1. 

The methods and detailed results of the individual site data comparisons are presented 

and discussed in Appendix G, while the combined-site analyses are summarized below. 

The results of this assessment showed the improved fatty acid profile and glyphosate 

tolerance trait did not unexpectedly alter MON 87705 compared to conventional soybean 

in terms of weediness potential, and the lack of differences in plant response to abiotic 

stressors, disease damage, arthropod damage, and arthropod pest and beneficial insect 

abundance further support the conclusion that the introduction of the modified oil profile 

trait is unlikely to increase plant pest potential. 

VIII.D.2.1. Field Phenotypic and Agronomic Characteristics 

A total of 14 phenotypic and agronomic characteristics were evaluated. For the 

combined-site analyses, no significant differences were detected between MON 87705 

and the control for seedling vigor, plant height, lodging, pod shattering, seed moisture, 

test weight, or yield (Table VIII-4). Four statistically significant differences were 

detected between MON 87705 and the control in the combined site analysis. 

MON 87705 was lower than the control for both early stand count (235.1 vs. 256.1 

plants/plot) and final stand count (216.3 vs. 239.3 plants/plot); however, mean counts of 

MON 87705 were within the reference soybean varieties range for both stand count 

evaluations. Furthermore, it is unlikely that decreased stand counts would contribute to 

increased weed potential of MON 87705 and the detected differences in stand count did 

not impact final yield. MON 87705 flowered approximately one day later than the 
Monsanto Company 09-SY-201U Page 123 of 471

control (198.1 vs. 196.9 days after 1 Jan. 2007) and the weight of 100 seeds was lower 

for MON 87705 compared to the control (15.6 vs. 16.1 g). The differences in days to 

50% flowering and 100 seed weight were relatively small in magnitude (0.6% and 3.2%, 

respectively), and the mean values of MON 87705 were within the range of the reference 

soybean varieties for both characteristics. Thus, the differences in days to 50% flowering 

and 100 seed weight are unlikely to be biologically meaningful in terms of increased 

weed potential. 

Flower color, plant pubescence, and plant growth stage data were categorical and were 

not statistically analyzed; however, at each site all plants of MON 87705 and the control 

had purple flowers and pubescence as expected. Furthermore, MON 87705 and the 

control were within the same range of plant growth stages for 113 out of the 114 growth 

stage observations among the sites. During a single observation at one site, MON 87705 

plants were at the V6 growth stage while the control plants were at V7; however, the 

growth stage of MON 87705 was within the range of growth stages observed for the 

reference soybean varieties (Appendix G; Table G-4). 

The phenotypic and agronomic characteristics evaluated in this study were used to 

provide a detailed description of MON 87705 compared to the nontransformed control 

(A3525). A subset of these characteristics was useful to assess the weediness potential of 

MON 87705 compared to the conventional soybean control. Based on the measured 

phenotypic and agronomic characteristics, the results support a conclusion of no 

unexpected changes in the phenotype and no increased plant pest potential of 

MON 87705 compared to the conventional soybean control. 
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Table VIII-3. Field Phenotypic Evaluation Sites for MON 87705 during 2007 

Location† Location 

Code 

USDA-APHIS 

Notification Number 

Jackson County, Arkansas * AR 07-043-105n 

07-115-103n 

Jefferson County, Iowa IA1 07-043-104n 

Benton County, Iowa IA2 07-043-104n 

Clinton County, Illinois IL1 07-043-104n 

Stark County, Illinois IL2 07-043-104n 

Warren County, Illinois IL3 07-043-105n 

Boone County, Indiana IN1 07-043-104n 

Parke County, Indiana IN2 07-043-105n 

Pawnee County, Kansas KS 07-043-105n 

Ottawa County, Michigan MI 07-043-105n 

Lincoln County, Missouri MO2 07-043-105n 

St. Louis County, Missouri MO3 07-043-105n 

Macon County, Missouri MO4 07-043-105n 

York County, Nebraska NE 07-043-104n 

Fayette County, Ohio OH 07-043-104n 

Berks County, Pennsylvania PA 07-043-104n 

Walworth County, Wisconsin WI 07-043-104n 

* The Arkansas site utilized two USDA-APHIS notifications. 07-043-105n was a release 

notification and 07-115-103n was a movement notification. 

†Shelby County, Missouri (USDA-APHIS notification number 07-043-105n) data not reported 

due to wild animal damage early in the season. 
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Table VIII-4. Plant Growth and Development Data across 17 Locations during 2007 

MON 87705* A3525 Reference Range1

Phenotypic Characteristic (units) Mean (S.E.) Mean (S.E.) Minimum Maximum Early stand count (#/plot) 235.1* (8.8) 256.1 (6.9) 142.3 267.2 Seedling vigor (1-9 scale) 3.0 (0.2) 2.9 (0.2) 2.3 5.4 Days to 50% flowering2 198.1* (1.1) 196.9 (1.1) 194.5 200.1 

Plant height (in) 34.2 (0.9) 35.1 (0.9) 26.6 43.0 

Lodging (0-9 scale) 1.8 (0.3) 1.6 (0.3) 0.4 2.3 

Pod shattering (0-9 scale) 0.2 (0.1) 0.2 (0.1) 0.0 0.5 Final stand count (#/plot) 216.3* (8.7) 239.3 (7.3) 145.0 258.2 

Seed moisture (%) 12.3 (0.3) 12.3 (0.3) 11.0 13.6 100 seed weight (g) 15.6* (0.4) 16.1 (0.4) 13.8 20.5 Test weight (lb/bu) 54.3 (0.6) 54.0 (0.7) 51.7 56.0 Yield (bu/ac) 54.3 (2.5) 54.8 (2.3) 38.7 63.8 

* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (A3525) (p < 0.05). 1 Reference range = Minimum and maximum mean values among the 17 commercially available reference soybean varieties.2 Calendar day number when approximately 50% of the plants in each plot were flowering. 

S.E. = standard error. Means based on n = 51 (except 100 seed weight where n = 48). 
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VIII.D.2.2. Environmental Interaction Analyses

APHIS considers the environmental interaction potential of the biotechnology-derived 

soybean compared to its conventional counterpart to determine the potential for increased 

weedy or invasive characteristics. Evaluations of environmental interactions were 

conducted as part of the plant characterization for MON 87705. In the 2007 U.S. field 

trials conducted for evaluation of phenotypic and agronomic characteristics of 

MON 87705, observational data on plant response to abiotic stressors (drought, wind, 

nutrient deficiency, etc.), disease damage, arthropod damage, and arthropod abundance 

(Appendix G; Tables G-5, G-6, G-7, G-8, and G-9, respectively) also were collected. 

These data are used as part of the environmental risk assessment to assess the plant pest 

potential and potential increased adverse impact on NTOs for MON 87705 compared to 

the conventional soybean control (see Section X and Section XI for additional 

discussion). In addition, multiple commercial soybean varieties were included in the 

analysis to establish a range of natural variability for each assessed characteristic. The 

environmental interactions evaluation included data collected in the phenotypic studies 

(plant-insect, plant-disease, and plant-environment interactions). The results of this 

assessment showed the improved fatty acid profile and glyphosate tolerance trait did not 

unexpectedly alter MON 87705 compared to conventional soybean in terms of pest 

potential. The lack of differences in plant response to abiotic stressors, disease damage, 

arthropod damage, and arthropod pest and beneficial insect abundance indicate that the 

introduction of the improved fatty acid profile and glyphosate tolerance trait is unlikely 

to be biologically meaningful in terms of increased pest potential. In these trials, the 

observations of plant response to abiotic stressors, disease damage, and arthropod 

damage were performed four times during the growing season at all 17 sites, and 

arthropod abundance was assessed from collections performed three times during the 

growing season at four of the 17 sites. The observed stressors were at natural levels (i.e., 

no artificial infestation or interference was used). Therefore, the same stressors were not 

necessarily observed at each field site. 

Environmental interactions were assessed qualitatively at 17 sites, and arthropod 

abundance data were collected quantitatively from four sites. For the plant-insect 

interactions, plant-disease interactions, and plant responses to abiotic stressors, the 

reported values represent the range of ratings observed across the three replications at 

each site. MON 87705 and the control were considered qualitatively different in 

response to a stressor if the ratings between MON 87705 and the conventional soybean 

control did not overlap across all replications for that particular stressor (e.g., “none” 

rating vs. “slight-moderate” rating). The ratings observed among the commercial 

reference soybean varieties provide qualitative assessment data common to soybean for 

each stressor assessed. 

In an assessment of abiotic stress response, disease damage, and arthropod damage, no 

differences were detected between MON 87705 and the conventional soybean control for 

574 of 579 comparisons (including 167 abiotic stress response, 206 disease damage, and 

206 arthropod damage comparisons) among all observations at the 17 sites (Appendix G; 

Tables G-5, G-6, and G-7). The five observed differences were in the disease and 

arthropod damage categories. MON 87705 had less damage than the control from 
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bacterial blight during three observations at a single site and from aphids and leafhoppers 

during one observation at a single site. For each of the five observed differences, the 

severity of damage in the MON 87705 plots was within the range of that for the reference 

soybean variety plots. Therefore, the detected differences in disease and arthropod 

damage ratings are unlikely to be biologically meaningful in terms of increased plant pest 

potential for MON 87705 compared to the conventional soybean control. 

In an assessment of pest and beneficial arthropod abundance, no statistical differences 

were detected between MON 87705 and the conventional soybean control for 95 out of 

96 comparisons (including 46 arthropod pest comparisons and 50 beneficial arthropod 

comparisons) among the collection intervals at the four sites (Appendix G; Tables G-8 

and G-9). The single detected statistically significant difference was for bean leaf beetle 

in a single collection from one site, and the mean abundance value from the MON 87705 

plots was within the range of that for the reference soybean variety plots. Thus, the 

differences are unlikely to be biologically meaningful in terms of increased plant pest 

potential. 

These results indicate that compared to conventional soybean, the environmental 

interactions between MON 87705 and arthropod pest and beneficial organisms, diseases, 

and abiotic stressors were not altered compared to conventional soybean. The lack of 

significant biological differences in plant response to abiotic stressors, disease damage, 

arthropod damage, and arthropod pest and beneficial insect abundance indicate that the 

improved fatty acid profile and glyphosate tolerance trait of MON 87705 is unlikely to be 

biologically meaningful in terms of increased plant pest potential. 

VIII.D.3. Pollen Characteristics 

APHIS considers the potential for gene flow to, and introgression of the biotechnologyderived trait into other soybean varieties and wild relatives to determine the potential for 

increased weedy or invasive characteristics of the receiving species. Pollen morphology 

and viability information are pertinent to this assessment and therefore were assessed on 

MON 87705. In addition, morphological characterization of pollen produced by 

MON 87705 and the conventional soybean control is relevant to the plant pest risk 

assessment because it adds to the detailed description of the phenotype of MON 87705 

compared to the conventional soybean. 

The purpose of this evaluation was to assess the morphology and viability of pollen 

collected from MON 87705 compared to a conventional soybean control. Pollen was 

collected from MON 87705, the control (A3525), and five commercially available 

reference soybean varieties grown under similar agronomic conditions in a field trial in 

Missouri. The trial was arranged in a randomized complete block design with three 

replications. A minimum of 20 flowers were collected from each plot. Pollen was 

extracted, combined among flowers collected from the same plot, and stained with 

Alexander’s stain (Alexander, 1980). Pollen viability was evaluated for each sample and 

pollen grain diameter was measured for ten representative viable pollen grains per 

replication. General morphology of the pollen was observed for each of the three 

replications of MON 87705, the control, and the reference soybean varieties (see 

Appendix H). 
Monsanto Company 09-SY-201U Page 128 of 471

No statistically significant differences were detected between MON 87705 and the 

control for percent viable pollen or pollen grain diameter (Table VIII-5). Furthermore, 

no visual differences in general pollen morphology were observed between MON 87705 

and the control. These results demonstrate that the introduction of the modified oil 

profile trait did not alter the overall morphology or viability of MON 87705 pollen 

compared to the conventional soybean control. The lack of statistically significant 

differences between the pollen collected from MON 87705 compared to the conventional 

soybean control for the assessed characteristics demonstrate that the observed values 

were within the range of observations expected for soybean. Thus, these data further 

support no change in plant pest potential for MON 87705 compared to the 

nontransformed control and other soybean varieties. 

Table VIII-5. Pollen Grain Diameter and Viability Analyses 

Pollen 

Characteristic 

MON 87705* A3525 Reference Range1

Mean (S.E.) Mean (S.E.) Minimum Maximum 

 

Viability (%) 98.3 (0.3) 96.7 (0.5) 97.2 99.6 

Diameter (μm) 25.7 (0.1) 25.0 (0.5) 24.3 25.8 

S.E. = Standard Error. Means based on n = 3. 

* No significant differences were detected between the MON 87705 and the control (p > 0.05). 

1

 Reference range is the minimum and maximum mean value observed among the five reference soybean 

varieties.
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VIII.D.4. Symbiont Interactions 

Members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae form a highly 

complex and specific symbiotic relationship with leguminous plants, including soybean 

(Gage, 2004). The nitrogen-fixing plant-microbe symbiosis results in the formation of 

root nodules, providing an environment in which differentiated bacteria called bacteroids 

are capable of reducing or fixing atmospheric nitrogen. The product of nitrogen fixation, 

ammonia, then can be utilized by the plant. In soybean, atmospheric nitrogen is fixed 

into ammonia through a symbiotic association with the bacterium Bradyrhizobium 

japonicum. As a result of this relationship, no nitrogen inputs are needed for agricultural 

production of soybean. 

As part of the plant pest risk assessment, APHIS considers the impact of the modified 

crop on pest potential and the environment as well as on agricultural or cultivation 

practices (CFR § 340.6). Changes in the symbiotic relationship with Rhizobiaceae and 

Bradyrhizobiaceae could directly impact pest potential, the environment, or cultivation 

practices (i.e., need to add additional nitrogen to soybean production). Thus, the purpose 

of this evaluation was to assess whether the B. japonicum-soybean symbiosis of 

MON 87705 had been altered as a result of the introduction of the improved fatty acid 

profile and glyphosate tolerance trait compared to a conventional soybean control. 

The relative effectiveness of the symbiotic association between a leguminous plant and 

its rhizobial symbiont can be assessed by various methods. Assessment of nodule 

number and mass along with plant growth and nitrogen status are commonly used to 

assess differences in the symbiotic association between a legume and its associated 

rhizobia (Israel et al., 1986). It should be noted, however, that nodule number relative to 

nodule dry weight may be variable in soybean experiments because some nodules may be 

larger in diameter and less numerous, while others are not as developed (smaller) but 

more abundant (Appunu and Dhar, 2006; Israel et al., 1986). 

MON 87705, a conventional soybean control (A3525), and six reference soybean 

varieties were produced from seed planted in pots containing nitrogen-deficient potting 

medium grown in a greenhouse. Seeds were inoculated with a solution of B. japonicum. 

The pots were arranged in a randomized complete block design with eight replicates. At 

six weeks after emergence, plants were excised at the surface of the potting medium, and 

then shoot and root plus nodule material were removed from the pots. Nodules were 

separated from roots prior to enumeration and determination of dry weight. Detailed 

information on materials and methods used for symbiont evaluation is presented in 

Appendix I. 

No significant differences were detected between MON 87705 and the control for each 

measured parameter, including nodule number, shoot total nitrogen (percent and mass), 

and biomass (dwt) of nodules, shoot material, and root material (Table VIII-6). 

Based on the assessed characteristics, the results support the conclusion that the 

introduction of the improved fatty acid profile and glyphosate tolerance trait does not 

alter the symbiotic relationship between B. japonicum and MON 87705 compared to 

conventional soybean. Thus, there is no increased plant pest potential and no expected 
Monsanto Company 09-SY-201U Page 130 of 471

impact to cultivation practices relative to nitrogen inputs for MON 87705 compared to 

the nontransformed control or other soybean varieties. 

Table VIII-6. Symbiont Interaction Assessment of MON 87705 and the Control 

Note: Pots were arranged in eight replicated blocks (n = 8) in a greenhouse using a randomized completed 

block design. S.E. = standard error. 

*No significant differences were detected between MON 87705 and the control (p>0.05). 

1

Reference range is the minimum and maximum mean value observed among six commercial reference 

soybean varieties. 

VIII.E. Overall Conclusions for Phenotypic, Agronomic, and Environmental 

Interactions Evaluation 

An extensive and robust set of information and data were used to assess whether the 

introduction of the improved fatty acid profile and glyphosate tolerance tolerance trait 

altered the plant pest potential of MON 87705 compared to the conventional soybean 

control A3525. Phenotypic and agronomic characteristics of MON 87705 were evaluated 

and compared to those of the conventional soybean control. These assessments included 

14 plant growth and development characteristics; five seed dormancy and germination 

parameters under six different temperature regimes; two pollen characteristics; 

observations for abiotic stressor, disease damage, arthropod damage and arthropod 

abundance; plant-symbiont interaction characteristics; and compositional evaluation 

(Section VII) of 67 different components (seven in forage, and 60 in seed). 

Results from the phenotypic and agronomic assessments demonstrate that MON 87705 

does not possess characteristics that would confer a plant pest risk compared to 

conventional soybean. Data on environmental interactions also indicate that MON 87705 

does not confer any biologically meaningful increased susceptibility or tolerance to 

specific disease, insect, or abiotic stressors, or changes in agronomic and phenotypic 

characteristics. Taken together, these data support the conclusion that MON 87705 is not 

likely to pose increased plant pest risk compared to conventional soybean. 

Measurement

Endpoint

Mean (S.E.)* Reference Range1

MON 87705 A3525 p-Value Min Max

Nodule Number

(per plant) 229 (43) 246 (39) 0.6625 140 235

Nodule Dry Wt (g/plant) 0.61 (0.08) 0.64 (0.03) 0.7160 0.52 0.73

Root Dry Wt (g/plant) 1.68 (0.11) 1.52 (0.10) 0.4228 1.54 2.22

Shoot Dry Wt (g/plant) 5.24 (0.59) 5.13 (0.38) 0.8839 5.76 7.71

Shoot Total Nitrogen

(% dwt) 3.26 (0.21) 3.54 (0.12) 0.0832 2.74 3.41

Shoot Total Nitrogen (g) 0.18 (0.02) 0.18 (0.02) 0.7957 0.18 0.23
Monsanto Company 09-SY-201U Page 131 of 471

IX. U.S. AGRONOMIC PRACTICES 

IX.A. Introduction 

As part of the plant pest assessment required by 7 CFR § 340.6(c)(4) impacts to 

agricultural and cultivation practices must be considered. This section provides a 

summary of current agronomic practices in the U.S. for producing soybean and is 

included in this petition as a baseline to assess possible impacts to agricultural practices 

due to the cultivation of MON 87705. Discussions include soybean production, seed 

production, growth and development, general management practices (including identity 

preservation practices), management of weeds, insects and diseases, soybean rotational 

crops, and volunteer soybean management. Information presented in the previous section 

demonstrated that MON 87705 is no more susceptible to diseases or pests than 

conventional soybean. Additionally data presented in Section VIII show that, with the 

exception of an improved fatty acid profile and tolerance to the herbicide glyphosate, 

MON 87705 is phenotypically equivalent to conventional soybean. Thus, there are no 

changes to the inputs needed for MON 87705, and no specific impacts to most of the 

agronomic practices employed for production of soybean. Where there is potential for 

impact on agronomic practices from the deregulation of MON 87705, discussion 

delineating the scope and magnitude of those impacts is provided. For example, 

MON 87705 will be produced under an identity preservation system requiring specific 

management practices to preserve the value of the oil. Therefore, emphasis is placed on 

anticipated impacts to agronomic practices used for production of specialty type soybean 

uponderegulation of MON 87705. Additionally MON 87705 has a glyphosate-tolerance 

trait so potential impacts to crop rotation practices are considered. 

Soybean is planted in over 30 states, demonstrating its wide adaptation to soils and 

climate. The soil, moisture, and temperature requirements for producing soybean are 

generally similar to those for corn, and thus the two crops share a similar cultivation area. 

Proper seedbed preparation, appropriate variety selection, appropriate planting dates and 

plant population, and good integrated pest management practices are important for 

optimizing the yield potential and economic returns of soybean. 

MON 87705 is expected to bring added value for growers, soybean handlers, crushers 

and food processors. An identity preservation system will be used for production, postharvest handling and processing to preserve the enhanced value of MON 87705. As such, 

production practices, post-harvest handling of soybean, and processing will fall under a 

separate production system and distribution channel termed for this specialty soybean. 

MON 87705 is expected to utilize existing agricultural practices employed for production 

and identity preservation of specialty soybean. 

Annual and perennial weeds are perceived to be the greatest pest problem in soybean 

production. Economic thresholds for controlling weeds in soybean require some form of 

weed management practice on all soybean acreage. Approximately 98% of the soybean 

acreage receives a herbicide application. Soybean insects and diseases generally are 
Monsanto Company 09-SY-201U Page 132 of 471

considered less problematic, although infestations can reach economic thresholds 

requiring treatment. 

Volunteer soybean, i.e., soybean plants that have germinated and emerged unintentionally 

in a subsequent crop, are not considered a significant concern in rotational crops 

primarily because of climatic conditions and adequate control from tillage practices. 

Additionally, mechanical and chemical control methods are available to manage the 

occasional volunteer soybean plant. Due to the lack of weediness potential, introduction 

of MON 87705 in the soybean production system would have a negligible impact on 

managing soybean volunteer plants in rotational crops such as corn, cotton, and rice, 

because control measures are available for volunteer soybean when they arise. Preplant 

tillage is the first management tool for control of emerging volunteer soybean in the 

spring. If volunteer soybean plants emerge after planting, shallow cultivation will control 

most of the plants and effectively reduce competition with the crop. Several 

postemergence herbicides also are available to control volunteer soybean (conventional 

or glyphosate-tolerant soybean) in each of the major rotational crops. 

As shown in Sections VII and VIII, with the exception of the improved fatty acid profile 

and glyphosate tolerance trait, no phenotypic, compositional, or environmental 

differences between MON 87705 and conventional soybean have been observed. 

Moreover herbicide-tolerant soybean is currently grown on 92% of soybean acres 

(USDA-NASS, 2008). Therefore, it is not anticipated that commercialization of 

MON 87705 in the U.S. would have a notable impact on current soybean cultivation 

practices, including the management of weeds, diseases, and insects. 

IX.B. Overview of U.S. Soybean Production 

IX.B.1. Soybean Production 

Soybean first entered North America in the 18th century (Hoeft et al., 2000). Sometime 

during the 1930s, soybean started to be processed industrially in the U.S. for edible oil 

and protein meal. Currently, the U.S. produces approximately 32% of the global soybean 

supply (ASA, 2008). In 2007, the U.S. exported 1 billion bushels (27.9 million metric 

tons) of soybean, which accounted for 37 percent of the world's soybean exports (ASA, 

2008). In total, the U.S. exported $12.9 billion USD worth of soybean and soybean 

products globally in 2007 (ASA, 2008). China is the largest export market for U.S. 

soybean with purchases totalling $4.1 billion. Japan is the second largest export market 

with sales of $1.1 billion in the same year. Other significant markets include the 

European Union and Mexico. 

The production of soybean is highly dependent upon soil and climatic conditions. In the 

U.S., the soil and climatic requirements for growing soybean are very similar to corn. 

The soils and climate in the Eastern, Midwestern and portions of the Great Plains regions 

of the U.S. provide sufficient water supplies under normal climatic conditions to produce 

a soybean crop. The general water requirement for a high-yielding soybean crop is 

approximately 20 inches of water during the growing season (Hoeft et al., 2000). Soil 

texture and structure are key components determining water availability in soils, where 

medium-textured soils hold more available water, allowing soybean roots to penetrate 
Monsanto Company 09-SY-201U Page 133 of 471

deeper in medium-textured soils than in clay soils. Irrigation is used on approximately 

9% of the acreage to supplement the water supply during dry periods in the western and 

southern soybean growing regions (ASA, 2008). 

Most of the soybean acreage is grown as a full-season crop. Approximately 8% of the 

soybean acres are planted in a double-crop system following winter wheat south of 35o 

North latitude (Boerma and Specht, 2004)). However, this percentage can vary 

significantly from year to year. The decision to plant double-crop soybean is influenced 

by both agronomic and economic factors. Agronomic factors include harvest date of the 

wheat crop, which determines the double-crop soybean planting date, and available soil 

moisture. Economic factors include expected soybean price and anticipated economic 

return (Boerma and Specht, 2004). 

The vast majority of soybean grown in the U.S. is grown for animal feed and is usually 

fed as soybean meal. However, soybean is also grown as a specialty soybean product for 

a specific market or use. 5 Examples of specialty soybean include high protein, tofu, high 

oil, high oleic, non-biotechnology-derived (also referred to as non-genetically modified), 

and organic soybean. 6 The uses of these soybean varieties include human consumption, 

food processing or specialty products. The soybean varieties used in the specialty market 

are typically specified by buyers and end-users of soybean for production, and a premium 

relative to commodity soybean is paid for delivering a product that meets purity and 

quality standards (Pritchett et al., 2002; Elberhi, 2007; Sundstrom et al., 2002; Lee and 

Herbek, 2004; Muth et al, 2003; and Smyth and Phillips, 2002) for the soybean variety. 

Product differentiation and market segmentation in the specialty soybean industry 

includes mechanisms to keep track of the soybean (traceability), methods for identity 

preservation (IDP), including closed-loop systems, and quality assurance processes (e.g., 

ISO9001-2000 certification), as well as contracts between growers and buyers that 

specify delivery agreements. MON 87705 is an improved fatty acid profile soybean oil 

product and will be considered a specialty soybean product and marketed in a manner 

similar to other high-value specialty crops. 

The U.S. soybean acreage in the past 10 years has varied from approximately 64.7 to 75.7 

million acres, with the lowest acreage recorded in 2007 and the highest in 2008 (Table 

IX-1). Average soybean yields have varied from 33.9 to 43.3 bushels per acre over this 

same time period. Soybean production ranged from 2.45 to 3.19 billion bushels over the 

past ten years, with 2006 being the largest production year on record. According to data 

from USDA-NASS (2009a,b), soybean was planted on approximately 75.7 million acres 

in the U.S. in 2008, producing 2.96 billion bushels of soybean (Table IX-1). Soybean 

acreage and production in 2007 was down significantly from 2006, mainly due to a large 

increase in corn acreage. The value of soybean reached $27.4 billion in the U.S. in 2008 

(USDA-NASS, 2009a,b). In comparison, corn and wheat values in 2008 were $47.37 

and $16.57 billion, respectively (USDA-NASS, 2009a,b). 

 

5

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For purposes of this agronomic practices discussion, soybean production is divided into 

three major soybean growing regions accounting for 99.1% of the 2008 U.S. soybean 

acreage: Midwest/Great Plains region (IL, IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, 

SD, and WI), Southeast region (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern 

Coastal region (DE, MD, NJ, NY, PA, and VA) (Table IX-2). The vast majority of 

soybean was grown in the Midwest region, representing 82.1% of the total U.S. acreage. 

The Southeast and Eastern Coastal regions represented 14.3% and 2.7% of the acreage, 

respectively. Among the three regions, the Midwest region produced the highest average 

yield at 38.6 bushels per acre in 2008, and average state yields in this region ranged from 

28.0 to 47.0 bushels per acre. The average yield in the Southeast region was 34.4 bushels 

per acre, with states within this region averaging from 30.0 to 40.0 bushels per acre. The 

average yield in the Eastern Coastal region was 34.1 bushels per acre, with individual 

state averages ranging from 27.5 to 46.0 bushels per acre. 

Managing input costs is a major component to the economics of producing a soybean 

crop. Key decisions on input costs include choosing what soybean varieties to plant, 

amounts of fertilizer to apply, and what herbicide program to use. The average operating 

cost for producing soybean in the U.S. in 2006 was $93.41 per acre, according to 

statistics compiled by the USDA-Economic Research Service (USDA-ERS, 2006). The 

value of the production less operating cost was reported to be $161.43 per acre. A 

summary of potential production costs and returns are presented in Table IX-3. 

Table IX-1. Soybean Production in the U.S., 1999 – 2008 

Year 

Acres 

Planted 

(×1000) 

Acres 

Harvested

(×1000) 

Average 

Yield 

(bushels/acre)

Total 

Production 

(×1000 bushels) 

Value 

(billions $) 

2008 

2007 

75,718 

64,741 

74,641 

64,146 

39.6 

41.7 

2,959,174 

2,677,117 

27.40 

26.97 

2006 75,522 74,602 42.7 3,188,247 20.42 

2005 72,142 71,361 43.3 3,086,432 16.93 

2004 75,208 73,958 42.2 3,123,686 17.89 

2003 73,404 72,476 33.9 2,453,665 18.01 

2002 73,963 72,497 38.0 2,756,147 15.25 

2001 74,075 72,975 39.6 2,890,682 12.61 

2000 74,266 72,408 38.1 2,757,810 12.47 

1999 73,730 72,446 36.6 2,653,758 12.21 

Source: USDA-NASS, 2009b. 

 
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Table IX-2. U.S. Soybean Production by Region and State in 2008

Region/State

Acres 

Planted1

(thousands)

Acres 

Harvested1

(thousands)

Average Yield1

(bushels/acre)

Total 

Production1

(×1000 bushels)

Value1

(billions $)

Midwest Region

Illinois 9,200 9,100 47.0 427,700 4.00

Indiana 5,450 5,430 45.0 244,350 2.27

Iowa 9,750 9,670 46.0 444,820 4.29

Kansas 3,300 3,250 37.0 120,250 1.03

Kentucky 1,390 1,380 34.0 46,920 0.42

Michigan 1,900 1,890 37.0 69,930 0.64

Minnesota 7,050 6,950 38.0 264,100 2.54

Missouri 5,200 5,030 38.0 191,140 1.72

Nebraska 4,900 4,860 46.5 225,990 2.12

North Dakota 3,800 3,760 28.0 105,280 0.96

Ohio 4,500 4,480 36.0 161,280 1.55

South Dakota 4,100 4,060 34.0 138,040 1.25

Wisconsin 1,610 1,590 35.0 55,650 0.51

Region Totals 62,150 61,450 38.6 2,495,450 23.30

Southeast Region

Alabama 360 350 35.0 12,250 0.12

Arkansas 3,300 3,250 38.0 123,500 1.09

Georgia 430 415 30.0 12,450 0.11

Louisiana 1,050 950 33.0 31,350 0.29

Mississippi 2,000 1,960 40.0 78,400 0.69

North Carolina 1,690 1,670 33.0 55,110 0.47

South Carolina 540 530 32.0 16,960 0.15

Tennessee 1,490 1,460 34.0 49,640 0.43

Region Totals 10,860 10,585 34.4 379,660 3.35

Eastern Coastal Region

Delaware 195 193 27.5 5,308 0.05

Maryland 495 485 30.0 14,550 0.13

New Jersey 92 90 29.0 2,610 0.02

New York 230 226 46.0 10,396 0.09

Pennsylvania 435 430 40.0 17,200 0.15

Virginia 580 570 32.0 18,240 0.16

Region Totals 2027 1994 34.1 68,304 0.60

1 Source: USDA-NASS, 2009b. 
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Table IX-3. U.S. Soybean Production Costs and Returns in 2006 

Production Cost or Return Category Itemized Costs

Return per 

Planted Acre

($ USD)

 

Total Gross Value of Production 254.84

Operating Costs: Seed 32.30

Fertilizer 13.05

Chemicals 14.46

Custom operations 6.01

Fuel, lube and electricity 13.51

Repairs 11.80

Purchased irrigation water 0.11

Interest on operating capital 2.17

Total, operating costs 93.41

 

Allocated overhead: Hired labor 1.78

Opportunity cost of unpaid 

grower’s labor

15.20

Capital recovery of machinery 

and equipment

60.38

Opportunity cost of land (rental 

rate)

86.17

Taxes and insurance 7.93

General farm overhead 13.22

Total, allocated overhead 184.68

 

Total cost listed 278.09

Value of production less total cost 

listed

(23.25)

 

Value of production less operating 

costs

161.43

Supporting Information: Yield = 46 bushels/acre, Price = $5.54/bushel, Enterprise size = 268 

planted acres, Irrigated = 9%, Dry land = 91%. 

Source: USDA-ERS, 2006. 
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IX.B.2. Specialty Soybean 

Commodity and specialty soybean are the two primary production and distribution 

systems for soybean produced in the U.S. The majority of soybean is commonly grown 

and marketed through commodity markets for the oil and protein content. Commodity 

soybean is not consumed directly, but is crushed for meal that is predominantly used for 

animal feed and as a minor protein source for food. The oil produced during the crushing 

of the soybean is used for cooking or food ingredients. The goal of the commodity 

supply chain is to supply a homogenous product to the enduser. The grower producing 

soybean for this chain has a choice from many different varieties for production; 

however, harvested soybean is viewed to be the same for all commodity soybean 

varieties. At harvest, the grower either delivers soybean to a handler or stores them on 

farm for later delivery. The handler is not interested in differentiating the commodity 

soybean for later use. Commodity soybean handlers typically have large volume storage 

capacity. Similarly, commodity soybean processors crush large volumes of soybean to 

produce homogeneous oil and meal products. The commodity system is designed to 

maximize efficiency at a low profit margin resulting in comingling of different sources of 

soybean that does not affect the price received for the final product. This production 

system has been in place in the U.S. since the production of soybean began in earnest in 

the 1960s (Sonka et al., 2004). 

In recent years, there has been an increased demand by consumers and food processers 

for soybean that has specific physical or chemical characteristics that are required by 

certain customers to meet specific food or feed needs. As a result a separate specialty 

soybean channel has developed. This production system and distribution channel is 

focused on value-added traits that involve much smaller volumes than commodity 

soybean (Sonka et al., 2004). Specialty soybean varieties are produced on approximately 

12% of the U.S. soybean acreage7

 (and according to the Midwest Shippers Association 

(MSA, 2009), this acreage could grow to over 25% of the crop acreage in certain states 

within the next decade. This supply chain typically consists of a specialty firm that 

contracts production of a specific variety and sets standards for quality of the harvested 

soybean (Lee and Herbek, 2004). In return, growers receive a premium over the price 

paid for commodity soybean. Growers may store harvested soybean on farm or deliver 

the product directly to a processor or to special containers for international shipment. 

The goal of this identity preserved system is to minimize handling so that value is 

maintained. The cost incurred from an identify preservation system is offset by the 

higher value received for the final product. 

According to the American Soybean Association (2009), specialty soybean can be 

grouped into ten broad categories: non-biotechnology-derived, certified seed, organic 

food-grade, low saturated fat, clear hilum, tofu, natto, high sucrose, high oleic, low 

 

7 http://usb.adayana.com:8080/usb/jsp/login.jsp: Percent U.S. soybean acreage estimate based on 

U.S. Domestic Consumption by Segment – 2008/09. D. Ludwig, personal communication, 2009.
Monsanto Company 09-SY-201U Page 138 of 471

linolenic and high protein. The categories refer to soybean with characteristics such as 

altered seed composition (e.g., low saturated fat, high sucrose, high oleic, low linolenic, 

and high protein), varieties of soybean with unique physical characteristics suited to their 

specific uses (e,g., clear hilum for direct human consumption), or refer to a production 

process (e.g., organic, certified seed). The categories are not meant to be exclusive; for 

example, soybean used to produce natto or tofu may employ organic production 

processes, and soybeans from all of the categories are often derived from varieties 

produced according to certified seed production practices. Tofu and soymilk produced 

from the tofu soybean category represent a large segment of the specialty soybean market 

and are produced from unique soybean varieties that have clear hilum and large seed size 

(Lee and Herbek, 2004). Tofu varieties also must be high in protein (40% or higher) and 

low in oil concentration compared to commodity soybean. Clear hilum and other 

characteristic are required for soybean used in the production of other soybean food 

products consumed directly by humans such as natto, soybean sprouts, edamame 

(vegetable soybean), and soy nuts (Lee and Herbek, 2004; UK, 2009). Organic foodgrade and non biotechnology-derived soybean varieties are identity protected and 

produced not to contain biotechnology-derived traits. Organic soybean have additional 

production restrictions requiring the soybean to be produced using no synthetic fertilizers 

or pesticides. In recent years, public and private soybean breeders have developed 

soybean varieties with improved nutritional characteristics (e.g., high sucrose, high oleic, 

low linolenic and high protein) and MON 87705 is considered part of this trend towards 

production of value-added soybean products. The characteristics or modifications in this 

group include reducing the need for hydrogenation of the soybean oil, increasing sugar 

concentration, increasing protein concentration, decreasing concentration of saturated and 

polyunsaturated fats, and lipoxygenase free soybean which removes some of the flavors 

that are objectionable to some consumers (Lee and Herbek, 2004). 

The majority of specialty soybean varieties are offered in Maturity Group II and early 

Group III varieties which are adapted to the upper Midwest/Great Plains region (Section 

IX.C) (Lee and Herbek, 2004). Maturity Group II and III soybean varieties are grown on 

approximately 42-45 million acres, occupying the largest percentage of soybean acreage 

(see Section IX.C). The varieties were developed for this area primarily due to proximity 

to processing facilities as well as international routes of shipment and to take advantage 

of efficiencies in soybean breeding programs (S. Joehl, personal communication, April 

2009). With a few exceptions, the agronomic or management practices for growing 

specialty soybean from planting to harvest are similar to commodity soybean (Lee and 

Herbek, 2004). Because special varieties are used in the production of each specialty 

soybean, the variety selection is dictated by the specialty soybean buyer or processor. 

Non biotechnology-derived and organic soybean by definition must be produced utilizing 

only conventional soybean varieties. Weed control is extremely important for specialty 

soybean to maintain a high yield potential and because weeds, such as nightshade 

(Solanum nigrum), can stain harvested soybean, which is particularly undesirable in foodgrade soybean (TCM, 2008). Because organic soybean must be grown without synthetic 

fertilizers or pesticides for three or more years prior to the current crop of soybean, 

fertilization and pest management is much more difficult (Lee and Herbek, 2004). Weed 

control in organic soybean relies on a combination of crop rotation, tillage, in-crop 
Monsanto Company 09-SY-201U Page 139 of 471

cultivation, and hand-weeding. Insect and disease control is managed primarily through 

crop rotation. Certain approved pesticides are permitted in this specialty soybean 

production. 

All equipment and storage facilities for specialty soybean must be clean of seed from 

other soybean varieties or plants, dirt, pathogens and other foreign material. Some 

soybean contracts may require a special inspection of the handling and storage facilities. 

The specialty soybean for soybean foods may require special harvesting equipment since 

some of these soybeans are harvested before full maturity (e.g., edemame or vegetable 

soybean). 

IX.B.3. Identity Preservation 

Identity Preservation (IDP) refers to a system of production, handling, and marketing 

practices that maintains the integrity and purity of agricultural commodities (Sundstrom 

et al., 2002). Commodity grains, on the other hand, are marketed in mass according to 

USDA grading standards. Specialty crops require some form of segregation or full-scale 

identity preservation to keep these grains separate from commodity grains (Elberhi, 

2007). This market segmentation within the grain channeling system is driven by the 

need to preserve market value or ensure a specified purity of the product. With certain 

specialty crops, IDP is required to prevent accidental or unintended commingling (e.g., 

non biotechnology, organic) or to segregate products that are approved only for certain 

uses (industrial use only). 

IDP grain production has been in existence for a long time. Agricultural producers have 

over many decades developed practices that allowed for differentiation between food vs. 

feed grain, or grain vs. seed production, or organic vs. non organic (Massey, 2002). Seed 

certification programs such as those used by the Association of Official Seed Certifying 

Agencies are often cited as the model of IDP systems. These programs date back to the 

1920s and 1930s when the certification process was implemented to verify the genetic 

purity of seed made available to growers (see Section IX.B.4). Standards were 

established to ensure production of seed from known pedigree with high purity and 

quality. Similarly, commodity grain traders, marketing organizations, and food 

processors have established purity and quality tolerances for specific end-product uses. 

The need for segregation and IDP production systems has increased with the 

development of specialty crops or crops with special output traits, such as high oil corn, 

high oleic sunflower, and low-linolenic soybean (Sundstrom et al., 2002). 

The production of IDP grains requires special processes in order for growers to meet 

buyers’ criteria for variety identification, composition, and quality. Buyers of IDP 

soybean typically contract for the production of seed-variety specific soybean and work 

directly with seed suppliers, growers, independent certification agencies, intermediate 

processors, and freight companies to deliver the preferred product within specified 

tolerances. 8 Contract specifications are written to ensure the delivery of the desired 

 

8

 United Soybean Board, International Buyer’s Guide at 

http://www.ussoyexports.org/ussoy/buyersguide/Chap6.pdf
Monsanto Company 09-SY-201U Page 140 of 471

attribute-specific product and that predetermined management practices are used 

(Massey, 2002). Lack of compliance with a product specification can lead either to a 

price discount or rejection of a shipment by the buyer. Depending on the end use, 

contracts can be extensive, defining many of the production, harvest and storage 

activities, or less stringent dealing more with pricing, quality specifications and only the 

most critical production practices. Premiums are paid to growers for the additional risk 

and management only when the grain meets the contract specifications. Premium prices 

provide the impetus to maintain the specialty grain’s purity and identity separate from 

commodity grains (Massey, 2002). Premiums are affected by various factors, including 

the proximity of suppliers to buyers and the cost and availability of substitutes 

(Sundstrom et al., 2002). For many trait-specific crops, price premiums rise or fall 

depending on the supply conditions for the generic commodity. 

Growers of IDP grains must have good managerial ability and implement certain 

management practices to produce and deliver grain possessing the desired physical and 

chemical characteristics. The production and marketing of trait-specific grains involves 

additional financial risks (Elberhi, 2007). The growers’ managerial ability can affect 

both yield performance and proficiency in meeting the contract specifications. From a 

soybean buyer’s perspective, contracts help the buyer meet the demand for specific 

product qualities, improve cost efficiencies of product processing, and reduce transaction 

costs. 

While specific IDP production practices vary depending on the characteristics of the 

product to be delivered, general elements are implemented to ensure that the end-user or 

processor receives the grain or end-product with the intended identity and desired quality. 

As mentioned above, many IDP systems were developed using the principles similar to 

those used in seed certification. IDP production begins with a system of standards, 

records, and auditing that are put in place throughout the entire crop production, 

harvesting, handling, and marketing process (Sundstrom et al., 2002). Some key 

considerations in the establishment of an IDP system include: 1) planted seed identity and 

tolerances, 2) appropriate field isolation, 3) inspection and clean-out of equipment and 

facilities, 4) end-product sampling and testing, and 5) record maintenance and identity 

labeling (Sundstrom et al., 2002). Each of these components are described in greater 

detailed below: 

Planting Seed Identity and Tolerances

The purity and identity of starting seed should be tested and confirmed. The purity of the 

seed stock should equal or exceed the purity standards of the desired final product. 

Single or multiple quality tolerances may be established in specialized IDP programs 

based on market-driven standards. 

Field Isolation

Crops must be isolated either spatially or temporally from pollen sources that could 

impact the quality or purity of the harvested seed. The amount of isolation depends on 

flower characteristics, sexual compatibility with neighboring crops, pollen quantity and 

viability, and mode of pollen dissemination. A self-pollinating crop, such as soybean, 

requires relatively small isolation distances to effectively preclude cross-pollination. 
Monsanto Company 09-SY-201U Page 141 of 471

Equipment and Facilities

Equipment used in production should be cleaned and inspected before and after use for 

IDP crop. Storage facilities and transporting vehicles are cleaned and inspected to assure 

that segregation is maintained and no physical contamination occurs. 

Sampling and Testing

In some cases, the IDP grain is sampled and tested at various stages to confirm the 

product identification, purity, and quality. Special consideration should be given to 

sampling and testing techniques that ensure reliable results.

Record Maintenance and Identity Labeling

Records typically are maintained on field designations, harvest amounts, storage bin 

locations, and product transfers. IDP products must be identified, segregated, and labeled 

in the market chain. 

Because value capture is also a vital part of IDP productions systems, growers must 

assure markets or buyers for these IDP crops are available, especially if the crop is not 

being grown under contract. The soybean industry has collaborated to foster the 

development and availability of several specialty soybean and soybean oil markets.9

 As 

an example, the soybean industry initiated a program called the Low-Linolenic Locater, 

an internet-based program to assist growers in locating the closest processor or elevator 

that contracts acreage and offers premiums for low-linolenic soybean. 

As with current IDP systems used for specialty soybean to preserve market value or 

ensure purity of the product, MON 87705 will employ an IDP system based on 

established practices. The IDP practices will be implemented to ensure that the end-user 

or processor receives the soybean with the identity, fatty acid composition of the oil, and 

desired quality for this value-added specialty soybean. 

IX.B.4. Soybean Seed Production 

Standardized seed production practices are responsible for maintaining high-quality seed 

stocks, an essential basis for U.S. agriculture. By the early 20th century, agronomists 

learned how to develop specific plant varieties with desirable traits. In the U.S., state 

agricultural experiment stations developed many seed varieties that were distributed to 

growers for use. Seed was saved by growers and later sold to neighbors; however, the 

desirable traits of the varieties often were lost through random genetic changes and 

contamination with other crop and weed seed (Sundstrom et al., 2002). The value of seed 

quality (including genetic purity, vigor, and presence of weed seed, seed-borne diseases, 

and inert materials, such as dirt) was quickly identified as a major factor in crop yields. 

States developed seed laws and certification agencies to ensure that purchasers who 

received certified seed could be assured that the seed met established seed quality 

standards (Bradford, 2006). The federal government passed the U.S. Federal Seed Act of 

1939 to recognize seed certification and official certifying agencies. Regulations first 

 

9

 Qualisoy: Low-linoleic locator at http://www.qualisoy.com\ 
Monsanto Company 09-SY-201U Page 142 of 471

adopted in 1969 under the Federal Seed Act recognize land history, field isolation, and 

varietal purity standards for foundation, registered, and certified seed. Under 

international agreements such as the Organization for Economic Co-Operation and 

Development (OECD) scheme, the U.S. and other countries mutually recognize 

minimum seed quality standards (Bradford, 2006). The Association of Official Seed 

Certifying Agencies represents state and private seed certification in the U.S., and 

includes international member countries in North and South America, Australia, and New 

Zealand. 

Soybean seed is separated into four seed classes: 1) breeder, 2) foundation, 3) registered, 

and 4) certified (AOSCA, 2009a). Breeder seed is seed directly controlled by the 

originating or sponsoring plant breeding organization or firm. Foundation seed is firstgeneration seed increased from breeder seed and is handled to maintain specific varietal 

purity and identity. Registered seed is the progeny of foundation seed that is handled to 

maintain satisfactory variety purity and identity. Certified seed is the progeny of breeder, 

foundation or registered seed, and is two generations from foundation seed. All soybean 

seed sold may not be officially certified; however, commercial soybean seed sold and 

planted for normal soybean production is produced predominately to meet or exceed 

certified seed standards. This section of the petition will provide a broad overview of the 

practices used in producing certified seed. 

Soybean seed breeders and producers have put in place practical measures to assure the 

quality and genetic purity of soybean seed varieties for commercial planting. The need 

for such systems arose from the recognition that the quality of improved soybean 

varieties quickly deteriorated in the absence of monitoring for quality and genetic purity 

(CAST, 2007). Seed certification programs were initiated in the early 1900s in the U.S. 

to preserve the genetic identity and variety purity of seed. There are special land 

requirements, seed stock eligibility requirements, field inspections and seed labeling 

standards for seed certification. Seed certification services are available through various 

state agencies affiliated with the Association of Official Seed Certifying Agencies. Large 

seed producers implement their own seed quality assurance programs. However, large 

seed producers often will utilize the services of state certifying agencies as a third party 

source to perform certain field inspections and audits. 

The U.S. soybean production for all purposes has varied from approximately 64.7 to 75.7 

million acres in the past ten years, with the lowest acreage recorded in 2007 and the 

highest in 2008 (USDA-NASS, 2009 b; Table IX-1). This range of soybean acreage 

would require between 105 - 125 million units (50 lbs/unit) of soybean seed. This seed 

volume includes allowances for seed losses due to weather, poor yields, and quality 

issues. Additional allowances are included for distribution excess, seed returns, replants, 

and potential increases in soybean acreage. Assuming an average soybean yield of 45 

bushels, or 54 units (50 lbs/unit) per acre, 1.9 to 2.3 million acres would be required to 

produce this volume of commercial certified soybean seed each year. 

Soybean seed is produced throughout most of the U.S. soybean-growing regions. 

Soybean varieties are developed and adapted to certain geographical zones and are 

separated into ten maturity groups – Group 00 to Group VIII (see Section IX.C.). Seed 

production for these maturity groups is grown in the respective geographical zone for 
Monsanto Company 09-SY-201U Page 143 of 471

each maturity group. However, the production areas generally are on the northern edge 

of the respective zone to minimize incidences of disease. 

Soybean seed is produced by companies that produce and sell seed, such as Monsanto 

Company, Pioneer Hi-Bred International, Syngenta Seeds, Kruger Seed Co., Becks 

Hybrids, and tollers, which are companies that produce but do not sell certified seed, such 

as Remington Seeds LLC and Precision Soya. Seed companies and tollers in turn 

contract acreage with growers to produce the required amount of soybean seed. 

Production or processing plants at these seed companies identify top soybean growers to 

produce the seed and also monitor and inspect seed fields throughout the growing season. 

The production plants also clean, condition, and bag the harvested soybean seed as well 

as monitor and inspect all the processes at the plant. Production plants typically produce 

between 100,000 units to 2,000,000 units of soybean seed. Production plants will 

produce the various soybean varieties in different climates or environments to spread 

production risks. 

The entire seed production process at the majority of the seed companies and tollers is 

International Organization for Standardization (ISO) certified and; therefore, includes 

internal and external audits (ISO, 2009). ISO standards ensure desirable characteristics 

of seeds and services, such as quality, safety, reliability, and efficiency. The ISO 

standards represent an international consensus on good management practices with the 

aim of ensuring that the organization can consistently deliver excellent product or 

services. The standards not only must meet the customer’s requirements and applicable 

seed regulatory requirements, but must aim to enhance customer satisfaction and achieve 

continual improvement of its performance in pursuit of these objectives. 

The field operations and management practices for producing soybean seed are similar to 

normal soybean production. However, special attention is needed in certain areas to 

produce seed with high quality, high germination rates, and high genetic purity (Helsel 

and Minor, 1993). General guidelines specific for seed production are discussed below. 

The seed production field should not have been planted with soybean the previous crop in 

order to avoid volunteer soybean plants (even though the risk of soybean volunteer plants 

is negligible) and to ensure genetic purity. 

Very early planting should be avoided because the seed produced from early planting 

often results in poorer quality seed (Helsel and Minor, 1993). Every effort must be made 

to eliminate weeds in a seed field through the use of herbicides and cultivation to prevent 

weed seed in the harvested soybean seed. Fields are scouted frequently for insect pests 

and insecticides are applied when insect pest infestations reach economical threshold 

levels. Foliar-applied fungicides should be considered when disease infestations are 

predicted in the area. Harvest should occur as soon as the mature soybean seed reaches 

13% moisture content. Harvesting soybean seed with less than 13% moisture can cause 

damage to the seed coat and result in split soybean seed that can affect germination and 

viability. Harvesting equipment must be adjusted to minimize or avoid seed damage. 

Harvesting equipment must be cleaned before entering the seed fields to minimize 

genetic contamination. Certain handling equipment, such as auger elevators, should be 

avoided because they can increase seed damage. 
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Field inspections are vital to ensure the soybean seed meets seed certification 

requirements, ISO certification standards, regulatory standards, and trait licensing 

agreement standards. Field inspections are conducted on seed production fields 

throughout the soybean growing season to evaluate variety purity, ensure soybean plants 

are developing properly, and fields are maintained free of weeds, insects, and diseases. 

The fields are also mapped to ensure the seed field has the minimum federal isolation 

requirement of five feet (AOSCA, 2009a). Some states and seed producers have a stricter 

isolation requirement of 10 feet. 

Production plant personnel make every effort to avoid mechanical damage to the 

harvested seed during the screening, cleaning, and bagging process. Specific methods are 

used to assure the genetic purity and identity of the seed is maintained throughout the 

handling and storage operation. Bin inspections and sample collections are conducted at 

storage locations at the plant to examine the physical characteristics of the soybean seed 

and to ensure proper bin cleanout. Seed is inspected for appearance, disease, 

discoloration, seed coat, mechanical damage, inert matter, and weed seed. Warm and 

cold germination tests are conducted on all seed lots to verify acceptable germination 

rates. Many seed companies will also conduct tetrazolium staining tests to assess seed 

viability. 

Commercially certified soybean seed must meet state and federal seed standards and 

labeling requirements. AOSCA standards for certified soybean seed are as follows: 98% 

pure seed (minimum), 2% inert matter (maximum), 0.05% weed seed (maximum, not to 

exceed 10 per lb.), 0.60% total of other crop seeds (maximum), 0.5% other varieties 

(maximum, includes off-colored beans and off-type seeds), 0.10% other crop seeds 

(maximum, not to exceed three per lb.), and 80% germination and hard seed (minimum) 

(AOSCA, 2009a). State seed certification standards vary slightly from state to state and 

can be more restrictive than the seed standards of AOSCA. 

MON 87705 seed will be produced in the same manner as commercially certified 

soybean seed, such that it will meet all state and federal seed standards and labeling 

requirements. 

IX.C. Production Management Considerations 

Pre-Season 

Crop rotation, tillage system, row spacing, planting equipment, seed or variety 

selection(s), and soil fertility require production decisions well in advance of planting the 

soybean crop. Many of the decisions in this area are made prior to or immediately after 

harvest of the previous crop. There are many benefits to crop rotation, with the majority 

of the soybean acreage planted in a two-year corn-soybean rotation (see Section IX.G.). 

Crop rotation is generally a long-term decision, but the rotation sequence can be modified 

to take advantage of a particular economic or market opportunity. The decision to plant 

soybean in a conservation tillage or no-till system may require special equipment and will 

be made long before planting. In addition, this decision usually will be a long-term 

commitment, provided the system is successful. A decision to change row spacing is a 

similar long-term commitment that generally requires new equipment. 
Monsanto Company 09-SY-201U Page 145 of 471

The benefits of conservation tillage or no-till systems are well documented and include 

reduced soil erosion, reduced fuel and labor costs, and conservation of soil moisture. In 

2004, approximately 29.3 million acres (38.6%) of soybean were planted in a no-till 

system (CTIC, 2004). Slow soybean emergence and growth plus lower yields have been 

some of the concerns associated with adoption of conservation tillage systems in 

soybean, especially no-till. Research in Wisconsin and Minnesota shows that soil 

temperatures can be four to five degrees colder in no-till than conventional tillage 

systems, which can slow emergence, but have little effect on soybean yield (Pedersen, 

2008a). Improved planters for establishment of good soybean populations and planting 

Roundup Ready soybean to effectively control weeds in no-till fields have made no-till a 

viable production system for soybean. Researchers still recommend some spring tillage 

on fine-textured and poorly drained soils for proper seedbed preparation (Pedersen, 

2008a). 

Most field crops, including soybean, respond well to fertilizer when planted in soils with 

low fertility levels. Soybean requires 16 essential elements for growth and development. 

Deficiencies in any of these elements can reduce yields (Hoeft et al., 2000). The primary 

or major essential nutrients are nitrogen, phosphorus and potassium. The soybean plant 

is a member of the legume family, like alfalfa and clover, and fixes a significant portion 

of its own nitrogen through the symbiotic relationship with the nitrogen-fixing 

Bradyrhizobia bacteria (Bradyrhizobium japonicum) that live in the nodules on its roots. 

Bradyrhizobia are unicellar, microscopic bacteria that invade the soybean plant through 

its root hairs (Hoeft et al., 2000). The plant responds to this invasion by forming nodules 

which contain colonies of bacteria. Once established on the soybean root, bacteria in the 

nodule take gaseous nitrogen from the atmosphere and fix it in forms easily used by the 

soybean plant. Since the bacteria are not native to U.S. soils and would not normally be 

found in these soils, inoculation of the soybean seed is recommended when soybean has 

not been grown in a field for three to five years. Nitrogen fertilizer applications at 

planting generally do not improve yield and decreases nodulation while increasing the 

plant’s dependency on the soil for nitrogen (Pedersen, 2008a). Therefore, nitrogen 

fertilizer is seldom applied prior to planting a soybean crop. 

Soil tests are the only reliable way to determine the pH, phosphorus, and potassium levels 

in the soil. Liming and fertilizer requirements subsequently are determined based on soil 

test results. Ideal soil test results for corn are also ideal for soybean (Scott and Aldrich, 

1970). In corn-soybean rotations in the Midwest, phosphorus and potassium fertilizers 

are applied prior to a corn crop in accordance with soil test recommendations, but are 

seldom applied prior to a soybean crop. However, in some of the southern growing areas, 

differences in crop rotations and soil types may require a fertilizer application prior to 

planting soybean. 

Although not common, deficiencies can occur in secondary nutrients (calcium, 

magnesium, and sulfur) or micronutrients (boron, chloride, copper, iron, manganese, 

molybdenum, and zinc). The availability of soil nutrients is dependent on soil acidity or 

pH level. Because soybean is adversely affected when the pH is below approximately 5.8 

(Hoeft et al., 2000), soil pH should be maintained at about 6.0 to 6.5 through the addition 

of limestone. 
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Soybean varieties are developed and adapted to certain geographical zones and are 

separated into ten maturity groups – Group 00 to Group VIII (Pimentel, 1991; Zhang et 

al., 2004). Groups 00 and 0 are the earliest maturity groups and are adapted best to the 

area north of latitude 46o north. Succeeding groups are adapted further south with 

Groups I and II within latitudes 41o and 46o north, and Group III within latitudes 38o and 

41o North. Group 00 through Group IV soybean varieties are planted in the Midwest and 

Eastern Coastal regions. Groups II, III and IV account for approximately 75% (24%, 

36%, and 16%, respectively) of the soybean planted in the U.S. (T. Schlueter, personal 

communication, August 2008). Groups IV through VIII are planted in the southern states 

with Groups V, VI and VII representing 7%, 2%, and 2% of the planted soybean, 

respectively (T. Schlueter, personal communication, August 2008). 

Soybean variety selection is crucial for high yield and quality, and is the foundation of an 

effective management plan (Pedersen, 2008a). Soybean characteristics to consider in 

selecting a variety include maturity, yield potential, disease and pest resistance, iron 

deficiency tolerance (chlorosis), lodging score, height, and specific soybean quality traits, 

such as protein and oil content. If a field has a history of a particular disease or pest, 

planting soybean varieties that have resistance or tolerance to these pests and diseases can 

be an effective and economical method of control. 

Row spacing is important to maximize soybean yield. Research in the Midwest over the 

past 20 years consistently shows that row spacing of less than 20 inches is preferred for 

soybean regardless of tillage system, rotation sequence or planting date (Pedersen, 

2008a). In the southern states, the advantage from narrow rows is less consistent and less 

beneficial. In 2000, approximately 40% of soybean was planted in row spacing of 10 

inches or less, 27% in 10.1 to 28.5 inches, and 33% in rows wider than 28.5 (Hoeft et al., 

2000). 

Planting and Early Season 

An understanding of the growth stages of soybean is also important for the proper timing 

of certain management practices, such as herbicide and insecticide applications. In 

addition, the impact of certain weather conditions, insect pests, and diseases on soybean 

yield is dependent on growth stage. The system of soybean growth stages divides plant 

development into vegetative (V) and reproductive (R) stages (Pedersen, 2008a). The 

vegetative stages begin with VE, which designates emergence. V stages continue and are 

numbered according to how many fully developed trifoliate leaves are present (i.e., V1, 

V2, etc.). The reproductive (R) stages begin at flowering (R1) and include pod 

development and plant maturation. Full maturity is designated as R8. 

Adequate soil moisture and warm temperatures facilitate rapid seed germination and 

emergence. The ideal soil temperature for soybean germination and emergence is 77 oF 

(Pedersen, 2008a). However, waiting for soils to reach this soil temperature will delay 

planting beyond the optimum planting date that will maximize yield. Soybean can 

germinate at a soil temperature of 50 oF when planted at a depth of two inches. However, 

emergence is slow and can take up to three weeks in northern climates. Because of 

fluctuations in soil temperature in early spring, soil temperature should not be the only 

criteria for optimum planting time. Planting into a good seedbed is the most important 
Monsanto Company 09-SY-201U Page 147 of 471

consideration. Planting into soil that is too wet will reduce emergence and plant 

population, and can lead to reduced yield. 

Planting date has the greatest impact on yield, according to research conducted in the 

Northern states (Hoeft et al., 2000). Highest yields are generally obtained when planting 

is in early to mid May. Yields begin to drop off quite rapidly when planting is delayed 

until late May. For example, the optimum planting dates for soybean in Iowa are the last 

week of April in the southern two-thirds of the state and the first week of May in the 

northern one-third of the state (Pedersen, 2008a). In the Southern U.S., planting adapted 

varieties before late April results in shorter plants and, in many cases, lower yields than 

when the same varieties are planted in May or early June. Planting after early June 

generally decreases plant height and yield due to water shortages in July and August. 

Variations in plant spacing through row spacing and plant population have a significant 

effect on canopy development and soybean yield. Soybean has the ability to produce 

good yield over a wide range of plant populations. Most soybean varieties have the 

ability to branch and adjust the number of pods on branches to compensate for large 

differences in seeding rate. Maximum yields generally require planting rates that result 

in about 2.5 to 5 plants per square foot (Hoeft et al., 2000). Therefore, a full stand of 

soybean is approximately eight to ten plants per foot of row at harvest for 40-inch rows, 

six to eight plants per foot of row in 30-inch rows, four to six plants in 20-inch rows, and 

two to three plants in 10-inch rows. This translates to 109,000 to 218,000 plants per acre 

at harvest. Higher populations are recommended in narrow rows for maximum yields 

because plants are more uniformly spaced in narrow rows. Seeding rates are generally 10 

to 25% higher than the desired harvest population, especially in no-till, to account for the 

losses in germination, emergence, and seedling diseases. The accuracy of the planting 

equipment also can impact the decision on seeding rate. Soybean seed traditionally has 

been sold by weight. Therefore, the farmer must know the number of seeds per pound for 

the particular soybean varieties being planted for accurate seeding rates. 

Treating soybean seed with a fungicide (e.g., pyraclostrobin, metalaxyl, or mefenoxam) 

to prevent damping-off diseases may be beneficial when planting in cold, wet soils, using 

reduced till and no-till planting systems, and when planting seed with a low germination 

rate (<80%) or low seed vigor. 

Annual and perennial weeds are considered to be the greatest pest problem in soybean 

production (Aref and Pike, 1998). In order to maximize yields, weeds must be controlled 

during the early growth stages of soybean because weeds compete with soybean for 

water, nutrients, and light. A combination of tillage and herbicides are used to control 

weeds throughout the growing season. 

Mid to Late Season 

Ideal daytime temperatures for soybean growth are between 75 oF and 85 oF (Hoeft et al., 

2000). Warmer temperatures result in larger plants and earlier flowering. Sustained 

temperatures below 75 oF will delay the beginning of flowering significantly. Seed set 

also is affected by temperature. Seed set is generally good when pollination follows 

night temperatures around 70 oF. Soybean varieties differ in their response and tolerance 

to temperatures. 
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Soybean is photoperiod sensitive, which means that it transitions from vegetative to 

flowering stage in direct response to length of daylight (Scott and Aldrich, 1970). Most 

soybean varieties begin flowering soon after the day length begins to shorten. Flowering 

of southern varieties is initiated by a shorter day than that of varieties adapted to the 

north. The extent of vegetative growth occurring after the initiation of flowering depends 

not only on environmental factors but also the growth habit. Soybean varieties are 

described as either indeterminate or determinate in their growth habit (Scott and Aldrich, 

1970). Indeterminate varieties increase their height by two to four times after flowering 

begins. These are grown in the northern and central U.S. Determinate varieties increase 

their height very little after flowering and generally are grown in the southern U.S. 

Indeterminate and determinate varieties also differ in flowering characteristics. 

Indeterminate plants generally bloom first at the fourth or fifth node and progress 

upward. Flowering on determinate plants begins at the eight or tenth node and progresses 

both downward and upward. 

The first appearance of flowers signals the beginning of the reproductive stage, namely 

the R1 stage (Hoeft et al., 2000). The reproductive period consists of flowering, pod set, 

and seed formation. Climatic conditions such as temperature and moisture supply during 

the flowering period will affect the number of flowers. The soybean plant does not form 

a pod for each flower. It is common for the soybean plant to have 75% of the flowers fail 

to develop a pod (Scott and Aldrich, 1970). This characteristic makes soybean less 

susceptible than corn to short periods of adverse weather during flowering. Under 

normal conditions, pod set occurs over about a three week period. Good soil moisture is 

most critical during the pod-filling stages to prevent pod abortion and to ensure high 

yields (Hoeft et al., 2000). Another critical period is during the seed-filling stages to 

assure high rates of photosynthesis. High humidity and temperatures during seed 

development and maturity can result in poor seed quality because these conditions 

promote the development of reproductive-stage diseases. 

Harvest Season 

When dry matter accumulation ends, the plant is considered to be physiologically mature. 

The seed moisture content is approximately 55 to 60% at this stage (Hoeft et al., 2000). 

At this stage, namely R7, at least one normal pod on the plant reaches the mature pod 

color. Under warm and dry weather conditions, seed moisture content will drop to 13 to 

14% in 10 to 14 days from physiological maturity (Hoeft et al., 2000). Soybean can be 

harvested when the moisture content drops below 15%. However, soybean should be at 

13% moisture to be stored without artificial drying (Scott and Aldrich, 1970). Moisture 

content below 12% may increase seed cracking and seed coat damage. 

Pre-harvest losses are influenced by variety, weather, and timeliness of harvest (Scott and 

Aldrich, 1970). Timely harvest when the moisture content is 13 to 14% also will 

minimize losses. Proper operation and adjustment of the combine is essential to 

minimizing harvest losses in the field. 
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IX.D. Weed Management 

Annual weeds are perceived to be the greatest pest problem in soybean production, 

followed by perennial weeds (Aref and Pike, 1998). Soybean insects and diseases are 

rated less problematic but may reach economic thresholds requiring treatment. Weed 

control in soybean is essential to optimizing yields. Weeds compete with soybean for 

light, nutrients, and soil moisture. Weeds can harbor insects and diseases, and also can 

interfere with harvest, causing extra wear on harvest equipment (Pedersen, 2008a). The 

primary factors affecting soybean yield loss from weed competition are the weed species, 

weed density, and the duration of the competition. When weeds are left to compete with 

soybean for the entire growing season, yield losses can exceed 75% (Dalley et al., 2001). 

Generally, the competition increases with increasing weed density. The time period that 

weeds compete with the soybean crop influences the level of yield loss. In general, the 

later the weeds emerge, the less impact the weeds will have on yield. Soybean plants 

withstand early-season weed competition longer than corn, and the canopy closes earlier 

in soybean than corn. In addition, canopy closure is much sooner when soybean is drilled 

or planted in narrow rows. The most common weeds in soybean for each of the three 

major growing regions are presented in Tables IX-4, IX-5 and IX-6. 

Crop rotations and environment have a significant impact on the adaptation and 

occurrence of weeds in soybean. Foxtail spp. (foxtail species group), pigweed, 

velvetleaf, lambs quarters, and cocklebur are common weeds in Midwest corn and 

soybean fields. However, growers consider giant ragweed (Ambrosia artemisiifolia), 

lambs quarters (Chenopodium album), Canada thistle (Cirsium arvense), cocklebur 

(Xanthium strumarium), and velvetleaf (Abutilon theophrasti) to be the top five most 

problematic weeds in corn and soybean because of the difficulty to control these weeds 

(Nice and Johnson, 2005). The most frequently reported common weeds in the Southeast 

region are morning glory (Ipomoea spp.), prickly sida (Sida spinosa), johnsongrass 

(Sorghum halepense), sicklepod (Cassia obtusifolia), and broadleaf signalgrass 

(Brachiaria platyphylla) (Webster et al., 2005). 
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Table IX-4. Common Weeds in Soybean Production: Midwest Region 

Foxtail spp. (12) 1 Ragweed, giant (3) Dandelion (1) 

Pigweed spp. (11) Shattercane (3) Johnson grass (1) 

Velvetleaf (11) Quackgrass (3) Milkweed, honeyvine (1) 

Lambsquarters (10) Buckwheat, wild (2) Nightshade, hairy (1) 

Cocklebur (9) Crabgrass spp. (2) Oats, wild (1) 

Ragweed, common (7) Kochia (2) Pokeweed, common (1) 

Smartweed spp. (6) Mustard, wild (2) Prickly sida (1) 

Morning glory spp. (5) Nightshade, Eastern black (2) Proso millet, wild (1) 

Sunflower, spp. (5) Palmer amaranth (2) Sandbur, field (1) 

Waterhemp spp. (5) Canada thistle (1) Venice mallow (1) 

Horseweed (marestail) (3) Chickweed (1) Volunteer cereal (1) 

Panicum, fall (3) Cupgrass, woolly (1) Volunteer corn (1) 

1 Number provided in parenthesis is the number of states out of the thirteen total states in the 

Midwest region reporting each weed as a common weed. 

Sources: 

IL: University of Illinois (2002) and Aaron Hager, Extension Weed Specialist, University of 

Illinois - Personal Communication (2006). 

IN: 2003-2005 Statewide Purdue Horseweed Weed Survey, Special database query and personal 

communication (2006), Bill Johnson, Extension Weed Specialist, Purdue University. 

IA, MN, OH, WI: WSSA, 1992. 

KS: Dallas Perterson, Extension Weed Specialist, Kansas State - Personal communication (2006). 

KY, MO: Webster et al., 2005. 

MI: Davis, et al., 2005. List is not ranked in order of importance or frequency.

NE: Alex Martin, Extension Weed Specialist, University of Nebraska – Personal communication 

(2006).

ND: Zollinger, 2000. 

SD: Michael Moechnig, Extension Weed Specialist, South Dakota State University – 

Personal communication (2006). 

Table IX-5. Common Weeds in Soybean Production: Mid-South Region 

Morning glory spp. (5) 1 Pigweed spp. (3) Ragweed, common (1) 

Prickly sida (5) Crabgrass spp. (2) Ragweed, giant (1) 

Johnson grass (4) Palmer amaranth (2) Red rice (1) 

Sicklepod (4) Cocklebur (1) Smartweed (1) 

Signalgrass, broadleaf (4) Copperleaf, hophorn (1) Spurge, nodding/hyssop (1) 

Barnyard grass (3) Florida pusely (1) Spurge, Prostrate (1) 

Hemp sesbania (3) Horseweed (marestail) (1) 

Nutsedge spp. (3) Poinsettia, wild (1) 

1 Number provided in parenthesis is the number of states out of the five total states in the MidSouth region reporting each weed as a common weed. 

Sources: 

AL, LA, MS, TN: Webster et al., 2005. 

AR: Ken Smith, Extension Weed Specialist, University of Arkansas - Personal communication 

(2006). 
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Table IX-6. Common Weeds in Soybean Production: Eastern Coastal Region 

Ragweed, common (8)1 Jimson weed (4) Dandelion (1) 

Cocklebur (7) Sicklepod (3) Goosegrass (1) 

Morning glory spp. (7) Florida pusely (2) Nightshade, Eastern black (1) 

Crabgrass spp. (6) Johnson grass (2) Panicum, Texas (1) 

Foxtail spp. (6) Palmer amaranth (2) Prickly sida (1) 

Lambsquarters (6) Quackgrass (2) Shattercane (1) 

Pigweed spp. (6) Arrowleaf sida (1) Signalgrass, broadleaf (1) 

Velvetleaf (6) Beggarweed, Florida (1) Smartweed spp. (1) 

Nutsedge spp. (5) Burcucumber (1) 

Panicum, fall (5) Canada thistle (1) 

1 Number provided in parenthesis is the number of states out of the eight total states in the 

Eastern Coastal region reporting each weed as a common weed. Data were not available for 

DE in soybean. 

Sources: 

DE, MD, NJ, PA: WSSA, 1992. 

GA, NC, SC: Webster et al., 2005. 

NY: Russell Hahn, Extension Weed Specialist, Cornell University – Personal Communication 

(2006). 

VA: Scott Hagood, Extension Weed Specialist, Virginia Tech – Personal Communication 

(2006). 

Cultural and mechanical weed control practices are important components of an effective 

weed management program (Baumann et al., 2008). Crop rotation, narrow row spacing 

and planting date are a few of the crop management practices that are implemented to 

provide the crop with a competitive edge over weeds. Although the primary purpose of 

tillage is for seedbed preparation, tillage is still used to supplement weed control with 

selective herbicides in soybean production. Approximately 98% of the soybean acreage 

received an herbicide application in 2006 indicating the importance of excellent weed 

control in maximizing soybean yield (USDA-ERS, 2007). Herbicide-tolerant soybean 

were introduced to provide growers with additional options to improve crop safety and/or 

improve weed control. The Roundup Ready soybean system, i.e., planting Roundup 

Ready soybean and applying glyphosate in crop, has become the standard weed control 

program in U.S. soybean production and is planted on 92% of the soybean acreage 

(USDA-NASS, 2008). 

Herbicides provide effective and economical control of weeds in soybean. The risk of 

weeds developing resistance to herbicides and the potential impact of resistance on the 

usefulness of a herbicide vary greatly across different mechanisms of action and are 

dependent on a combination of factors, such as selection pressure, herbicide soil residual 

activity, herbicide chemistry, prolific seed production and high genetic variation in 

plants. Weed-resistance management programs that integrate the use of herbicides with 

different mechanisms of action and short residual activity times in soil reduce selection 

pressure (Prather et al., 2000). In conjunction with crop rotation, which may allow the 

grower to manipulate planting times to avoid early-season weed germination (Jordan et 

al., 1995) and to use mechanical as well as chemical weed control methods, these 

practices impede the development of herbicide resistance in weeds. Monsanto invests 
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considerably in research to understand the proper uses and stewardship of the glyphosate 

molecule. This research includes an evaluation of some of the factors that can contribute 

to the development of weed resistance. Detailed information regarding glyphosate weed 

resistance management is presented in Appendix L. 

IX.E. Management of Insects 

Although insects are rated as less problematic than weeds in U.S. soybean production, 

management of insect pests during the growth and development of soybean is important 

for protecting the yield of soybean (Aref and Pike, 1998). Understanding the impact of 

insects on soybean growth is essential for proper management (Higley and Boethel, 

1994). It is important to understand the way that insects injure soybean as well as how 

the soybean plant responds to insect injury. Insect injury can impact yield, plant 

maturity, and seed quality. Injury is defined as a stimulus producing an abnormal change 

in plant physiological processes. Injury may produce stress, which is a departure from 

optimal physiological conditions (Higley and Boethel, 1994). The ultimate impact of 

injury is damage: a measurable reduction in plant growth development or reproduction. 

Insect injury in soybean seldom reaches levels to cause an economic loss, as indicated by 

the low percentage (16%) of soybean acreage that receives an insecticide treatment 

(USDA-NASS, 2007). 

Characterizing soybean responses to insect injury is essential in establishing economic 

injury levels (Higley and Boethel, 1994). Most often, soybean insects are categorized or 

defined by the plant parts they injure, namely root-feeding, stem-feeding, leaf-feeding, or 

pod-feeding insects. The root- and stem-feeding insect groups are often the hardest to 

scout and typically are not detected until after they have caused their damage. The leaffeeding insects comprise the biggest group of insects, but not necessarily the most 

damaging insects. Recent research on defoliation has determined that a major effect of 

injury is to reduce light interception by the soybean canopy which in turn can have a 

significant effect on yield (Higley and Boethel, 1994). Soybean has an extraordinary 

capacity to withstand considerable defoliation early in the season without significant 

yield loss. By contrast, defoliation during the flowering and pod filling stages poses a 

greater threat to yield because the soybean plant has less time to compensate for injury 

compared to other growth stages. Research indicates that the soybean plant can sustain a 

35% leaf loss prior to the pre-bloom period without lowering yield (NDSU, 2002). 

However, from pod-set to maturity, the plant can tolerate only a 20% defoliation level 

before yield is impacted. 

IX.F. Management of Diseases and Other Pests 

More than 100 pathogens are known to affect soybean, of which 35 are considered to be 

of economic importance (Heatherly and Hodges, 1999). The estimated yield losses to 

soybean diseases in the U.S. were 10.9, 11.9, and 14.0 million metric tons in 1996, 1997, 

and 1998, respectively (Wrather et al., 2000). Pathogens can affect all parts of the 

soybean plant, resulting in reduced quality and yield. The extent of losses depends upon 

the pathogen, the state of plant development and health when infection occurs, the 
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severity of the disease on individual plants, and the number of plants affected (Heatherly 

and Hodges, 1999). 

One or more diseases can generally be found in fields wherever soybean is grown 

(Heatherly and Hodges, 1999). However, a pathogen may be very destructive one season 

and difficult or impossible to find the next season. The extent and severity of soybean 

diseases depend on the degree of compatibility between the host and the pathogen and the 

influence of the environment. 

According to field surveys conducted in fifteen soybean-producing states during 1996 to 

1998, soybean cyst nematode (Heterodera gylcines) caused the greatest soybean yield 

losses (Wrather et al., 2000). Phytophthora root and stem rot (Phytophthora sojae), 

brown stem rot (Phialophora gregata), sclerotinia stem rot (Sclerotinia sclerotiorum), 

and seedling diseases followed in economical importance. As expected, yield losses 

varied by region. Sclerotinia stem rot caused yield losses in several Northern states, but 

not in other states. Rhizoctonia foliar blight losses were greatest in Arkansas, Louisiana, 

and Texas where humidity and temperature conditions are suitable for disease 

development. 

Selecting resistant varieties is the primary tool growers have for disease control 

(Heatherly and Hodges, 1999). Resistant varieties may have morphological or 

physiological characteristics that provide immunity, resistance, tolerance or avoidance to 

certain pathogens. Cultural practices play an important role in disease management by 

reducing initial inoculums or reducing the rate of disease development (Heatherly and 

Hodges, 1999). Preplant tillage can bury crop residue, which encourages the 

decomposition of fungal-resting structures. Crop rotation is routinely recommended as a 

disease-management strategy. Rotating crops interrupts the disease cycle and allows time 

for the decomposition of inoculums. One exception is Rhizontonia, a soil-inhabitant 

pathogen that grows on a wide variety of crops and can survive sufficiently in the soil to 

make crop rotation as a means of controlling this pest impractical. Row spacing, plant 

population, and planting date also can be changed to manage soybean diseases. 

Soybean cyst nematode (SCN) is one of the most damaging pathogens of soybean 

throughout the soybean growing regions of the U.S. (Pedersen, 2008b). Losses have 

been estimated to be at about $1.5 billion in the U.S. (Pedersen, 2008a). SCN can cause 

yield losses up to 50%, where this pest in 2004 alone caused an estimated loss of 50 

million bushels in Iowa (Pedersen, 2008b). Soybean cyst nematodes feed on the roots, 

causing severely stunted and yellow plants. The simplest, least expensive method to 

reduce populations of this pest is to rotate soybean with a non-host crop such as corn, 

small grains, or sorghum. Planting resistant varieties is regarded as the best and most 

effective management practice to prevent losses from this pest. Several public and 

private soybean varieties offer sources of resistance to certain races of nematode. 

Alternating varieties with different sources of resistance also is beneficial. 

High-quality seed is essential for controlling seedling diseases. The most important 

seedling diseases in soybean are Phytophthora, Pythium, Rhizoctonia, and Fusarium 

(Pedersen, 2008a). Many soybean varieties demonstrate resistance to specific taxonomic 

races of Phytophthora. Treating soybean seed with a fungicide (e.g., pyraclostrobin, 

metalaxyl, mefenoxam) is effective against damping-off disease (seedling blight) caused 
Monsanto Company 09-SY-201U Page 154 of 471

by common soil fungi, such as Phytophthora and Pythium. Fungicide seed treatments are 

recommended where there is a history of these seedling diseases. 

Asian soybean rust is a foliar fungal disease that typically infests soybean during 

reproductive stages of development and can cause defoliation and reduce yields 

significantly in geographies such as Brazil (Dorrance et al., 2007). Soybean rust is 

caused by the fungus Phakopsora pachyrhizi. This disease in the U.S. was first detected 

in Louisiana in 2004. At this time, foliar application of fungicides is the standard 

disease-management practice to limit yield losses due to soybean rust. 

Foliar fungicide applications can effectively reduce the incidence of many diseases 

(Heatherly and Hodges, 1999). However, the economic return from a fungicide 

application may be limited to select production programs; for example, high-yield 

environments or when producing soybean seed. According to USDA-NASS statistics, 

fungicides were applied on approximately 4% of the soybean acreage in 2006 (2007). 

IX.G. Crop Rotation Practices in Soybean 

The well-established farming practice of crop rotation is still a key management tool for 

growers. The purposes of growing soybean in rotation are to improve yield and 

profitability of one or both crops over time, decrease the need for nitrogen fertilizer on 

the crop following soybean, increase residue cover, mitigate or break disease, insect, and 

weed cycles, reduce soil erosion, increase soil organic matter, improve soil tilth and soil 

physical properties, and reduce runoff of nutrients, herbicides, and insecticides (Boerma 

and Specht, 2004; Al-Kaisi et al., 2003). According to USDA Economic Research 

Service, 95% of the soybean-planted acreage has been in some form of a crop rotation 

system since 1991 (USDA-ERS, 2001). Corn- and wheat-planted acreage has been 

rotated at a slightly lower level of 75% and 70%, respectively. Although the benefits of 

crop rotations can be substantial, the grower must make cropping decisions by evaluating 

both the agronomic and economic returns on various cropping systems. Crop rotations 

also afford growers the opportunity to diversify farm production in order to minimize 

market risks. 

Continuous soybean production is not a common practice in the Midwest and is 

discouraged by most extension soybean specialists to reduce the risk of diseases and 

nematodes (Hoeft et al., 2000; Al-Kaisi et al., 2003). Corn and soybean occupy more 

than 80% of the farmland in many of the Midwestern states, and the two-year cropping 

sequence of soybean-corn is used most extensively in this region. However, a soybean 

crop sometimes is grown after soybean and then rotated to corn in a 3-year rotation 

sequence (soybean-soybean-corn) in the Midwest. Compared to corn, soybean shows a 

greater yield response to being grown after a number of years without soybean. The yield 

of both corn and soybean is approximately 10% higher when grown in rotation than when 

either crop is grown continuously (Hoeft et al., 2000). 

A combination of conservation tillage practices and crop rotation has been shown to be 

very effective in improving soil physical properties. Long-term studies in the Midwest 

indicate that the corn-soybean rotation improves yield potential of no-till systems 

compared to continuous corn production (Al-Kaisi, 2001). The reduction in yield of 
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continuous corn production in no-till systems is attributed to low soil temperature during 

seed germination, which is evident on poorly drained soils under no-till practices. 

Crop rotations may change over a long period of time due to economic conditions and 

market opportunities. Roundup Ready soybean has provided growers more profit 

opportunities than conventional soybean primarily by reducing input costs (Gianessi, 

2005). In addition, Roundup Ready soybean has provided growers greater flexibility to 

grow soybean in fields with weed infestations, which previously were considered to be 

too problematic or unproductive for growing. 

Unique to the southern portion of the Midwest and the Mid-South regions, soybean is 

grown in a double-cropping system. Double-cropping refers to the practice of growing 

two crops in one year. This practice can improve income and reduce soil and water 

losses by having the soil covered with a plant canopy most of the year (Hoeft et al., 

2000). In the Midwest, winter wheat is harvested in late June or July, and then soybean is 

planted into the wheat residue in a no-till system to conserve moisture. Due to the 

uncertainty of double-cropping yields, growers sometimes do not plant if soils are too dry 

at the time of wheat harvest. Soybean typically is grown in a corn-wheat-soybean 

rotation sequence when soybean is grown in a double-cropping system. In the northern 

soybean growing areas, wheat will typically follow soybean in the rotation. 

Agronomic practices such as rotations for soybean vary from state to state. However, 

there are similarities among states within certain growing regions. This section provides 

a detailed description and quantitative assessment of the rotational cropping practices 

immediately following soybean production, by state. This assessment accounts for about 

99% of the total soybean acreage. These data are presented in Tables IX-7 through IX10. 

The majority of the U.S. soybean acreage (68.6%) is rotated to corn (Table IX-7). The 

second largest rotational crop following soybean is soybean. Approximately 14.5% of 

the soybean acreage is rotated back to soybean the following year. Wheat follows 

soybean on approximately 11.2% of the U.S. soybean acreage, with cotton, rice, and 

sorghum the next largest rotational crops following soybean. However, these three crops 

were planted on only 4.6% of the soybean acreage. Other minor rotational crops that 

follow soybean production are listed in Tables IX-7 through IX-10. 

Column J of each table provides the percentage of soybean acreage as a function of the 

total rotational crop acreage to indicate the level that soybean is the primary crop 

preceding the rotational crops. For the entire country (Table IX-7), this percentage is 

35.3% indicating that soybean is a major crop preceding these rotational crops. The 

percentage of soybean as a preceding crop varies widely in different states, which ranges 

from 16.8% (KS) to 95.2% (NJ). In the Midwest region where 82% of the soybean is 

grown, 34.6% of the rotational crop area was planted with soybean during the previous 

growing season. 

One rotation choice available to growers is to plant another Roundup Ready crop 

following the production of glyphosate-tolerant soybean. To determine the likelihood 

that the rotational crops planted after MON 87705 will be another Roundup Ready crop, 

an assessment also has been provided in Tables IX-7 through IX-10. This assessment is 

based on current agronomic practices following soybean production. Roundup Ready 
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alfalfa, canola, corn, cotton, soybean, and sugar beets have been nonregulated by the 

USDA and were considered as potential Roundup Ready crops following soybean 

production. For the purposes of this assessment, the adoption rates used for Roundup 

Ready corn, cotton, and soybean in 2008 were obtained from the USDA-NASS Acreage 

Summary report (2008c). The percentages for Roundup Ready corn, cotton, and soybean 

in the following tables were assumed to be the total percentage of herbicide-tolerant 

crops because the USDA-NASS report does not show the percentages of each individual 

herbicide-tolerant trait. Therefore, this is a slight overstatement for Roundup Ready corn, 

cotton, and soybean since other herbicide-tolerant traits are planted. Roundup Ready 

sugar beets were commercially introduced in 2008, although the USDA-NASS report 

does not provide planting information for this biotechnology-derived crop. Therefore, a 

90% adoption rate will be assumed for future plantings. Considering Roundup Ready 

alfalfa was nonregulated by the USDA, but is currently not available in the marketplace, 

a future market adoption estimate will be used. An adoption rate of 50% will be assumed 

for Roundup Ready alfalfa. 

This assessment showed that the percentage of the total rotational crop acreage that may 

be rotated from Roundup Ready soybean to another Roundup Ready crop (Table IX-7 - 

Column K) is estimated to be 21.0% in the U.S. and ranges from 7.1% (KS) to 78.3% 

(MS) across the soybean growing states. The percentage is 20.0% in the Midwest region, 

which is the largest soybean growing region. 

 
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Table IX-7. Rotational Practices in the U.S. Following Soybean Production

A B C D E F G H I State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in the U.S.1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

United States 

75037 Corn 

Soybean Sorghum Cotton Wheat Barley  Oats Rice Alfalfa Sugar Beets Potatoes Dry Beans Dry Peas Millet Flax Other9

80130 75037 4020 3767 37414 2159 1995 2301 1864 830 334 1183 520 250 345 452 

Total: 212601 

64.3 14.5 20.9 41.7 22.4 1.9 4.9 45.3 8.7 17.3 9.6 3.0 7.3 16.4 22.0 34.3 

51500 10866 841 1570 8396 41 98 1042 162 144 32 35 38 41 76 155 

Total: 75037 

68.6 14.5 1.1 2.1 11.2 0.05 0.1 1.4 0.2 0.19 0.04 0.05 0.05 0.05 0.10 0.2 

64.5 92.6 NA 79.3 NA NA NA NA 50 90 NA NA NA NA NA NA 

33214 10062 1245 81 129 Total: 44731 

The U.S. summary (Table IX-7) was developed by compiling the data from all three regional summaries. NA denotes not applicable. A1000s of acres. 1 Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2008); “other” crop and newly seeded alfalfa acreag

data from the Individual States data which was obtained from Quick Stat searches on http://www.nass.usda.gov/Data_and_Statistics/Quic

2 Column E is obtained by dividing Column F by Column D. 3 Column F is obtained by multiplying Column B by Column G. 4 Column G is obtained by dividing Column F by Column B. 5 Column H is obtained by dividing Column I by Column F. 6 Column I is obtained by compiling the data from all three regional summaries. 7 Column J is obtained by dividing Column B by Column D Total. 8 Column K is obtained by dividing Column I Total by Column D Total. 9 Various vegetables. 
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Table IX-8. Rotational Practices Following Soybean Production in the Midwest Region 

A B C D E F G H I J State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

APMR

Region 62150 Corn 

Soybean Sorghum Cotton Wheat Barley Oats Rice Alfalfa9

Sugar Beets Potatoes Dry Beans Dry Peas Millet Flax Other10

72260 62150 3553 341 32039 1929 1590 200 1617 830 278 1166 520 250 345 342 Total: 179410

65.7 7.9 18.8 22.9 25.3 2.1 6.2 91.0 10.0 17.3 11.6 3.0 7.3 16.4 22.0 25.3 

47480 4885 670 78 8102 41 98 182 162 144 32 3538417687Total: 62150

76.47.91.10.113.00.070.20.30.30.20.050.060.060.070.10.1

65.4 92.6 NA 87.2 NA NA NA NA 50 90 NA NA NA NA NA NA 

31040 4523 68 81 129 Total: 35841 3

IL 9200 Corn 

Soybean Sorghum Wheat 

12100 9200 80 1200 Total: 22580

71 3 92 28 

8556 230 74 340Total: 9200

93.0 2.5 0.8 3.7 

67% 87% NA NA 

5733 200 Total: 5933 4

IN 5450 Corn 

Soybean Wheat 

5700 5450 580 Total: 11730 

86 5 47 

4905 273 273 Total: 5450 

90 5 5 

71% 96% NA 

3483 262 Total: 3744 4
Monsanto Company 09-SY-201U Page 159 of 471

Table IX-8 (cont.). Rotational Practices Following Soybean Production in the Midwest Region 

A B C D E F G H I State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

IA 9750 Corn 

Soybean Alfalfa9

13300 9750 125 Total: 23175

71 2 78 

9458 195 98 Total: 9750

97 2 1 

68 95 50 

6431 185 49 Total: 6665 

KS 3300 Corn 

Soybean Sorghum Wheat 

3850 3300 2900 9600 Total: 19650

43 10 6 12 

1650 330 165 1155 Total: 3300

50 10 5 35 

65 95 NA NA 

1073 314 Total: 1386 

KY 1390 Corn 

Soybean Wheat 

1210 1390 580 Total: 3180

98 10 12 

1182 139 70 Total: 1390

85 10 5 

54 87 NA 

638 121 Total: 759 

MI 1900 Corn 

Soybean Wheat 

2400 1900 730 Total: 5030

55 5 65 

1330 95 475 Total: 1900

70 5 25 

57 84 NA 

758 80 Total: 838 

MN 7050 Corn 

Soybean Wheat Sugar beets Dry Beans 

Other11

7700 7050 1925 440 150 203 Total: 17468

70 3 66 24 24 35 

5358 212 1269 106 35 71 Total: 7050

76 3 18 1.5 0.5 1 

69 91 NA 90 NA NA 

3697 192 95 Total: 3984 
Monsanto Company 09-SY-201U Page 160 of 471

Table IX-8 (cont.). Rotational Practices Following Soybean Production in the Midwest Region 

A B C D E F G H I J State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

% APMR

MO 5200 Corn 

Soybean Sorghum Cotton Wheat Rice 

2800 5200 90 306 1250 200 Total: 9846

98 30 116 25 42 91 

2756 1560 104 78 520 182 Total: 5200

53 30 2 1.5 10 3.5 

43 92 NA 87 NA NA 

1185 1435 68 Total: 2688 5

NE 4900 Corn 

Soybean Sorghum Wheat 

8800 4900 300 1750 Total: 15750

42 10 82 28 

3675 490 245 490 Total: 4900

75 10 5 10 

59 97 NA NA 

2168 475 Total: 2644 3

ND 3800 Corn 

Soybean Wheat Sugar Beets Dry Peas Flax 

2550 3800 9230 208 520 335 Total: 16643

45 21 19 18 7 23 

1140 798 1710 38 38 76 Total: 3800

30 21 45 1 1 2 

65 94 NA 90 NA NA 

741 750 34 Total: 1525 2

OH 4500 Corn 

Soybean Wheat 

3300 4500 1120 Total: 8920

95 10 80 

3150 450 900 Total: 4500

70 

10 20 

54 89 NA 

 1701 401 Total: 2102 5
Monsanto Company 09-SY-201U Page 161 of 471

Table IX-8 (cont.). Rotational Practices Following Soybean Production in the Midwest Region 

A B C D E F G H I State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

SD 4100 Corn 

Soybean Sorghum Wheat Barley Oats Millet 

4750 4100 170 3661 63 220 110 Total: 13074

62 2 48 22 65 37 37 

2952 82 82 820 41 82 41 Total: 4100

72 2 2 20 1 2 1 

88 97 NA NA NA NA NA 

2598 80 Total: 2677 

WI 1610 Corn 

Soybean Wheat Oats Alfalfa9

Potatoes Other12

3800 1610 373 270 420 63.5 139.2 Total: 6676

36 2 22 6 15 51 12 

1369 32 81 16 64 32 16 Total: 1610

85 2 5 1 4 2 1 

61 90 NA NA 50 NA NA 

835 29 32 Total: 896 

The Midwest region summary (Table IX-8) was developed by compiling the data from all the states within the region. Unlike the individual state data, the d

summary were obtained by dividing Column F by Column B and the data in Column H were obtained by dividing Column I by Column F. NA denotes 

expressed as 1000s of acres. 

1 Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2008); “other” crop and newly seeded alfalfa acreages are based 

Individual States data which were obtained from Quick Stat searches on http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp. 

2 Column E is obtained by dividing Column F by Column D. 3 Column F is obtained by multiplying Column B by Column G. 4 The rotational crop percentages are based on estimates from personal communications (2006) with individual state Extension Crop Production Spec

Soybean, Corn and Cotton; Extension Weed Control Specialist on Soybean and Corn; and/or Monsanto Technology Development Representatives. 

5 Roundup Ready rotational crop adoption rates for corn, soybean and cotton are based on 2008 planting data (USDA-NASS, 2009a). The percentages for 

soybean represent the percentages for total herbicide-tolerant traits. Percentages of herbicide-tolerant alfalfa and sugar beets are future market adoption est

6 Column I is obtained by compiling the data from all the states within the region. 7 Column J is obtained by dividing Column B by Column D Total. 8 Column K is obtained by dividing Column I Total by Column D Total. 9 Newly seeded alfalfa. 10 Various vegetables. 11 Sweet corn and green peas. 12 Sweet corn, green peas and onions.
Monsanto Company 09-SY-201U Page 162 of 471

Table IX-9. Rotational Practices Following Soybean Production in the Mid-South Region 

A B C D E F G H I J State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

% AcPrMRo

Region 8200 Corn 

Soybean Sorghum Cotton Wheat Rice 

2630 8200 368 1860 2850 2101 Total: 18009 

43.1 63.0 46.5 32.6 9.4 40.9 

1132 5164 171 606 267 860 Total: 8200

13.8 63.0 2.1 7.4 3.3 10.5 

54.0 93.3 NA 82.8 NA NA 

612 4820 502 Total: 5934 45

AL 360 Corn 

Soybean Cotton Wheat 

260 360 290 240 Total: 1150

48 5 62 15 

126 18 180 36 Total: 360

35 5 50 10 

54 87 80 NA 

68 16 144 Total: 228 31

AR 3300 Corn 

Soybean Sorghum Wheat Rice 

440 3300 125 1070 1401 Total: 6336

53 64 53 22 47 

231 2112 66 231 660 Total: 3300

7 64 2 7 20 

54 94 NA NA NA 

125 1985 Total: 2110 52

LA 1050 Corn 

Soybean Sorghum Cotton 

520 1050 120 300 Total: 1990

20 65 88 53 

105 683 105 158 Total: 1050

10 65 10 15 

54 87 NA 79 

57 594 124Total: 775 52
Monsanto Company 09-SY-201U Page 163 of 471

Table IX-9 (continued). Rotational Practices Following Soybean Production in the Mid-South Regi

A B C D E F G H I J State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

% APMR

MS 2000 Soybean 

Rice

2000 230 Total: 2230

90 87 

1800 200 Total: 2000

90 10 

97 NA 

1746 Total: 1746 8

TN 1490 Corn 

Soybean Cotton 

690 1490 285 Total: 2465

97 37 94 

671 551 268 Total: 1490

45 37 18 

54 87 87 

362 480 233 Total: 1075 6

The Mid-South region summary (Table IX-9) was developed by compiling the data from all the states within the region. Unlike the ind

Column G for this regional summary were obtained by dividing Column F by Column B and the data in Column H were obtained by divi

NA denotes not applicable. All acreages are expressed as 1000s of acres. 

1 Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2008); “other” crop and newly seeded alfalfa acreag

data from the Individual States data which were obtained from Quick Stat searches on http://www.nass.usda.gov/Data_and_Statistics/Qui

2 Column E is obtained by dividing Column F by Column D. 3 Column F is obtained by multiplying Column B by Column G. 4 The rotational crop percentages are based on estimates from personal communications (2006) with individual state Extension Crop Pro

Agronomists – Soybean, Corn and Cotton; Extension Weed Control Specialist on Soybean and Corn ;and/or Monsanto Technology Devel

5 Roundup Ready rotational crop adoption rates for corn, soybean and cotton are based on 2008 planting data (USDA-NASS, 2009a). T

Ready corn, cotton and soybean represent the percentages for total herbicide-tolerant traits. Percentages of herbicide-tolerant alfalfa and 

adoption estimates. 

6 Column I is obtained by compiling the data from all the states within the region. 7 Column J is obtained by dividing Column B by Column D Total. 8 Column K is obtained by dividing Column I Total by Column D Total. 
Monsanto Company 09-SY-201U Page 164 of 471

Table IX-10. Rotational Practices Following Soybean Production in the Eastern Coastal Region 

A B C D E F G H I State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

Region 4687 Corn 

Soybean Cotton Wheat Other9

5240 4687 1566 2525 110 Total: 14128

55.2 17.6 56.6 1.1 49.5 

2892 827 887 27 54 Total: 4687

61.7 17.6 18.9 0.6 1.2 

54.0 87.0 76.1 NA NA 

1562 719 675 Total: 2956 

DE 195 Corn 

Soybean 

160 195 Total: 355

98 20 

156 39 Total: 195

80 20 

54 87 

84 34 Total: 115 

GA 430 Corn 

Cotton 

370 940 Total: 1310

12 41 

43 387 Total: 430

10 90 

54 78 

23 302 Total: 325 

MD 495 Corn 

Soybean 

460 495 Total: 955

97 10 

446 50 Total: 495

90 10 

54 87 

241 43Total: 284 

NJ 92 Corn 

Other10

85 11.6 Total: 97

97 79 

83 9 Total: 92

90 10 

54 NA 

45 Total: 45 

NY 230 Corn 

Other11

1090 33.5 Total: 1124

20 34 

219 12 Total: 230

95 5 

54 NA 

118 Total: 118 

NC 1690 Corn 

Soybean Cotton Other12

900 1690 430 65 Total: 3085

90 25 98 52 

811 423 423 34 Total: 1690

48 25 25 2 

54 87 76 NA 

438 368 321 Total: 1127 
Monsanto Company 09-SY-201U Page 165 of 471

Table IX-10 (continued). Rotational Practices Following Soybean Production in the Eastern Coasta

A B C D E F G H I J State 

Total Soybean Acres1

Major Crops Following Soybean In Rotation 

Total Acreage of Rotational Crop in States1

% Rotational Crop Following Soybean2

Rotational Crop Acres Following Soybean3

% Rotational Crop of Total Soybean4

% Roundup Ready Rotational Crop Option5

Acreage of Roundup Ready Rotational Crop Option6

% APMR

 PA 435 Corn 

Soybean 

1350 435 Total: 1785

32 2 

426 9 Total: 435

98 2 

54 87 

230 8 Total: 238 2

SC 540 Corn 

Soybean Cotton Wheat

355 540 135 220 Total: 1250

84 30 40 12 

297 162 54 27 Total: 540

55 30 10 5 

54 87 68 NA 

160 141 37 Total: 338 4

VA 580 Corn 

Soybean Cotton 

470 580 61 Total: 1111

88 25 38 

412 145 23 Total: 580

71 25 4 

54 87 68 

222 126 16 Total: 364 5

The Eastern Coastal region summary (Table IX-10) was developed by compiling the data from all the states within the region. Unlike the in Column G for this regional summary were obtained by dividing Column F by Column B and the data in Column H were obtained by divNA denotes not applicable. All acreages are expressed as 1000s of acres. 1 Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2008); “other” crop and newly seeded alfalfa acreag

data from the Individual States data which were obtained from Quick Stat searches on http://www.nass.usda.gov/Data_and_Statistics/Qui

2 Column E is obtained by dividing Column F by Column D. 3 Column F is obtained by multiplying Column B by Column G. 4 The rotational crop percentages are based on estimates from personal communications (2006) with individual state Extension Crop Pro

Agronomists – Soybean, Corn and Cotton; Extension Weed Control Specialist on Soybean and Corn; and/or Monsanto Technology Devel

5 Roundup Ready rotational crop adoption rates for corn, soybean and cotton are based on 2008 planting data (USDA-NASS, 2009a). T

Ready corn, cotton and soybean represent the percentages for total herbicide-tolerant traits. Percentages of herbicide-tolerant alfalfa and 

adoption estimates. 

6 Column I is obtained by compiling the data from all the states within the region. 7 Column J is obtained by dividing Column B by Column D Total. 8 Column K is obtained by dividing Column I Total by Column D Total. 9 Sweet corn and other vegetables. 10 Sweet corn, and other vegetables. 11 Sweet corn and onions. 12 Cucumbers and sweet potatoes. 
Monsanto Company 09-SY-201U Page 166 of 471

IX.H. Soybean Volunteer Management 

Volunteer soybean is defined as a plant that has germinated and emerged unintentionally 

in a subsequent crop. Soybean seeds can remain in a field after soybean harvest as a 

result of pods splitting before or during harvest. Soybean seeds also can remain in a field 

when pod placement on the plants is too close to the ground for the combine head to 

collect all the pods or when the combine is improperly adjusted for efficient harvesting. 

Volunteer soybean in rotational crops is not a concern in the Midwest region because the 

soybean seed is typically not viable after the winter period (Carpenter et al., 2002; 

OECD, 2000). In southern soybean growing areas of the U.S. where the winter 

temperatures are milder, it is possible for soybean seed to remain viable over the winter 

and germinate the following spring. 

Volunteer soybean normally is not a concern in rotational crops, such as corn, cotton, 

rice, and wheat, that are the significant rotational crops following soybean due to control 

measures that are available for volunteer soybean when they arise (Carpenter et al., 2002; 

OECD, 2000). Preplant tillage is the first management tool for control of emerging 

volunteer soybean in the spring. If volunteer soybean should emerge after planting, 

shallow cultivation will control most of the plants and effectively reduce competition 

with the crop. Several postemergence herbicides also are available to control volunteer 

soybean (conventional or glyphosate tolerant soybean) in each of the major rotational 

crops. Table IX-11 provides control ratings on volunteer glyphosate-tolerant soybean for 

several herbicides used in the major rotational crops. 

To provide control of volunteer soybean in corn, postemergence applications of AAtrex 

(atrazine), Clarity (dicamba), Distinct (diflufenzopyr + dicamba), Hornet (flumetsulam + 

clopyralid) and Widematch (clopyralid + fluroxypyr) provide excellent control 

(Zollinger, 2005). In wheat, Bronate Advanced (bromoxynil), Clarity (dicamba) and 

Widematch postemergence provide excellent control of volunteer soybean (Zollinger, 

2005). 

Volunteer soybean in cotton is normally not a concern. However, hurricanes or other 

extreme weather conditions can damage a soybean crop preceding cotton production in 

the Mid-South states, where the unharvested soybean seed can produce volunteer plants. 

Preplant applications of paraquat or herbicide mixtures containing paraquat will 

effectively control volunteer glyphosate-tolerant soybean (Montgomery et al., 2002; 

Murdock et al., 2002). Recent research in North Carolina indicates Envoke 

(trifloxysulfuron) will provide excellent postemergence control of soybean containing 

traits for glyphosate and sulfonylurea herbicide tolerance in Roundup Ready cotton (York 

et al., 2005). 

Volunteer soybean in rice is rarely a concern due to the combination of preplant tillage, 

flooding practices, and herbicides used in producing rice. If volunteer plants should 

emerge in rice, the postemergence applications of Grasp (penoxsulam), Permit 

(halosulfuron) and Regiment (bispyribac) typically used for weed control in rice will 

effectively alleviate competition from volunteer soybean (Dillon et al., 2006). 
Monsanto Company 09-SY-201U Page 167 of 471

Table IX-11. Ratings for Control of Volunteer Glyphosate-Tolerant Soybean in 

Labeled Rotational Crops1

Product 

Rate 

(Product/Acre)

Soybean 

V2 – V3 

Soybean 

V4- V6 

Corn2 

AAtrex 0.38 qts E P 

0.50 qts E F 

Clarity 4 fl oz E E 

5 fl oz E E 

Distinct 1 oz E G 

2 oz E E 

Hornet 1 oz E F 

2 oz E F-G 

Widematch 0.25 pt E G 

Wheat2 

Bronate Advanced 0.8 pt E E 

Clarity 4 fl oz E E 

5 fl oz E E 

Widematch 0.25 pt E G 

Cotton3 

Envoke 0.1 oz E E 

Rice4 

Grasp 2 oz E NA 

Permit 1 oz E NA 

Regiment 0.4 oz E E 

NA denotes “not applicable.” 

1 Weed control ratings: E = Excellent (90 to 99% control), G = Good (80 to 90% control), F = 

Fair (65 to 80 control), and P = Poor (40 to 65% control). 

2

 Zollinger, 2005. 

3 York et al., 2005. 

4 Dillon et al., 2006. 
Monsanto Company 09-SY-201U Page 168 of 471

 

IX.I. Stewardship of MON 87705 

Monsanto Company is firmly committed to its legal, ethical, and moral obligation to 

ensure that its products and technologies are safe and environmentally responsible. 

Monsanto demonstrates this commitment by implementing product stewardship processes 

throughout the lifecycle of a product and by participation in the Excellence Through 

StewardshipSM Program (http://www.excellencethroughstewardship.org/). These policies 

and practices include rigorous field compliance and quality management systems and 

verification through auditing.

As with all of our products, Monsanto is committed to the rigorous product stewardship 

of MON 87705. In keeping with past practice, Monsanto will seek regulatory approval 

for MON 87705 in all key soybean import countries with a functioning regulatory system 

to assure global compliance and support the flow of international trade. Monsanto 

continues to monitor other countries that are key importers of soybean from the U. S., for 

the development of formal biotechnology approval processes. If new functioning 

regulatory processes are developed, Monsanto will make appropriate and timely 

regulatory submissions. 

As with other value-added specialty soybean, once appropriate approvals are received, 

MON 87705-derived varieties will be grown using an appropriate IDP system based on 

established practices as described in Section IX.B.3. IDP practices are implemented for 

value-added specialty soybean to capture the enhanced value of the product and ensure 

that the end-user or processor receives the soybean with the identity, fatty acid 

composition of the oil, and desired quality. 

Prior to obtaining all key global regulatory approvals, Monsanto will conduct seed 

testing, variety development and production, and oil manufacturing, testing and 

commercial activities leading to the commercial introduction of soybean varieties 

developed with MON 87705. Monsanto will work in a closed loop system under contract 

with partners to plant, harvest and process MON 87705 and MON 87705-derived 

soybean for the purpose of producing low saturated fat, high oleic oil. 

A closed loop system will utilize appropriate processes for containment, documentation 

and traceability of seed production, planting, harvest, processing and use of the product. 

Grain production processes will include mass balance and accounting of all planting, 

harvested seed, secured storage facilities, labeling of all soybean, training of personnel, 

identification and audit of field production sites and acreage, spatial isolation of fields, 

equipment clean out procedures and documentation. Soybean processing will include 

segregation, control and traceability of handling, processing, packaging, and shipping of 

products and co-products. 

Before implementing a closed loop system, Monsanto will dialogue with the appropriate 

value chain stakeholders in the countries of production and of use of the product to gain 

feedback on and confirm the robustness and validity of the closed loop production 

system. Monsanto will not employ the closed loop system without adequate assurance 

that the system will be effective in containing, preventing the escape of, and controlling 
Monsanto Company 09-SY-201U Page 169 of 471

the disposition of MON 87705 and MON 87705-derived soybean so it is not comingled 

with commodity soybean. As part of this process, Monsanto will continue to provide 

regular updates on MON 87705 to key members of the soybean industry grain trade, 

processing industry and food industry throughout the regulatory and product development 

process. 

IX.J. Impact of the Introduction of MON 87705 on Agricultural Practices 

Introduction of MON 87705 is expected to have no impact on current cultivation and 

management practices for soybean. The Roundup Ready soybean system, i.e., planting 

Roundup Ready soybean and applying glyphosate in crop, has become the standard weed 

control program in U.S. soybean production. Currently Roundup Ready soybean is 

planted on 92% of U.S. soybean acres (USDA-NASS, 2008). Therefore from an 

agricultural perspective, MON 87705 is similar to the commercial Roundup Ready 

soybean products used in the U.S. since 1996. For an overview of the cumulative 

impacts on agricultural practices (weed control, tillage and crop rotation) from 

deregulation of glyphosate-tolerant crops see Appendix N. 

Changes in the MON 87705 soybean oil profile are not anticipated to impact agricultural 

practices. MON 87705 is a nutritionally improved soybean product that is expected to 

bring added value to consumers that will result in the soybean being sold at a premium. 

The added value of MON 87705 as a specialty soybean is expected to bring higher 

returns to growers compared to commodity soybean. As such, MON 87705 will be 

harvested, processed and marketed using well-established IDP methods to maintain its 

integrity, purity and value. 

 
Monsanto Company 09-SY-201U Page 170 of 471

X. ENVIRONMENTAL CONSEQUENCES AND IMPACT ON AGRONOMIC 

PRACTICES 

X.A. Introduction 

This section provides a brief review and assessment of the plant pest potential of 

MON 87705 and its impact on agronomic practices. USDA-APHIS has responsibility, 

under the Plant Protection Act (7 U.S.C. § 7701-7772), to prevent the introduction and 

dissemination of plant pests into the U.S. APHIS regulation 7 CFR § 340.6 provides that 

an applicant may petition APHIS to evaluate submitted data to determine that a particular 

regulated article does not present a plant pest risk and no longer should be regulated. If 

APHIS determines that the regulated article does not present a plant pest risk, the petition 

can be granted, thereby allowing unrestricted introduction of the article. 

The definition of “plant pest” in the Plant Protection Act (PPA) includes living organisms 

that could directly or indirectly injure, damage, or cause disease in any plant or plant 

product (7 U.S.C. § 7702[14]). Information in this petition related to plant pest risk 

characteristics includes the mechanism of action and changes to plant metabolism and 

composition, expression and characteristics of the gene products (CP4 EPSPS protein and 

the FATB1-A and FAD2-1A RNA), weediness of the regulated article, impacts to NTOs, 

disease and pest susceptibilities, impacts on agronomic practices, any impacts on the 

weediness of any other plant with which it can interbreed, and the transfer of genetic 

information to organisms with which it cannot interbreed. 

The regulatory endpoint under the PPA for biotechnology-derived crop products is not 

zero risk, but rather a determination that deregulation of the regulated article is not likely 

to pose a plant pest risk. The plant pest risk assessment of MON 87705 is based 

primarily on eight lines of evidence: (1) insertion of a single functional copy of the 

inserted expression cassette, (2) characterization of the CP4 EPSPS protein expressed in 

MON 87705 and the improved fatty acid profile, (3) safety of the CP4 EPSPS protein and 

the improved fatty acid profile, (4) compositional equivalence of harvested MON 87705 

soybean seed as compared to conventional soybean, (5) phenotypic and agronomic 

characteristics demonstrating no increased plant pest potential, (6) negligible risk to 

NTOs and threatened or endangered species, (7) modern soybean has inherently low 

plant pest potential, and (8) no greater likelihood to impact agronomic practices, 

including land use, cultivation practices, or the management of weeds, diseases, and 

insects than conventional soybean. 

Phenotypic and agronomic characteristics of MON 87705 were analyzed to determine if 

it will become any more weedy or invasive relative to soybean varieties currently on the 

market approved for unconfined release. Additional evaluations included the propensity 

of MON 87705 to become a greater reservoir of plant pests (insects or pathogens) when 

compared to conventional plants. An assessment for potentially adverse impacts of 

MON 87705 on NTOs, including symbiotic soil microorganisms, also was performed. 

The potential for, and consequences of, gene flow and introgression of the genetic 

construct into sexually compatibile plants or wild relatives, were also evaluated to 

determine the potential for increased weedy or invasive characteristics in sexually 

compatible plant species. 
Monsanto Company 09-SY-201U Page 171 of 471

Molecular characterization of MON 87705 by Southern blot analyses demonstrated that a 

single copy of the T-DNA I and T- DNA II sequences from the transformation vector 

PV-GMPQ/HT4404 was integrated into the soybean genome at a single locus. There 

were no additional genetic elements, including backbone sequences, from the 

transformation vector PV-GMPQ/HT4404 detected, linked or unlinked to the intact DNA 

insert, in MON 87705. The assessment of weediness potential and gene flow indicated 

that MON 87705 is no more likely to become a weed than conventional soybean, and 

MON 87705 is expected to be similar to conventional soybean regarding the potential for 

and impact from gene flow. Due to lack of sexually compatible relatives in the U.S., 

pollen-mediated gene flow is expected to occur only within cultivated soybean. Given 

the reproductive biology of soybean, pollen-mediated gene flow is expected to be 

negligible within cultivated soybean. The probability for horizontal gene flow is 

exceedingly small. Even if it were to occur, the consequences would be negligible 

because the CP4 EPSPS protein which confers herbicide tolerance has no meaningful 

toxicity to humans and NTOs under the conditions of use. Transfer of the tandem 

FATB1-A and FAD2-1A gene segments is equally unlikely. As with the cp4 epsps gene, 

even if transfer were to occur, the result would be negligible because the FATB1-A and 

FAD2-1A gene segments do not encode for a protein, nucleic acids are generally 

recognized as safe (GRAS), and the resulting change in fatty acids results in an oil profile 

that is similar in composition to other plant-derived oils. An assessment of current 

agronomic practices in the U.S. indicates that the introduction of MON 87705 will not 

impact current U.S. soybean cultivation practices or the management of weeds, diseases, 

or insects (Section IX). 

Using the assessment above, the data and analysis presented in this petition leads to a 

conclusion that MON 87705 is unlikely to be a plant pest and, therefore, no longer should 

be subject to regulation under 7 CFR §340. 

APHIS recently has proposed amendments to 7 CFR § 340 that include its noxious weed 

authority. Because the data show that MON 87705 has no potential to cause injury or 

damage to protected interests under the noxious weed authority, MON 87705 also would 

not be considered a “noxious weed” as defined by the Plant Protection Act. 

X.B. Plant Pest Assessment of the MON 87705 Insert and Expressed Substances 

X.B.1. Characteristics of the Genetic Insert and the Expressed Protein 

Genetic Insert 

MON 87705 was produced by Agrobacterium-mediated transformation of soybean with 

PV-GMPQ/HT4404, which is a binary vector containing 2T-DNAs (Table IV-1). TDNA I contains a cp4 epsps expression cassette and a partial suppression cassette 

containing the sense segments of the FAD2-1A intron and FATB1-A 5' UTR. T-DNA II 

contains a partial suppression cassette that consists of the antisense segments of FAD2-1A

and FATB1-A. During plant transformation, the two T-DNAs co-integrated at one locus 

in the soybean genome, creating a single DNA insert that contains the single cp4 epsps

cassette and a single FAD2-1A and FATB1-A suppression cassette. The chimeric 

promoter P-FMV/TSF1 consists of enhancer sequences from the 35S promoter of the 
Monsanto Company 09-SY-201U Page 172 of 471

figwort mosaic virus (FMV) and the promoter from the Tsf1 gene of the plant 

Arabidopsis thaliana encoding elongation factor EF-1 alpha. The FMV promoter has a 

history of safe use in transgenic plants (USDA-APHIS, 2006b) and is highly unlikely to 

promote plant disease. The inserted T-DNA in MON 87705 contains left and right 

border sequences from Agrobacterium tumefaciens, a plant pest. These sequences are 

well characterized, are noncoding regions and will not cause MON 87705 to promote 

plant disease (Section IV). 

Molecular analyses demonstrate that MON 87705 contains one copy of the insert at a 

single integration locus. No additional elements from the transformation vector were 

detected in the genome of MON 87705, including backbone sequence from plasmid PVGMPQ/HT4404. Additionally, the data confirm the organization and sequence of the 

insert, demonstrate the stability of the insert over several generations, and demonstrate 

that the genomic DNA sequences flanking the 5' and 3' ends of the insert are native to the 

soybean genome. On the basis of these data, it is concluded that the expected CP4 

EPSPS protein and dsRNA are produced from the inserted DNA. 

Mechanism of Action 

Monsanto Company has developed biotechnology-derived soybean with an improved 

soybean oil profile. Compared to conventional soybean oil, MON 87705 soybean oil 

fatty acid (FA) levels are lower for saturated fats (6% vs. 15% FA), and higher for oleic 

acid (76% vs. 23% FA) (USDA-NND database at http://www.nad.usda.gov). The 

increase in monounsaturated fatty acid (oleic) is accompanied by an overall decrease in 

polyunsaturated fatty acids (17% vs 60% FA). As described earlier in Section I.C., the 

improved fatty acid profile in MON 87705 soybean oil is achieved through the use of 

endogenous soybean (Glycine max L.) gene segments configured to suppress FATB and 

FAD2 gene expression. MON 87705 contains FATB1-A and FAD2-1A gene segments 

under the control of a seed promoter, limiting oil composition modification to this tissue. 

The assembled gene transcript has an inverted repeat that produces double stranded RNA 

(dsRNA) that, via the RNA interference (RNAi) pathway, suppresses endogenous FATB 

and FAD2 gene expression, thereby producing the desired fatty acid phenotype. Acylacyl carrier protein (ACP) thioesterases (referred to herein as FATB enzymes) are 

localized in plastids and hydrolyze saturated fatty acids from the ACP-fatty acid moiety. 

The suppression of FATB results in a decrease in the transport of the saturated fats out of 

the plastid, thus retaining their availability for desaturation to 18:1 oleic acid (see Figure 

E-1). Therefore, suppression of FATB decreases saturated fat content in the oil as well as 

increasing oleic acid. Subsequently, this increased amount of oleic acid is either 

delivered to the oil body or endoplasmic reticulum for further desaturation. Delta-12 

desaturases (referred to as FAD2 enzymes) desaturate 18:1 oleic acid to 18:2 linoleic 

acid. The suppression of FAD2 in soybean seed causes reduced desaturation of oleic to 

linoleic acid thus contributing further to the increase in oleic while reducing linoleic acid 

content in the oil. Therefore, the overall result of the suppression of these two enzymes is 

a reduction in saturated 16:0 palmitic and 18:0 stearic fatty acids, an increase in 

monounsaturated 18:1 oleic acid, and lower levels of polyunsaturated 18:2 linoleic acid 

relative to commodity soybean. 
Monsanto Company 09-SY-201U Page 173 of 471

The RNA-based suppression of FATB and FAD2 soybean genes in MON 87705 is 

mediated by dsRNA molecules. Double stranded RNAs are commonly found in 

eukaryotes, including plants, for endogenous gene suppression and are composed of 

nucleic acids. Nucleic acids have a long history of safe consumption and are considered 

GRAS by the U.S. Food and Drug Administration. There is no evidence to suggest 

dietary consumption of RNA is associated with toxicity or allergenicity. Moreover, 

analysis of the DNA segments encoding this dsRNA showed that they lack the sequences 

required for translation initiation and protein synthesis. The production of a protein from 

the dsRNA encoded by the insert in MON 87705 is highly unlikely. Several 

biotechnology-derived plant products previously deregulated by APHIS were developed 

using RNA-based suppression mechanisms, including virus-resistant papaya and squash, 

high oleic soybean, FLAVR SAVR tomatoes, and plum trees resistant to Plum pox virus 

(FDA, 1994; USDA-APHIS 1994; USDA-APHIS, 1997; USDA-APHIS, 2006; USDAAPHIS, 2007). Based on this information, it is concluded that the inserted DNA and 

resulting dsRNA are safe and unlikely to produce a protein or polypeptide. As a result, 

the RNA-based suppression technology used in MON 87705 poses no novel risks from a 

food, feed or environmental perspective. 

MON 87705 incorporates the cp4 epsps coding sequence derived from the common soil 

bacterium Agrobacterium sp. strain CP4. The cp4 epsps coding sequence directs the 

production of the 5-enolpyruvylshikimate-3-phosphate synthase (termed CP4 EPSPS 

protein) that is less sensitive to inhibition by glyphosate compared to endogenous EPSPS 

in plants. The CP4 EPSPS protein renders Roundup Ready soybean tolerant to 

glyphosate, the active ingredient in Roundup agricultural herbicides. The CP4 EPSPS 

protein has been assessed previously as a component in multiple Roundup Ready crops 

and has not been found to confer a selective advantage which would lead to increased 

plant pest risk. 

Expressed Protein Safety 

The CP4 EPSPS protein produced in MON 87705 is equivalent to the CP4 EPSPS 

proteins consumed in food and feed derived from other Roundup Ready crops, such as 

Roundup Ready soybean, that have a history of safe use. It is structurally homologous to 

EPSPS proteins that are part of the amino acid synthesis pathway of all plants (Devine et 

al., 1993). The safety of CP4 EPSPS proteins present in biotechnology-derived crops has 

been extensively evaluated by Harrison et al. (1996). EPA also has reviewed the safety 

of the CP4 EPSPS protein and has established a tolerance exemption for the protein and 

the genetic material necessary for its production in or on all raw agricultural commodities 

(40 CFR § 174.523). This exemption was based on a safety assessment that included 

rapid digestion in simulated gastric fluids, lack of homology to known toxins and 

allergens, and lack of toxicity in an acute oral mouse gavage study. A history of safe use 

is supported by the lack of any documented reports of adverse effects since the 

introduction of other Roundup Ready crops in 1996. Therefore, it is concluded that the 

CP4 EPSPS protein poses no risk to human or animal health. 

 
Monsanto Company 09-SY-201U Page 174 of 471

Protein Expression Levels 

CP4 EPSPS mean protein expression levels in MON 87705 samples range from 77 to 530 

μg/g dry weight for root, forage, harvested seed and overseason leaf (see Section VI). 

These levels are comparable to other commercialized CP4 EPSPS protein-containing 

soybean products and confer tolerance to glyphosate found in the agricultural herbicide 

Roundup. The inverted repeat encoded by the insert in MON 87705 does not code for 

any proteins. Thus, no new proteins are produced in soybean containing the MON 87705 

trait. 

X.B.2. Compositional Characteristics 

MON 87705 soybean seed was expected to be compositionally equivalent to 

conventional soybean except for the intended changes in their fatty acid composition 

brought about by the suppression of FATB and FAD2 RNAs. Compositional analyses 

were conducted on seed and forage from MON 87705 samples collected at multiple sites. 

Additional non biotechnology-derived conventional soybean varieties also were included 

in the analysis to establish a range of natural variability for each analyte. 

The compositional analyses confirmed MON 87705 seed had the intended change in fatty 

acid composition; that is, decreased levels of the saturated fatty acids, elevated levels of 

18:1 oleic acid, and a corresponding decrease in 18:2 linoleic acid. Other components 

analyzed in MON 87705 seed and forage were compositionally equivalent to that of 

conventional soybean control (Section VII). Collectively, the data established that, with 

the exception of intended changes in fatty acid levels, MON 87705 is compositionally 

equivalent to conventional soybean. 

Similarly, fatty acid analysis of MON 87705 refined, bleached and deodorized oil 

confirmed the intended differences for saturated fatty acids, oleic acid, and linoleic acid. 

Food and feed derived from MON 87705 soybean oil will be lower in saturated fats than 

food and feed derived from commodity soybean oil, and is considered nutritionally 

improved. Other components analyzed in MON 87705 processed fractions were 

compositionally equivalent to that of conventional soybean control. Collectively, the 

data established that, with the exception of intended changes in fatty acid levels, 

MON 87705 is compositionally equivalent to conventional soybean. 

X.B.3. Phenotypic and Agronomic and Environmental Interaction Characteristics 

An extensive and robust set of information and data were used to assess whether the 

introduction of the improved oil profile trait altered the plant pest potential (OECD, 

1993) of MON 87705 compared to the conventional soybean control, which had a genetic 

background similar to MON 87705 but did not possess the improved fatty acid profile 

trait. Phenotypic and agronomic characteristics of MON 87705 were evaluated and 

compared to those of the conventional soybean control. These assessments included seed 

dormancy and germination parameters, plant growth and development characteristics, 

plant responses to abiotic stressors, pollen morphology and viability and plant symbiont 

interactions (Section VIII). Results from the phenotypic and agronomic assessments 

demonstrate that MON 87705 does not possess characteristics that would confer a plant 

pest risk compared to conventional soybean. Data on environmental interactions also 
Monsanto Company 09-SY-201U Page 175 of 471

indicate that MON 87705 does not confer any biologically meaningful increased 

susceptibility or tolerance to specific disease, insect, or abiotic stressors, or changes in 

agronomic and phenotypic characteristics. Taken together, these data support the 

conclusion that MON 87705 is not likely to pose increased plant pest risk compared to 

conventional soybean. 

Seed Dormancy and Germination 

Seed dormancy and germination characterization indicated that MON 87705 seed had 

germination characteristics similar to that of the conventional soybean control. In 

particular, the lack of hard seed, a well-accepted characteristic often associated with 

plants that are weeds, supports a conclusion of no increased weediness potential of 

MON 87705 when compared to conventional soybean. 

Plant Growth and Development 

Evaluations of plant growth and development characteristics in the field are useful for 

assessing potential weediness characteristics such as lodging and pod shattering, 

indicated there were no significant differences between MON 87705 and the 

conventional control. Of the remaining 12 growth and development characteristics 

assessed, there were four significant differences (early stand count, final stand count, 

days to flowering and 100 seed weight). The differences in these parameters were 

relatively small in magnitude, and the mean values of MON 87705 were within the range 

of the reference varieties for these characteristics. Thus, the differences in these 

parameters are unlikely to be biologically meaningful in terms of increased weed 

potential. 

Response to Abiotic Stressors 

Comparative field observations between MON 87705 and its conventional control and 

their response to abiotic stressors, such as drought, heat stress, high winds, nutrient 

deficiency, etc., found no differences. Therefore, these factors indicate no increased 

weediness potential. 

Pollen Morphology and Viability 

Evaluations of pollen morphology and viability from field-grown plants may be 

indicative of increased plant pest potential as they relate to the potential for gene flow to, 

and introgression of the biotechnology-derived trait into other soybean varieties and wild 

relatives. These evaluations demonstrated no statistically significant differences between 

MON 87705 and the conventional control. Taken together, these comparative 

assessments indicate that MON 87705 is not likely to have increased weed or plant pest 

potential compared to conventional soybean. 

Interactions with NTOs 

Evaluation of MON 87705 for potential adverse impacts on NTOs is a component of the 

plant pest risk assessment. The nature of MON 87705 as a product with no pesticidal 

activity leads to a conclusion that all exposed organisms are considered to be NTOs. In a 

2007 U.S. phenotypic and agronomic study, observational data on environmental 

interactions were collected at select sites for MON 87705 and a conventional soybean 

control. In addition, multiple commercial conventional soybean varieties were included 
Monsanto Company 09-SY-201U Page 176 of 471

in the analysis to establish a range of natural variability for each characteristic. The 

environmental interactions evaluation (Section VIII) included data collected on plantinsect, plant-disease, and plant-environment interactions. The results of this assessment 

indicated that the presence of the improved fatty acid profile and herbicide tolerance 

traits did not alter plant-insect interactions, including beneficial arthropods and insect 

pests, nor did it alter disease susceptibility of MON 87705 compared to conventional 

soybean. The lack of differences in plant response to disease damage, arthropod damage, 

and arthropod pest and beneficial insect abundance demonstrate that the introduction of 

the improved fatty acid profile trait is unlikely to be biologically meaningful in terms of 

increased pest potential. 

In the field, soybean forms a complex symbiotic relationship with members of the 

bacterial family Rhizobiaceae and Bradyrhizobiaceae. This symbiosis results in the 

formation of root nodules in which the bacteria reduce or fix atmospheric nitrogenproducing ammonia that can be used by the plant. No significant differences were 

detected between MON 87705 and the control for the parameters measured, indicating no 

impact on either the symbiotic relationship or the symbiotic nitrogen-fixing bacteria. A 

lack of altered pest potential of MON 87705, compared to conventional soybean, is 

further supported by an assessment demonstrating that the symbiosis between nitrogen 

fixing bacteria and soybean was not altered as a result of the introduction of the improved 

oil profile and herbicide tolerance traits (Section VIII). Consequently, there is no 

increased plant pest potential due to the nutritionally improved soybean oil profile or 

herbicide tolerance. 

These results provide evidence that when compared to conventional soybean, the 

environmental interactions between MON 87705 and arthropod pests, beneficial 

arthropods, diseases, and nitrogen-fixing bacteria were not altered. The lack of 

significant biological differences in plant response to environmental interactions indicates 

that it is unlikely the improved fatty acid profile and herbicide-tolerance traits of 

MON 87705 will be biologically meaningful in terms of increased plant pest potential. 

X.C. Weediness Potential of MON 87705 

The commercial Glycine species in the U.S. (Glycine max L.) does not exhibit weedy 

characteristics and is not effective in invading established ecosystems. Soybean is not 

listed as a weed in the major weed references (Crockett, 1977; Holm et al., 1979; 

Muenscher, 1980), nor is it present on the lists of noxious weed species distributed by the 

federal government (7 CFR § 360). Soybean does not possess any of the attributes 

commonly associated with weeds (Baker, 1965), such as long persistence of seed in the 

soil, the ability to disperse, invade, and become a dominant species in new or diverse 

landscapes or the ability to compete well with native vegetation. Due to the lack of 

dormancy, a trait that has been removed through commercial breeding, soybean seed can 

germinate quickly under adequate temperature and moisture and potentially can grow as 

volunteer plants. However, volunteer plants likely would be killed by frost during 

autumn or winter of the year they were produced. If they did become established, 

volunteer plants would not compete well with the succeeding crop, and could be 

controlled readily by either mechanical or chemical means (OECD, 2000). In addition, 

since wild populations of Glycine species are not known to exist in the U.S., the potential 
Monsanto Company 09-SY-201U Page 177 of 471

does not exist for MON 87705 to outcross to wild or weedy relatives and to alter 

weediness potential. 

In comparative studies between MON 87705 and a conventional soybean control, 

phenotypic, agronomic, and environmental interaction data were evaluated (Section VIII) 

for changes that would impact the plant pest potential and, in particular, plant weediness 

potential. Results of these evaluations show that there is no fundamental difference 

between MON 87705 and the conventional control for traits potentially associated with 

weediness. Furthermore, comparative field observations between MON 87705 and its 

conventional control and their response to abiotic stressors, such as drought, heat stress, 

and high winds, indicated no differences and, therefore, no increased weediness potential. 

Collectively, these findings support the conclusion that MON 87705 has no increased 

weed potential compared to conventional soybean. Data on environmental interactions 

also indicate that MON 87705 does not confer any biologically meaningful increased 

susceptibility or tolerance to specific disease or insect stressors. 

X.D. Potential for Pollen mediated Gene Flow 

Gene introgression is a process whereby one or more genes successfully integrate into the 

genome of a recipient plant. Introgression is affected by many factors, including the 

frequency of the initial pollination event, environmental factors, sexual compatibility of 

pollen donor and recipient plants, pollination biology, flowering phenology, hybrid 

stability and fertility, selection, and the ability to backcross repeatedly. Because gene 

introgression is a natural biological process, it does not constitute an environmental risk 

in and of itself (Sutherland and Poppy, 2005). Gene introgression must be considered in 

the context of the transgenes inserted into the biotechnology-derived plant, and the 

likelihood that the presence of the transgenes and their subsequent transfer to recipient 

plants will result in increased plant pest potential. The potential for gene introgression 

from MON 87705 is discussed below. 

X.D.1. Hybridization with Cultivated Soybean Glycine max

Although soybean is a largely self-pollinated species, low levels of natural crosspollination can occur (Caviness, 1966; OECD, 2000; Ray et al., 2003; Yoshimura et al., 

2006). In studies with cultivated soybean where conditions have been optimized to 

ensure close proximity and flowering synchrony, natural cross-pollination generally has 

been found to be very low. Most outcrossing occurred with surrounding plants, and 

cross-pollination frequencies vary depending on growing season and genotype. Insect 

activity does increase the outcrossing rate, but soybean generally is not a preferred plant 

for pollinators (Erickson, 1975; Erickson, 1984). 

Numerous studies on soybean cross-pollination have been conducted, and the published 

results, with and without supplemental pollinators, are summarized in Table X-1. Under 

natural conditions, cross-pollination among adjacent plants in a row or among plants in 

adjacent rows ranged from 0 to 6.3%. In experiments where supplemental pollinators 

(usually bees) were added to the experimental area, cross-pollination ranged from 0.5 to 

7.74% in adjacent plants or adjacent rows. However, cross-pollination does not occur at 

these levels over long distances. Cross-pollination rates decrease to less than 1.5% 
Monsanto Company 09-SY-201U Page 178 of 471

beyond one meter from the pollen source, and rapidly decrease with greater distances 

from the source. The following cross-pollination rates at extended distances have been 

reported: 0.02% at 8.2 m of separation (Caviness, 1966), 0.05% at 5.4 m (Ray et al., 

2003), and 0% at 6.5 m (Abud et al., 2003). 

The potential for cross-pollination is limited. This is recognized in certified seed 

regulations for foundation seed in the U.S., which permit any distance between different 

soybean cultivars in the field as long as the distance is adequate to prevent mechanical 

mixing (USDA-APHIS, 2006a). 

The ecological risk associated with gene flow and introgression from MON 87705 

derives from the presence of the cp4 epsps gene and the FAD2-1A and FATB1-A gene 

segments. The consequence of introgression of the cp4 epsps gene and the FAD2-1A and 

FATB1-A gene segments from MON 87705 into other soybean is negligible since, as data 

presented in this petition confirm, they confer no increased plant pest potential to 

soybean. 

X.D.2. Hybridization with Wild Annual Species within Subgenus Soja 

The subgenus Soja includes the cultivated soybean Glycine. max and the wild annual 

species Glycine soja. Glycine soja is found in China, Taiwan, Japan, Korea, and Russia 

and can hybridize naturally with the cultivated soybean, G. max (Hymowitz, 2004). 

Hybridization between female G. soja and male G. max was less successful than 

hybridization in the opposing direction (Dorokhov et al., 2004), where frequency of 

spontaneous cross pollination in reciprocal combinations of G. max and G. soja varied 

from 0.73 (♀ G. soja × ♂ G. max) to 12.8% (♀ G. max × ♂ G. soja). Species 

relationships in the subgenus Soja indicated that F1 hybrids of G. max and G. soja carry 

similar genomes and are fertile (Singh and Hymowitz, 1989). Abe et al. (1999) note that 

“natural hybrids between G. max and G. soja are rare ... and hybrid swarms involving 

both species have never been reported.” This is also supported by work from Kuroda et 

al (2008) in which molecular markers was used and no gene flow from G. max to G.soja 

was detected. Many barriers exist to natural hybridization between soybean and wild 

relatives, including the highly selfing nature of both plants, required proximity of wild 

soybean to cultivated soybean, synchrony of flowering, and presence of pollinators. As 

such, it is highly unlikely that naturally occurring, pollen-mediated gene flow and 

transgene introgression into wild soybean relatives from incidentally released 

biotechnology-derived soybean will occur at any meaningful frequency. 

As described earlier, the subgenus Soja also contains an unofficial species, G. gracilis

(Hymowitz, 2004). G. gracilis is known only from Northeast China, and is considered to 

be a weedy or semi-wild form of G. max, with some phenotypic characteristics 

intermediate to those of G. max and G. soja. G. gracilis may be a hybrid between G. soja

and G. max (Hymowitz, 1970). Interspecific fertile hybrids formed by intentional crosses 

between G. max and G. soja and between G. max and G. gracilis have been easily 

obtained (Dorokhov et al., 2004). Although hybridization between G. max and members 

of the subgenus G. soja can take place, G. soja is not found in North or South America, 

and it is highly unlikely that gene transfer will occur. 
Monsanto Company 09-SY-201U Page 179 of 471

X.D.3. Hybridization with the Wild Perennial Species of Subgenus Glycine 

Wild perennial species of the Glycine subgenus occur in Australia; West, Central and 

South Pacific Islands; China; Papua, New Guinea; Philippines; and Taiwan. Therefore, 

the only opportunities for inter-subgeneric hybridization would occur in areas where 

those species are endemic (Hymowitz et al., 1992; Hymowitz and Singh, 1992). 

Nonetheless, the likelihood of interspecific hybridization between G. max and the wild 

perennial Glycine species is extremely low because they are genomically dissimilar (see 

Table II-2) and pod abortion is common. From time to time, immature seeds of the 

crosses have been germinated aseptically in vitro, but the resulting F1 hybrids are slowgrowing, morphologically weak, and completely sterile. Their sterility is due to poor 

chromosome pairing. Furthermore, species distantly related usually produce nonviable 

F1 seeds that either have premature death of the germinating seedlings or suffer from 

seedling and vegetative lethality (Kollipara et al., 1993; Singh and Hymowitz, 1989). In 

North and South America, it is not possible for gene transfer to occur between cultivated 

soybean and wild perennial species of Glycine subgenera because these wild species do 

not exist in these regions. 

X.D.4. Transfer of Genetic Information to Species with which Soybean Cannot 

Interbreed (Horizontal Gene Flow) 

Monsanto is not aware of any reports regarding the unaided transfer of genetic material 

from soybean species to other sexually incompatible plant species. The likelihood for 

horizontal gene flow to occur is exceedingly small. The ecological risk associated with 

gene flow and introgression from MON 87705 derives from the presence of the cp4 epsps

gene and the FAD2-1A and FATB1-A gene segments. The consequence of introgression 

of the cp4 epsps gene and the FAD2-1A and FATB1-A gene segments from MON 87705 

into other soybean is negligible since, as data presented in this petition confirm, they 

confer no increased plant pest potential to soybean. 

X.E. Summary of Plant Pest Assessments 

Plant pests are defined in the Plant Protection Act as certain living organisms that can 

directly or indirectly injure, cause damage to, or cause disease to any plant or plant 

product (7 U.S.C. § 7702[14]). Characterization data presented in Sections III through 

IX of this petition confirms MON 87705 is not meaningfully different from conventional 

soybean in terms of pest potential in its phenotypic, agronomic and environmental 

interaction characteristics, with the exception of the nutritionally improved fatty acid 

profile and glyphosate tolerance. Thus, MON 87705 is similar to conventional soybean 

in its plant pest potential. Monsanto is not aware of any study results or observations 

associated with MON 87705 that would suggest an increased plant pest risk would result 

from its introduction. 

The plant pest assessment was based on multiple lines of evidence developed from a 

detailed characterization of MON 87705 compared to conventional soybean, followed by 

a risk assessment on detected differences. The risk assessment considered various factors 

including: (1) insertion of a single functional copy of the inserted expression cassette, (2) 

characterization of the CP4 EPSPS protein expressed in MON 87705 and the improved 
Monsanto Company 09-SY-201U Page 180 of 471

sfatty acid profile, (3) safety of the CP4 EPSPS protein and the improved fatty acid 

profile, (4) compositional equivalence of harvested MON 87705 soybean seed as 

compared to conventional soybean, (5) phenotypic and agronomic characteristics 

demonstrating no increased plant pest potential, (6) negligible risk to NTOs and 

threatened or endangered species, (7) modern soybean has inherently low plant pest 

potential, and (8) no greater likelihood to impact agronomic practices, including land use, 

cultivation practices, or the management of weeds, diseases, and insects than 

conventional soybean. 

Based on the data and information presented in this petition, it is concluded that, like 

currently deregulated soybean, MON 87705 is highly unlikely to be a plant pest. 

Therefore, Monsanto Company requests a determination from APHIS that MON 87705 

and any progeny derived from crosses between MON 87705 and other commercial 

soybean be granted nonregulated status under 7 CFR § 340. 

Table X-1. Summary of Published Literature on Soybean Cross Pollination 

Distance 

from Pollen 

Source

% CrossPollination Comments Reference

0.3 m 0.04% 

(estimated per 

pod)

Interspaced plants within a row. Experiment 

conducted in a single year. Single male and 

female parental varieties. Percent 

outcrossing calculated per pod rather than 

per seed. 

Woodworth, 

1922

0.8 m 0.07 to 0.18% Adjacent rows. Experiment conducted over 

two years. Several male and female parental 

varieties. 

Garber and 

Odland, 1926

0.1 m 0.38 to 2.43% Adjacent plants within a row. Experiment 

conducted in a single year. Several male and 

female parental varieties.

Cutler, 1934

0.1 m 0.2 to 1% Adjacent plants within a row. Experiment 

conducted in single year at two locations. 

Several male and female parental varieties.

Weber and 

Hanson, 1961

0.9 m

2.7 – 4.6 m

6.4 – 8.2 m

10 – 15.5 m

0.03 to 0.44 %

0.007 to 0.04% 

0 to 0.02%

0 to 0.01%

Frequency by distance was investigated. 

Experiment conducted over three years. 

Single male and female parental varieties.

Caviness, 1966

0.8 m 0.3 to 3.62% Various arrangements within and among 

adjacent rows. Experiment conducted over 

three years. Several male and female 

parental varieties.

Beard and 

Knowles, 1971

One row

(undefined)

1.15 to 7.74% Bee pollination of single-row, small-plots of 

pollen receptor surrounded by large fields 

(several acres) of pollen donor soybean. 

Soybean is not a preferred flower for 

honeybee. 

Abrams et al., 

1978
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Table X-1 (cont.) Summary of Published Literature on Soybean Cross Pollination 

Distance 

from Pollen 

Source

% CrossPollination Comments Reference

0.1 – 0.6 m 0.5 to 1.03% 

(depending on 

planting 

design)

Bee pollination of soybean grown in various 

spatial arrangements. Experiment conducted 

over four years. Several soybean cultivars. 

Chiang and 

Kiang, 1987

1.0 m 0.09 to 1.63% Adjacent rows. Experiment conducted over 

two years. Several male and female parental 

varieties. 

Ahrent and 

Caviness, 1994

0.5 m

1.0 m

6.5 m

0.44 to 0.45%

0.04 to 1.4%

none detected

Frequency by distance was investigated. 

Experiment conducted in a single year. 

Single male and female parental varieties.

Abud et al., 

2003

0.9 m

5.4 m

0.29 to 0.41%

0.03 to 0.05%

Frequency by distance was investigated. 

Experiment conducted in a single year. 

Single male and female parental varieties.

Ray et al., 2003

0.15 m 0.65 to 6.32%

1.8%

Interspaced plants within a row. Experiment 

conducted in a single year. Single male and 

female parental varieties.

Ray et al., 2003

0.7 m

1.4 m

2.1 m

2.8 m

3.5 m

7.0 m

10.5 m

0 to 0.19%

0 to 0.04%

0 to 0.05%

0 to 0.08%

0 to 0.04%

0 to 0.04%

0

Interspaced plants within a row arranged in 

small plots. Experiment conducted in a four 

year period. Single male and two female 

parental varieties.

Yoshimura, et 

al., 2006

 
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XI. ADVERSE CONSEQUENCES OF INTRODUCTION 

Monsanto knows of no study results or observations associated with MON 87705, the 

improved fatty acid profile or the CP4 EPSPS protein, indicating that there would be an 

adverse environmental consequence from the introduction of MON 87705. MON 87705 

soybean oil contains a reduced level of saturated fats, an increase in oleic acid and an 

associated decrease in linoleic acid levels. The decrease in saturated fats and 

polyunsaturated fatty acids in MON 87705 soybean oil provides important options for 

food companies to develop foods with lower saturated fat and greater food functionality. 

In addition, these attributes provide key enhancements for biodiesel and industrial 

applications. MON 87705 also contains the CP4 EPSPS protein that renders the soybean 

plant tolerant to glyphosate, the active ingredient in the Roundup family of agricultural 

herbicides. As demonstrated by field results and laboratory tests, the only phenotypic 

difference between MON 87705 and conventional soybean is the improved soybean fatty 

acid profile and glyphosate tolerance. 

The data and information presented in this petition demonstrate that MON 87705 is 

unlikely to pose an increased plant pest potential or to have an adverse environmental 

consequence compared to conventional soybean. This conclusion is reached based on 

multiple lines of evidence developed from a detailed characterization of the product 

compared to conventional soybean, followed by risk assessment on detected differences. 

The characterization evaluations included molecular and protein analyses, which 

confirmed the insertion of a single functional copy of the cp4 epsps and partial 

suppression cassette at a single locus within the soybean genome; that the suppression of 

FATB and FAD2 RNAs result in the improved soybean fatty acid profile, and that the 

CP4 EPSPS protein was expressed in tissues at levels that resulted in tolerance to the 

herbicide glyphosate. Extensive characterization of the plant phenotype, including 

compositional analysis of key nutrients and antinutrients also indicated MON 87705, with 

the exception of the intended fatty acid changes, was unchanged compared to 

conventional soybean. A history of safe use of the CP4 EPSPS protein is supported by 

the lack of any documented reports of adverse effects since the introduction of other 

Roundup Ready crops in 1996. The EPA previously reviewed and established a tolerance 

exemption for the CP4 EPSPS protein and the genetic material necessary for its 

production in or on all raw agricultural commodities. 

An endangered species risk assessment concluded MON 87705 is unlikely to have 

adverse effects on these organisms. Therefore, the risks for humans, animals, and other 

NTOs from MON 87705 are negligible under the conditions of use. Additionally the 

introduction of MON 87705 will not adversely impact cultivation practices or the 

management of weeds, diseases, and insects in soybean production systems. 

Successful adoption of MON 87705 will provide growers with an opportunity to produce 

this value-added specialty soybean that produces soybean oil with improved stability and 

a healthier profile to help meet needs in food and industrial markets. 

 
Monsanto Company 09-SY-201U Page 183 of 471

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APPENDICES 
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Appendix A. USDA Notification 

Field trials of MON 87705 were conducted in the U.S. since 2005. The protocols for 

these trials include field performance, breeding and observation, agronomics, and 

generation of field materials and data necessary for this petition. In addition to the 

phenotypic assessment data provided for MON 87705, observational data on pest and 

disease stressors were collected from these product development trials. The majority of 

the final reports have been submitted to the USDA. However, some final reports, mainly 

from the 2007-2008 seasons, are still in preparation. A list of trials conducted under 

USDA notification and the status of the final reports for these trials are provided in 

Table A-1. 

 
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Table A-1. USDA Notifications Approved for MON 87705 and Status of Trials 

Conducted under These Notifications 

USDA No. 

Effective 

Date Release Site (State) Trial Status 

2005 

05-220-03n 9/28/2005 PR Submitted to USDA 

05-242-05n 9/28/2005 HI Submitted to USDA 

 

2006 

06-033-05n 5/18/2006 HI(3) Submitted to USDA 

06-045-13n 5/18/2006 HI(5) Submitted to USDA 

06-045-18n 5/18/2006 PR(3) Submitted to USDA 

06-109-04n 6/14/2006 PR(3) Submitted to USDA 

06-201-102n 9/11/2006 IA, PR(2) Submitted to USDA 

06-033-01n 3/14/2006 IL(7), KS(5) Submitted to USDA 

06-033-02n 3/23/2006 IA(7), IL(5), IN(2) Submitted to USDA 

06-223-111n 9/11/2006 PR(4) Submitted to USDA 

06-319-102n 12/15/2006 PR(3) Submitted to USDA 

 

2007 

07-038-117n 4/9/2007 IA(2) Submitted to USDA 

07-046-108n 4/1/2007 IL(2) Submitted to USDA 

07-054-103n 3/25/2007 IA, IL(2), NE, OH Submitted to USDA 

07-043-104n 3/20/2007 

IA(2), IL(2), IN(2), MO, NE, 

OH, PA, WI Submitted to USDA 

07-094-105n 5/4/2007 WI Submitted to USDA 

07-043-105n 4/17/2007 

AR(2), IL, IN, KS, MI, 

MO(5) Submitted to USDA 

07-137-104n 6/16/2007 PR(2) Submitted to USDA 

07-247-108n 10/4/2007 PR(2) Submitted to USDA 

07-211-102n 8/31/2007 PR(2) Submitted to USDA 

07-254-103n 10/11/2007 IA, IL, IN, MO, NE Submitted to USDA 

07-031-109n 3/21/2007 IL(10), IN(3), MO Submitted to USDA 

07-031-112n 3/20/2007 AR, IA(7), KS(6), MD Submitted to USDA 

07-352-101rm 3/26/2008 IA(8), IL(16), IN(4), KS(6) Submitted to USDA 

07-031-102n 3/18/2007 PR(2) Submitted to USDA 

07-032-101n 4/5/2007 IL(3) Submitted to USDA 

 
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Table A-1 (cont.) USDA Notifications Approved for MON 87705 and Status of 

Trials Conducted under These Notifications 

2008 

08-056-109n 3/28/2008 AR, IL(3), MD, WI Submitted to USDA 

08-058-113n 3/28/2008 

IA(2), IL(2), IN(2), OH, 

PR(2) Submitted to USDA 

08-045-118n 3/15/2008 PR(2) Submitted to USDA 

08-049-101n 3/19/2008 IL, MD, WI Submitted to USDA 

08-058-105n 3/28/2008 IL, IN, MI, MO, WI(2) Submitted to USDA 

08-080-114n 4/19/2008 IA, PR(2) Submitted to USDA 

08-079-101n 4/17/2008 IA(3) Submitted to USDA 

08-084-102n 4/24/2008 IA, NE Submitted to USDA 

08-137-101n 6/15/2008 PR Submitted to USDA 

08-170-102n 7/18/2008 PR(2) Submitted to USDA 

08-182-101n 8/1/2008 PR(2) Submitted to USDA 

08-270-101n 10/26/2008 PR In Progress 

08-301-103n 11/26/2008 HI, PR In Progress 

08-357-101rm 3/17/2009 

IA(11), IL(14), KS(5), IN(3), 

MO, NE In Progress 

08-323-101n 12/18/2008 PR(3) In Progress 

09-007-106n 2/25/2009 PR(2) In Progress 

08-042-101n 3/14/2008 AR, IA, IL(2), IN, MI, NE(2) Submitted to USDA 

08-042-102n 3/12/2008 IA(2), IL(8), IN(4), MO Submitted to USDA 

08-042-107n 3/12/2008 IA(7), KS(5) Submitted to USDA 

 

2009 

09-050-117n 3/21/2009 

HI, IA(4), IL(4), IN(2), 

OH(2), PR, WI In Progress 

09-033-101n 3/1/2009 IA(8), KS(5), NE In Progress 

09-061-108n 4/1/2009 

AR, IA, IL(5), IN(2), KS, 

MI, MO, NE, WI(2) In Progress 

09-050-134n 3/21/2009 HI, PR In Progress 

09-068-110n 4/8/2009 IL(5) In Progress 

09-099-102n 5/9/2009 PR(2) In Progress 

09-124-105n 5/13/2009 IA In Progress 

09-135-103n 6/14/2009 IL In Progress 

09-135-104n 6/14/2009 IL In Progress 

09-030-105n 3/1/2009 IA(3), IL(4), IN, KS, NE In Progress 

09-030-104n 3/1/2009 IA(3), IL(8), IN(3), MO In Progress 

09-036-103n 3/7/2009 IA(2), IL(2), IN, MS, NE(2) In Progress 
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Appendix B. Materials and Methods Used for Molecular Analyses of MON 87705 

B.1 Materials 

The DNA used in molecular analyses was isolated from leaf tissue of MON 87705 

collected in 2008 harvested from Production Plan 07-01-83-30 (Seed lot: GLP 0704 

18620-S). Additional DNA extracted from various MON 87705 generations of leaf 

tissues were used in generation stability analyses. The control substance was 

conventional soybean variety A3525 which has the same genetic background as the test 

substance. The reference substance, plasmid PV-GMPQ/HT4404 (Figures IV-1 and 2), 

was used as the transformation vector to develop MON 87705. The plasmid was digested 

and used as a positive hybridization control in Southern analyses. Probe templates 

generated from this plasmid also served as positive hybridization controls. As additional 

reference standards, the 1 kb DNA extension ladder and λ DNA/Hind III segments from 

Invitrogen (Carlsbad, CA) were used for size estimations on Southern blots and agarose 

gels. The 500 bp ladder from Invitrogen and GeneRuler 1 kb Plus Ladder from 

Fermentas (Hanover, MD) were used for size estimations for PCR analyses. 

B.2 Characterization of the Materials 

The quality of the source materials from MON 87705 and A3525 were verified by PCR 

analysis to confirm the presence or absence of MON 87705 except the materials used in 

the generational stability analyses where the identity of the materials was confirmed by 

the generation stability Southern blots themselves. The stability of the genomic DNA 

was confirmed in each Southern analysis by observation of the digested DNA sample on 

an ethidium bromide-stained agarose gel. 

B.3 DNA Isolation for Southern Blot and PCR Analyses 

Genomic DNA from the test and control substances was extracted prior to the initiation 

of the study using a hexadecyltrimethylammonium bromide (CTAB)-based method 

according to SOP BR-ME-1153-01. DNA extractions were stored in a 4 °C refrigerator 

or a -20 °C freezer. 

B.4 Quantification of Genomic DNA 

Extracted genomic DNA and plasmid DNA were quantified prior to the initiation of the 

study using Hoefer’s DyNA Quant 200 Fluorometer according to SOP BR-EQ-0065-02. 

Molecular size marker IX (Roche, Indianapolis, IN) was used as the DNA calibration 

standard. 

B.5 Restriction Enzyme Digestion of Genomic DNA 

Approximately 10 or 20 μg of genomic DNA extracted from the test and control 

substances were used for restriction enzyme digestions. When digesting genomic DNA 

with Nco I (New England BioLabs, Beverly, MA), 10X NE buffer 3 (New England 

BioLabs) was used. When digesting genomic DNA with the Spe I (Roche, Indianapolis, 

IN), 10X Tango buffer (Fermentas) was used. All digests were performed at 37°C 

according to SOP BR-ME-0316-01 in a total volume of ~500 μl using ~25-100 units of 

the appropriate restriction enzyme(s). 
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B.6 DNA Probe Preparation for Southern Blot Analyses 

Probe template DNA containing sequences of plasmid PV-GMPQ/HT4404 was prepared 

by PCR amplification according to SOP BR-ME-0486-01 and gel purified according to 

SOP BR-ME-0889-01. The probes were designed based on the nucleotide content 

(%GC) so that the entire probe would be hybridized under the conditions appropriate for 

the sequence. Approximately 25 ng of each probe template were radiolabeled with 

32P-deoxyadenosine triphosphate (dATP) (6000 Ci/mmol) using the random priming 

method (RadPrime DNA Labeling System, Invitrogen) according to SOP BR-ME-0611-

01. Approximately 1×106

 cpm of labeled probe per ml of hybridization solution was 

hybridized to the Southern blot. Probe locations relative to the genetic elements in 

plasmid PV-GMPQ/HT4404 are depicted in Figures IV-1 and IV-2. 

B.7 Southern Blot Analyses of Genomic DNA 

Digested genomic DNA isolated from test and control material, in addition to predigested 

control material mixed with appropriate positive hybridization controls were evaluated 

using Southern blot analyses according to SOP BR-ME-0317-02. The plasmid DNA was 

digested and then added to the predigested conventional soybean genomic DNA to serve as 

a positive hybridization control. Probe templates were added to predigested control 

material to serve as an additional positive hybridization control. The DNA was then 

separated by agarose gel electrophoresis. Southern blots were hybridized and washed at 

55° C or 60° C depending on the calculated melting temperature (Tm) of the probes used. 

The table below lists the hybridization conditions of the probes used in this study. 

Multiple exposures of each blot were then generated using Kodak Biomax MS film in 

conjunction with one Kodak Biomax MS intensifying screen in a -80°C freezer. 

 
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Probe DNA Probe 

Element Sequence Spanned by 

DNA Probe 

Hybridization/Wash 

Temperature (°C) 

1 T-DNA I Probe 1A Left Border + P-FMV/Tsf1 + L-Tsf1 + 

I-Tsf1 (portion) 55

2 T-DNA I Probe 2B I-Tsf1 (portion) + TS-CTP2 (portion) 55

3 T-DNA I Probe 3C TS-CTP2 (portion) + 

CS-cp4 epsps (portion) 60

4 T-DNA I Probe 4D CS-cp4 epsps (portion) + T-E9 + 

P-7Sα' + FAD2-1A (portion) 55

5 T-DNA I Probe 5E FAD2-1Ap (portion)+ FATB1-Ap

(portion) + Right Border

55

6 T-DNA II Probe 1A T-H6 + FAD2-1Ap + FATB1-Ap 55

7 Backbone Probe 1 Backbone Sequence 60

8 Backbone Probe 2 Backbone Sequence 60

9 Backbone Probe 3 Backbone Sequence 60

10 Backbone Probe 4 Backbone Sequence 60

B.8 DNA Sequence Analyses of the Insert 

Overlapping PCR products were generated that span the insert in MON 87705. These 

products were sequenced to determine the nucleotide sequence of the insert in 

MON 87705, as well as determining the nucleotide sequence of the genomic DNA 

flanking the 5' and 3' ends of the insert. The PCR analysis was performed according to 

SOP BR-ME-0486-01. 

The PCR analyses were conducted using ~96-100 ng of genomic DNA template or ~12 

ng of plasmid DNA in a 50 μl reaction volume or ~48-50 ng of genomic DNA template 

in a 25 μl reaction volume containing a final concentration of 1 mM MgSO4, 1 M 

Betaine, 0.8 μM of each primer, 0.2 mM each dNTP, and 0.02 units of KOD Hot Start 

DNA polymerase from Novagen (Gibbstown, NJ). The amplification of Products A, D, 

and E was performed under the following cycling conditions: 94°C for 2 minutes; 35 

cycles at 94° C for 45 seconds, 65° C for 45 seconds, 72° C for 5 minutes; one cycle at 

72°C for 10 minutes. The amplification of Products B, C, and F was performed under the 

following cycling conditions: 94° C for 2 minutes; 35 cycles at 94° C for 55 seconds, 

68°C for 60 seconds, 72° C for 5 minutes; and one cycle at 72° C for 10 minutes. 

Aliquots of each PCR product were separated on 0.8% (w/v) agarose E-gel® (Invitrogen) 

or separated on a 0.8% agarose gel according to SOP BR-ME-0315-02. Prior to 

sequencing, the PCR products were visualized by ethidium staining to verify the products 

were of the expected size prior to sequencing. To remove excess primers following PCR 

amplification, products were treated with a mixture of 0.1 unit Exonuclease I, designated 

as EXO, from USB (Cleveland, OH) and 0.1 unit Shrimp Alkaline Phosphatase, 

designated as SAP (USB) per 5 μl of PCR product and cycled at the following 

conditions: one cycle at 37° C for 15 minutes, one cycle at 80° C for 15 minutes. As 

documented in the raw data, not all products were treated with EXO-SAP prior to 

sequencing. The PCR products were sequenced using multiple primers, including 

primers used for PCR amplification. In addition, primers internal to the amplified PCR 
Monsanto Company 09-SY-201U Page 206 of 471

sequences were used to sequence the regions of the amplicon. All sequencing was 

performed by the Monsanto Genomics Sequencing Center using BigDye terminator 

chemistry (ABI, Foster City, CA). 

B.9 PCR and DNA Sequence Analysis to Examine the MON 87705 Insertion Site 

To demonstrate that the DNA sequences flanking the insert in MON 87705 are native to 

the soybean genome and to examine the MON 87705 insertion site in conventional 

soybean, PCR and sequence analyses were performed on genomic DNA from both 

MON 87705 and conventional soybean. The primers used in this analysis were designed 

from the DNA sequences flanking the insert in MON 87705. One primer designed from 

the genomic DNA sequence flanking the 5' end of the insert was paired with a second 

primer located in the genomic DNA sequence flanking the 3' end of the insert. The PCR 

analysis was performed according to SOP BR-ME-0486-01. 

The PCR analyses were conducted using ~96-100 ng of genomic DNA template in a 50 

μl reaction volume or ~50 ng of genomic DNA template in a 25 μl reaction volume 

containing a final concentration of 1 mM MgSO4, 1 M Betaine, 0.8 μM of each primer, 

0.2 mM each dNTP, and 0.02 units of KOD Hot Start DNA polymerase (Novagen). The 

amplification of the product was performed under the following cycling conditions: 94°C 

for 2 minutes; 35 cycles at 94°C for 55 seconds, 68°C for 60 seconds, 72°C for 5 

minutes; one cycle at 72°C for 10 minutes. Aliquots of each PCR product were separated 

on 0.8% (w/v) agarose E-gel® (Invitrogen) and visualized using the UV transilluminator 

to verify that the products were of the expected size prior to sequencing. To remove 

excess primer following 

PCR amplification, the PCR product containing the conventional soybean template was 

treated with a mixture of 0.1 unit EXO and 0.1 unit SAP per 5μl of PCR product and 

cycled as follows: one cycle at 37°C for 15 minutes, one cycle at 80°C for 15 minutes. 

The PCR products were sequenced using multiple primers, including primers used for 

PCR amplification. In addition, primers internal to the amplified PCR sequences were 

used to sequence the regions of the amplicon. All sequencing was performed by the 

Monsanto Genomics Sequencing Center using BigDye terminator chemistry (ABI). 

 
Monsanto Company 09-SY-201U Page 207 of 471

Figure B-1. Overlapping PCR Analysis across the Insert in MON 87705 

PCR analyses demonstrating the linkage of the individual genetic elements within the insert in MON 87705 

were performed on MON 87705 genomic DNA extracted from leaf (Lanes 3, 6, 10, 14, 17, and 20). Lanes 

2, 5, 9, 13, 16, and 19 contain reactions with conventional soybean DNA while lanes 4, 8, 12, 15, 18, and 

21 are reactions containing no template DNA. Lanes 7 and 11 contain reactions with PV-GMPQ/HT4404 

plasmid control DNA. Lanes 1and 22 contain Fermentas GeneRuler 1 kb Plus DNA Ladder. Lanes are 

marked to show which product has been loaded and is visualized on the agarose gel. The expected product 

size for each amplicon is provided in the illustration of the insert in MON 87705 that appears near the 

bottom of the figure. Three to fifteen microliters of each of the PCR products was loaded on the gel. This 

figure is representative of the data generated in the study; however the PCR amplicons reported in this 

figure were not necessarily used in sequencing. 

Lane 1: GeneRulerTM 1 kb Plus DNA Ladder 12: No template DNA control 

2: Conventional soybean DNA 13: Conventional soybean DNA 

 3: MON 87705 genomic DNA 14: MON 87705 genomic DNA 

 4: No template DNA control 15: No template DNA control 

5: Conventional soybean DNA 16: Conventional soybean DNA 

6: MON 87705 genomic DNA 17: MON 87705 genomic DNA 

7: PV-GMPQ/HT4404 control DNA 18: No template DNA control 

8: No template DNA control 19: Conventional soybean DNA 

9: Conventional soybean DNA 20: MON 87705 genomic DNA 

10: MON 87705 genomic DNA 21: No template DNA control 

11: PV-GMPQ/HT4404 control DNA 22: GeneRuler 1 kb Plus DNA Ladder 

 Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium stained gel. 

* Symbol denotes partial sequences from Left and Right Border after integration into MON 87705. 
Monsanto Company 09-SY-201U Page 208 of 471

Appendix C. Materials, Methods and Results for Characterization of CP4 EPSPS 

Protein Produced in MON 87705 

C.1 Materials 

The MON 87705-produced CP4 EPSPS protein (Orion lot 10002253) was purified as 

described below from harvested seed of MON 87705 prior to the initiation of this study. 

The identity of the harvested seed containing MON 87705 was confirmed by eventspecific PCR; a copy of the Certificate of Analysis (COA) for this seed lot is archived in 

the Monsanto Regulatory archives with the records documenting protein isolation. The 

purified MON 87705-produced protein was stored in a -80 oC freezer in a buffer solution 

containing 50 mM Tris-HCl, pH 7.5, 50mM KCl, 2mM DTT, 1mM benzamidine-HCl 

and 25% glycerol. The records describing the purification of this MON 87705-produced 

protein are archived under the Orion lot 10002253. 

The E. coli-produced CP4 EPSPS reference protein (Orion lot 10000739) was purified 

from the fermentation of E. coli transformed with a plasmid containing the cp4 epsps

gene. The DNA sequence encoding this CP4 EPSPS reference protein was confirmed 

both prior to and following fermentation of E. coli. Records pertaining to the purification 

of this E. coli-produced reference protein are archived under Orion lot 10000739. The E. 

coli-produced CP4 EPSPS protein reference standard was previously characterized (APS 

Characterization Plan 20-100015) and a copy of the COA is included in the Monsanto 

archives. The E. coli-produced CP4 EPSPS protein was stored in a -80 °C freezer in a 

buffer solution (50 mM Tris-Cl, 1 mM benzamidine-HCl, 50 mM KCl, 2 mM DTT, and 

25% (v:v) glycerol, pH 7.5) at a total protein concentration of 3.8 mg/ml with a purity of 

97%. 

The E. coli-produced CP4 EPSPS protein was used as a reference protein for the 

immunoblot assay, the functional activity assay, and the purity and molecular weight 

evaluation, and as a negative control in the glycosylation analysis. 

C.2 Description of Assay Controls 

Protein molecular weight standards (BioRad, Hercules, CA) were used to calibrate SDSPAGE gels and verify protein transfer to PVDF membranes. A peptide mixture 

(CalMix2 from the Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster 

City, CA) was used to calibrate the MALDI-TOF mass spectrometer for tryptic mass 

analysis. A PTH-amino acid standard mixture (Applied Biosystems) was used to 

calibrate the sequencer for N-terminal sequence analysis. Dilutions of BSA standard 

(BioRad, Hercules, CA) were used to generate a standard curve for determining total 

protein concentration. Transferrin and horseradish peroxidase (both from Sigma, St. 

Louis, MO) were used as positive controls for glycosylation analysis. CandyCane 

Glycoprotein Molecular Weight Standards (Molecular Probes, Eugene, OR) were used as 

molecular weight markers and positive and negative controls for glycosylation analysis. 

C.3 Protein Purification 

The plant-produced CP4 EPSPS protein was purified from seed of MON 87705 prior to 

initiation of this characterization plan. The purification procedure was not performed 

under a GLP plan; however, all procedures were documented on worksheets and, where 
Monsanto Company 09-SY-201U Page 209 of 471

applicable, SOPs were followed. The CP4 EPSPS protein was purified from an extract of 

ground seed using a combination of ammonium sulfate fractionation, hydrophobic 

interaction chromatography, anion exchange chromatography, and cellulose phosphate 

affinity chromatography. All protein extraction and chromatography steps were 

performed at ~4°C. A detailed description of the purification process was filed under 

Orion Lot 10002253, and is briefly described below. 

Approximately 100 g of pre-chilled seed of MON 87705 were ground using a Perten 

Laboratory Mill 3100. The ground powder (~ 100 g) was defatted 3 times with 500 ml 

each of Hexanes (w:v, EMD, Gibbstown, NJ) prewarmed to 37 oC, air-dried, and stored 

in a -80 oC freezer prior to extraction of the CP4 EPSPS protein. The portion of the 

defatted seed powder (50 g) was mixed with an extraction buffer (100 mM Tris-HCl, pH 

7.5, 2 mM EDTA, 2 mM Benzamidine-HCl, 4 mM DTT, 2 mM Phenylmethylsulfonyl 

fluoride, 1% Polyvinylpolypyrrolidone and 10% glycerol) for 2 hours at approximately a 

1:10 powder weight to extraction buffer volume ratio. The slurry was centrifuged at 

23,500 × g for 20 minutes at ~ 4 oC. The resultant 430 ml supernatant was subjected to 

40% ammonium sulfate protein fractionation by addition of 97 g of ammonium sulfate 

over one hour in the cold room (2 oC to 8 oC). The solution was stirred for two hours at 

~ 4 oC and centrifuged at 23,500 X g for 20 minutes. Another 89.8 g of ammonium 

sulfate was added to the supernatant (480 ml) over a period of one hour to 70% 

saturation. The solution was stirred for two hours in a 4 oC cold room and the pellet was 

collected by centrifugation at 23,500 X g for 30 min. The pellet was resuspended in 150 

ml of buffer designed as PS(A) [50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10% glycerol 

(v:v), 1.5 M (NH4)2SO4]. The resuspended sample was loaded onto a 140 ml column of 

Phenyl Sepharose Fast Flow (5 cm X 7 cm column) (GE Healthcare, Piscataway, NJ) 

equilibrated with the buffer PS(A). Proteins were eluted with a linear salt gradient that 

decreased from 1.5 to 0 M (NH4)2SO4 in the buffer PS(A) over a volume of 1400 ml. 

Fractions containing the CP4 EPSPS protein, identified based on western blot analysis, 

were pooled to a final volume of ~250 ml. The pooled sample was desalted by dialysis 

against 4 L of buffer designed as QS(A) (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM 

benzamidine-HCl, 4 mM DTT) for 20 h at ~4 °C (with three additional buffer changes) 

using dialysis tubing [Molecular Weight Cutoff (MWCO), 12 to 14 kDa] (Spectrum 

Laboratories, Inc. Rancho Dominguez, CA). 

The desalted sample (337 ml) was loaded onto a 60 ml column of Q Sepharose Fast Flow 

anion exchange resin column (2 cm X 20 cm) (GE Healthcare, Piscataway, NJ ), which 

was equilibrated with the QS(A) buffer. Bound CP4 EPSPS protein was eluted with a 

linear salt gradient that increased from 0 M to 0.4 M potassium chloride in the QS(A) 

buffer over 600 ml. Fractions containing CP4 EPSPS protein, identified by western blot 

analysis, were pooled to a final volume of ~ 140 ml. The pooled sample was place into 

dialysis tubing (MWCO, 12 to 14 kDa, Spectrum Laboratories, Inc. Rancho Dominguez, 

CA) and dialyzed against buffer designed as CP(A) [50 mM MES, pH 5.8, 10% glycerol 

(v:v), 1 mM benzamidine-HCl and 1 mM DTT] for 18 hours at ~4 °C in three buffer 

changes. 

The dialyzed sample (100 ml) was then loaded onto a 19 ml cellulose phosphate P11 

cation exchange column (1.6 X 9.5 cm) pre-equilibrated with the CP(A) buffer. Bound 

CP4 EPSPS protein was eluted with the CP(A) buffer containing 0.5 mM 
Monsanto Company 09-SY-201U Page 210 of 471

phosphoenolpyruvate (PEP) and 0.5 mM S3P. Fractions containing CP4 EPSPS protein, 

based on SDS PAGE analysis, were pooled (~14.5 ml). This pooled sample was 

concentrated to 10 ml at ~4 °C using a slide-A-lyzer dialysis cassette (MWCO: 10 kDa, 

size: 3 to 12 ml, Pierce, Rockford, IL) and covering it in a water absorbing polymer 

powder (Aquacide I, EMD, Gibbstown, NJ). After concentration, the cassette was placed 

into 2 L of dialysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM DTT, 1 mM 

benzamidine-HCl) and dialyzed for a total of 20 hours at ~ 4 °C in three 2 L buffer 

changes. The dialyzed sample in the cassette was further concentrated to 5 ml using 

Aquacide I as described above. This 5 ml sample was mixed with 5 ml dialysis buffer 

containing 50% glycerol to final volume of 10 ml. Final buffer composition of the 

sample was 50 mM Tris-HCl, pH 7.5, 50mM KCl, 2mM DTT, 1 mM benzamidine-HCl 

and 25% glycerol. This CP4 EPSPS protein purified from seed of MON 87705 was 

aliquoted (100 μl each), assigned APS lot 10002253 and stored at ~ -80 °C. 

C.4 Molecular Weight and Purity Estimation-SDS-PAGE 

SDS-PAGE analysis was performed to determine the molecular weight of CP4 EPSPS 

protein purified from MON 87705 and to compare the molecular weight of the 

MON 87705- produced and E coli-produced CP4 EPSPS proteins. 

An aliquot of the test substance was mixed with 5 X sample loading buffer (LB) 

[312 mM Tris-HCl, 20% (v:v) 2-mercaptoethanol, 10% (w:v) SDS, 0.025% (w:v) 

bromophenol blue, 50% (v:v) glycerol, pH 6.8)], to a final total protein concentration of 

0.168 μg/μl. Molecular weight markers (Bio-Rad broad-range) and reference substance 

were diluted to a final total protein concentration of 0.9 and 0.2 μg/μl, respectively. The 

test substance was analyzed in duplicate at 1, 2, and 3 μg protein per lane. The E. coliproduced CP4 EPSPS protein reference standard (Orion lot 10000739) was analyzed at 1 

μg total protein. All samples were heated at ~ 100 °C for three minutes and loaded onto a 

pre-cast Tris glycine 4 to 20% polyacrylamide gradient 10-well mini-gel (Invitrogen, 

Carlsbad, CA). Electrophoresis was performed at a constant voltage of 150 V for 95 

minutes. Proteins were fixed by placing the gel in a solution of 40% (v:v) methanol and 

7% (v:v) acetic acid for 30 minutes, stained for 18 hours and 35 minutes with Brilliant 

Blue G-Colloidal stain (Sigma-Aldrich, St. Louis, MO), destained 30 seconds with a 

solution containing 10% (v:v) acetic acid and 25% (v:v) methanol, and finally destained 

with 25% (v:v) methanol for 6.5 hours. Analysis of the gel was performed using a BioRad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, 

Hercules, CA). Molecular weight markers were used to estimate the apparent molecular 

weight of each observed band. All visible bands within each lane were quantified using 

Quantity One software. Apparent molecular weight and purity were reported as an 

average of all six loadings containing the MON 87705-produced CP4 EPSPS protein. 

C.5 Immunoblot Analysis-Immunoreactivity 

Immunoblot analysis was performed to confirm the identity of the CP4 EPSPS protein 

purified from MON 87705 and to compare the immunoreactivity of the MON 87705- 

produced and E coli-produced CP4 EPSPS proteins. 

The MON 87705- produced and E. coli-produced CP4 EPSPS proteins were both loaded 

onto the same gel at equal loads of 1, 2, and 3 ng per well. Aliquots of each protein were 

diluted in water and 5 X LB heated at 100.5 °C for 3 min, and applied to a pre-cast Tris 
Monsanto Company 09-SY-201U Page 211 of 471

glycine 4 to 20% polyacrylamide gradient 15-well gel (Invitrogen, Carlsbad, CA). Three 

amounts of each protein were loaded in duplicate on the gel. Electrophoresis was 

performed at a constant voltage of 150 V for 88 minutes. Pre-stained molecular weight 

markers (SeeBlue Plus2 Prestained, Invitrogen, Carlsbad, CA) were loaded in parallel to 

verify electrotransfer of the proteins to the membrane and estimate the size of the 

immunoreactive bands observed. Electrotransfer to a 0.45 μm nitrocellulose membrane 

(Invitrogen, Carlsbad, CA) was performed for 90 minutes at a constant voltage of 25 V.

For immunodetection, the membrane was blocked for 1 hour with 5% (w:v) Non-Fat 

Dried Milk (NFDM) in 1 X Phosphate Buffered Saline containing 0.05% (v:v) Tween-20 

(PBST). The membrane was then probed with a 1:1,000 dilution of goat anti-CP4 EPSPS 

antibody (lot 10000787, aliquot # 20) in 5% (w:v) NFDM in PBST for one hour. Excess 

antibody was removed using three 10 minutes washes with PBST. Finally, the membrane 

was probed with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (Thermo, 

Rockford, IL) at a dilution of 1:10,000 in 5% (w:v) NFDM in PBST for one hour. Excess 

HRP-conjugate was removed using three 10 minutes washes with PBST. All incubations 

were performed at room temperature. Immunoreactive bands were visualized using the 

ECL detection system (GE, Healthcare, Piscataway, NJ) and exposed (10, 30, and 60 s) 

to Amersham Hyperfilm (GE, Healthcare, Piscataway, NJ). Films were developed using 

a Konica SRX-101A automated film processor (Tokyo, Japan).

The immunoreactive bands of the MON 87705-produced CP4 EPSPS protein in each lane 

migrating to the same position as the reference standard were quantified and compared to 

the signals corresponding to the E. coli CP4 EPSPS protein reference substance. 

Quantification of the blot was performed using a Bio-Rad GS-800 densitometer with the 

supplied Quantity One software (version 4.4.0, Hercules, CA) using the lane finding and 

contour tool. The raw data was exported to a Microsoft Excel [2007 (12.0.6324.5001) 

SP1 MSO (12.0.6320.5000] file for the pair wise comparison of all the loads. An average 

absolute difference was calculated for each comparison to determine the 

immunoreactivity equivalence. 

C.6 MALDI-TOF Tryptic Mass Map Analysis 

MALDI-TOF MS was used to confirm the identity of the MON 87705-produced CP4 

EPSPS protein. Since the protein was determined to be pure (100%) based on pre-study 

data, it was not deemed necessary to separate the protein by SDS-PAGE prior to 

trypsinization. 

An ethanol precipitation was performed to concentrate the MON 87705-produced CP4 

EPSPS protein sample and remove any buffer components that may interfere with the 

analysis. Twenty five μl of the MON 87705-produced CP4 EPSPS sample (0.21 mg/ml) 

was concentrated to approximately 20 μl with a Speed-Vac concentrator and then mixed 

with 200 μl prechilled 95% ethanol. After overnight incubation at -20 °C, the mixture 

was centrifuged at 13,000 X g for 30 minutes at ~4 °C. The pellet was collected, washed 

twice with 200 μl of prechilled acetone and then twice with 200 μl of water. Ten μl of 

trypsin solution [20 μg/ml trypsin (Promega, Madison, WI) in a 25 mM ammonium 

bicarbonate buffer, pH 7.8] was added and incubated overnight at 37 °C. Trypsin 

digested samples (0.3 μl) were added directly onto the analysis plate in triplicate and 

followed by the addition of ~ 0.75 μl of three matrices, 2,5-dihydroxybenzoic acid 
Monsanto Company 09-SY-201U Page 212 of 471

(DHB), α-cyano-4-hydroxycinnamic acid (α-Cyano) , and Sinapinic acid (Waters Corp., 

Milford, MA) on separate spots. The sample in DHB matrix was analyzed in the 300 to 

7500 dalton range using 100 shots at a laser intensity setting of 2480 (a unit-less MALDITOF instrument specific value) while samples in α-Cyano and Sinapinic acid were 

analyzed in the 500 to 7500 dalton range using 100 shots at a laser intensity setting of 

1980 and 2380, respectively. Protonated (MH+) peptide masses were observed 

monoisotopically in reflector mode (Aebersold, 1993; Billeci and Stults, 1993). Calmix 2 

was used as the external calibrant (Sequazyme Peptide Mass Standards kit, Applied 

Biosystems, Foster City, CA) for the analysis. GPMAW32 software (Applied 

Biosystems, version 4.23) was used to generate a theoretical trypsin digest of the CP4 

EPSPS protein sequence based upon the nucleotide sequence. Masses were calculated 

for each theoretical peptide and compared to the raw mass data. Experimental masses 

(MH+) were assigned to peaks in the 300 to 7500 Da range if they met the following 

criteria: resolved monoisotopic peak; with at least one additional associated ion peak for 

masses < 1000 Da and at least two associated ion peaks for masses > 1000 Da; peak 

height greater than twice the baseline noise; and did not overlap with a stronger mass 

signal (±2 daltons from the mass analyzed). Known autocatalytic segments from trypsin 

digestion were identified in the raw data. The list of experimental masses was then 

compared to the theoretical list from the GPMAW software. Those experimental masses 

within one Da of a theoretical mass were matched. All matching masses were tallied and 

a coverage map was generated. The tryptic mass map coverage was considered 

acceptable if ≥ 40 % of the protein sequence was identified by matching experimental 

masses observed for the tryptic peptide segments to the expected masses for the 

segments. 

C.7 MALDI-TOF Mass Analysis of MON 87705-produced CP4 EPSPS Protein 

MALDI-TOF mass spectrometry was used to confirm the molecular weight of the 

MON 87705-produced CP4 EPSPS protein. Since the protein was determined to be pure 

(100%) prior to this analysis, it was not deemed necessary to separate the protein by 

SDS-PAGE.

MALDI-TOF MS was used to further characterize the molecular weight of the 

MON 87705-produced CP4 EPSPS protein. Prior to analysis, the MON 87705-produced 

CP4 EPSPS protein was dialyzed using drop dialysis (Görisch, 1988). Briefly, a 25 mm 

Millipore microdialysis disk (type VSWP, 0.025 μm pore size, Bedford, MA) was floated 

on HPLC-grade water, spotted with 2 μl of the sample, and dialyzed for 45 minutes. A 

portion of the MON 87705-produced CP4 EPSPS and BSA protein samples (0.125 and 

0.25 μl) was spotted on an analysis plate, mixed with 0.375 and 0.75 μl of 3,5-

dimethoxy-4-hydroxycinnamic acid (Sinapinic acid) solution, respectively, and air-dried. 

Mass spectral analysis of the MON 87705-produced CP4 EPSPS protein was performed 

using an Applied Biosystems Voyager DE-Pro Biospectrometry Workstation MALDITOF instrument with the supplied Data Explorer software (version 4.0, Foster City, CA). 

Mass calibration of the instrument was performed using a BSA protein standard. The 

sample was analyzed in the 10,000 to 100,000 dalton range using 100 shots at a laser 

intensity setting of 2983 (a unit-less MALDI-TOF instrument specific value). Average 

protonated (MH+) protein masses were observed in linear mode (Aebersold, 1993; Billeci 

and Stults, 1993). GPMAW32 software (Applied Biosystems, version 4.23, Foster City, 
Monsanto Company 09-SY-201U Page 213 of 471

CA) was used to generate a theoretical mass of the expected CP4 EPSPS protein 

sequence based upon the nucleotide sequence. The mass of the MON 87705-produced 

CP4 EPSPS protein was reported as an average of three separate mass spectral 

acquisitions. 

C.8 N-Terminal Sequencing 

N-terminal sequencing using automated Edman degradation chemistry was used to 

confirm the identity of the MON 87705-produced CP4 EPSPS protein. 

Because the protein was determined to be 100% pure based on pre-study data, it was not 

necessary to separate the protein by SDS-PAGE. Therefore, an aliquot of the 

MON 87705-produced CP4 EPSPS protein was used for N-terminal sequence analysis. 

The analysis was performed for 15 cycles using automated Edman degradation chemistry 

(Hunkapillar et al., 1983). An Applied Biosystems 494 Procise Sequencing System with 

140C Microgradient system and 785 Programmable Absorbance Detector and Procise 

Control Software (version 1.1a) were used. Chromatographic data were collected using 

Atlas99 software (version 3.59a, LabSystems, Altrincham, Cheshire, England). A PTHamino acid standard mixture (Applied Biosystems, Foster City, CA) was used to 

chromatographically calibrate the instrument for each analysis. This mixture served to 

verify system suitability criteria such as percent peak resolution and relative amino acid 

chromatographic retention times. A control protein (10 picomoles of β-lactoglobulin, 

Applied Biosystems, Foster City, CA) was analyzed before and after the analysis of the 

CP4 EPSPS protein to verify that the sequencer met performance criteria for repetitive 

yield and sequence identity. Identity was deemed to be established if ≥8 amino acids, 

consistent with the predicted sequence of the N-terminus of the MON 87705-produced 

CP4 EPSPS protein were observed during analysis. 

C.9 Glycosylation Analysis 

Glycosylation analysis was performed to determine whether the MON 87705-produced 

CP4 EPSPS protein was post-translationally modified with covalently bound 

carbohydrate moieties. Aliquots of the MON 87705-produced CP4 EPSPS protein, the 

E.-coli-produced CP4 EPSPS reference standard protein, and the positive controls, 

transferrin (~ 76 – 81kDa, Sigma-Aldrich, St. Louis, MO), and horseradish peroxidase (~ 

40 kDa, Pierce, Rockford, IL) were each diluted with water and mixed with 5× LB. 

These samples were heated at 97.6 °C for 5 minutes, cooled, and loaded on a Tris glycine 

4-20% polyacrylamide gradient 10-well mini-gel. Each sample was loaded at 50 and 100 

ng per lane. SeeBlue Plus2 pre-stained protein molecular weight markers (Invitrogen, 

Carlsbad, CA) were loaded to verify electrotransfer of the proteins to the membrane and 

the CandyCane Glycoprotein Molecular Weight Standards (Molecular Probes, Eugene, 

OR) were loaded as positive/negative controls and markers for molecular weight. 

Electrophoresis was performed at a constant voltage of 150 V for 80 minutes. 

Electrotransfer to a 0.45 μm PVDF membrane (Invitrogen, Carlsbad, CA) was performed 

for 90 minutes at a constant voltage of 25 V.

Carbohydrate detection was performed directly on the PVDF membrane using the Pro-Q 

Emerald 488 Glycoprotein Gel and Blot Stain Kit (Molecular Probes). The 

manufacturer’s protocol was followed. All steps were performed at room temperature. 

The PVDF membrane was fixed in 25 ml of a solution containing 50% methanol and 5% 
Monsanto Company 09-SY-201U Page 214 of 471

glacial acetic acid for one hour, the solution was then changed and the membrane was 

incubated overnight. Two, 15 minutes washes (50 ml each) with 3% (v:v) glacial acetic 

acid (wash solution) were followed by a 20 minutes oxidation in 25 ml of an oxidizing 

solution containing periodic acid (Component C from kit). The membrane was washed 

three times, 10 minutes each, in 50 ml of wash solution. The membrane was then 

incubated in 25 ml of Pro-Q Emerald Staining Solution that was prepared using the kit 

reagents. After 40 minutes of staining in the dark, one 15 minutes, 50 ml wash cycle was 

followed by two 30 minutes, 50 ml wash cycles. The final wash cycles included two 50 

ml, one minute deionized water washes followed by three five minutes methanol 

washes(EMD, San Diego, CA). The blot was then scanned using the BioRad Molecular 

Imager FX using the Alexa 488 illumination setting (Quantity One software; version 4.6, 

build 036) in order to visualize the fluorescently labeled glycosylated proteins. 

After glycosylation analysis the blot was stained to visualize the proteins present on the 

membrane. Proteins were stained for one minute using Coomassie Brilliant Blue R-250 

staining solution (Bio-Rad, Hercules, CA) and then destained with 1× destain solution 

(Bio-Rad, Hercules, CA) for 5 min. After washing with water, the blot was scanned 

using Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 

4.4.0, Hercules, CA) in order to visualize total proteins. 

C.10 Functional Activity Assay 

In order to assess the functional activity of the MON 87705-produced CP4 EPSPS 

protein and to compare its activity to the E. coli-produced CP4 EPSPS reference standard 

protein, aliquots of the MON 87705-produced CP4 EPSPS protein and E. coli-produced 

CP4 EPSPS reference standard protein were analyzed for their ability to release inorganic 

phosphate from phosphoenolpyruvate (PEP). The specific activity is expressed in units 

per mg of protein (U/mg), where a unit (U) is defined as 1 μmole of inorganic phosphate 

released from PEP per minute at 25 °C. 

The assay was carried out on a micro titer plate. Prior to functional activity analysis, both 

test and reference proteins were diluted to a purity corrected concentration of 50 μg/ml 

with a 50 mM HEPES, pH 7.0 buffer. Assays for both proteins were conducted in 

triplicate. Each assay replicate was subsequently analyzed spectrophotometrically in 

duplicate. Briefly, the reactions containing the CP4 EPSPS enzyme with S3P were 

initiated by the addition of PEP. The reactions were performed in a mixture of 50 mM 

HEPES (pH 7.0), 0.1 mM ammonium molybdate, 2 mM S3P, 1 mM PEP and 5 mM 

potassium fluoride for two minutes at 25.2 °C. The reactions were quenched with 

malachite green (phosphate assay reagent) and fixed after two minutes with 33% (w:v) 

sodium citrate. The release of inorganic phosphate from PEP was determined at a 

wavelength of 660 nm using a PowerWave Xi (Bio-Tek, Richmond, VA) microplate 

reader, and quantitated relative to a standard curve of inorganic phosphate treated with 

the malachite green (phosphate assay) reagent and 33% (w:v) sodium citrate.

C.11 Results of CP4 EPSPS Protein Molecular Weight Equivalence 

The equivalence in apparent molecular weight of the purified MON 87705-produced and 

the E. coli-produced CP4 EPSPS protein was demonstrated using SDS-PAGE (Figure 

C-1). The MON 87705-produced CP4 EPSPS protein migrated with a molecular weight 
Monsanto Company 09-SY-201U Page 215 of 471

indistinguishable to that of the E. coli-produced protein standard analyzed concurrently 

(Table C-1). Based on comparable electrophoretic mobilities, the MON 87705-produced 

and E.coli-produced CP4 EPSPS proteins were determined to have equivalent apparent 

molecular weights. 

The predicted mass of the MON 87705-produced CP4 EPSPS protein was also confirmed 

by MALDI-TOF MS. The average mass obtained for CP4 EPSPS was 47,396 Da. This 

experimentally obtained mass differs from the theoretical mass calculated for the CP4 

EPSPS reference standard protein minus the N-terminal methionine by only 0.18%. The 

difference between the expected and the observed mass for MON 87705-produced CP4 

EPSPS is minimal and within the acceptable error for MALDI-TOF mass determination. 

The absence of the N-terminal methionine was confirmed by N-terminal sequencing. 

Table C-1. Molecular Weight Difference Between the MON 87705- and E. coli -

produced CP4 EPSPS Proteins. 

Molecular Weight 

of MON 87705-Produced 

CP4 EPSPS Protein1

Molecular Weight of E. coliproduced CP4 EPSPS Protein2

% Difference from E. coli -

produced CP4 EPSPS 

Protein3

44.6 kDa 43.8 kDa 1.8% 

1 Reference Table C-1 for the molecular weight of the full-length MON 87705-produced protein. 

2

 Reference the Orion 10000739 COA for the molecular weight of the full-length E. coli -

produced reference protein. 

3 Percent difference was calculated as follows: 100 % 1.8%

44.6

44.6 43.8

× = −

 
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Figure C-1. Molecular Weight and Purity Analysis of the MON 87705-Produced 

CP4 EPSPS Protein 

Aliquots of the MON 87705- produced and the E. coli-produced CP4 EPSPS proteins 

were separated on a 4 to 20% Tris glycine polyacrylamide gradient gel and then stained 

with Brilliant Blue G-Colloidal stain. Approximate MWs (kDa) are shown on the left 

and correspond to the markers loaded in Lanes 1 and 9. 

Lane Sample Amount loaded (μg) 

1 Broad Range molecular weight markers 4.5

2 E. coli -produced CP4 EPSPS protein 

standard

1

3 MON 87705-produced CP4 EPSPS protein 1

4 MON 87705-produced CP4 EPSPS protein 1

5 MON 87705-produced CP4 EPSPS protein 2 

6 MON 87705-produced CP4 EPSPS protein 2 

7 MON 87705-produced CP4 EPSPS protein 3 

8 MON 87705-produced CP4 EPSPS protein 3 

9 Broad Range molecular weight markers 4.5 

 

200

97.4

66.2

45

31

21.5

6.5

14.4

116.3

kDa

Lane # 1 2 3 4 5 6 7 8 9 
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C.12 Results of CP4 EPSPS Protein Immunoreactivity Equivalence 

A western blot analysis using goat anti-CP4 EPSPS serum was conducted to determine 

the relative immunoreactivity of the MON 87705-produced CP4 EPSPS protein and the 

E. coli-produced CP4 EPSPS protein reference standard. The results demonstrated that 

the anti-CP4 EPSPS antibody recognized the MON 87705-produced CP4 EPSPS protein 

that migrated to a similar position as the E. coli-produced reference standard protein 

(Figure C-2). Furthermore, the immunoreactive signal increased with increasing levels of 

CP4 EPSPS protein loaded. The observed immunoreactivities between the MON 87705-

produced and E. coli-produced proteins were similar based on densitometric analysis of 

the western blot (Table C-2). Based on the above analysis, the MON 87705-produced 

and E. coli-produced CP4 EPSPS proteins demonstrated equivalent immunoreactive 

properties, which confirmed the identity and equivalence of the two proteins. 

 
Monsanto Company 09-SY-201U Page 218 of 471

Figure C-2. Western Blot Analysis of MON 87705-produced and E. coli -produced 

CP4 EPSPS Proteins 

Aliquots of the purified, MON 87705-produced and E. coli-produced CP4 EPSPS 

proteins were separated by SDS-PAGE, and electrotransferred to a PVDF membrane. 

The membrane was probed with goat anti-CP4 EPSPS antibodies and developed using an 

ECL system (GE Healthcare). Approximate molecular weights (kDa) of markers loaded 

in Lane 1 are shown on the left side of the blot. The 30 second exposure is shown. *: 

Non-assigned molecular weight in marker.

Lane Sample 

Amount 

Loaded 

(ng) 

1 See Blue® Plus2 Pre-Stained molecular weight markers - 

2 E. coli -produced CP4 EPSPS reference standard 1 

3 E. coli -produced CP4 EPSPS reference standard 1 

4 E. coli- produced CP4 EPSPS reference standard 2 

5 E. coli -produced CP4 EPSPS reference standard 2 

6 E. coli -produced CP4 EPSPS reference standard 3 

7 E. coli -produced CP4 EPSPS reference standard 3 

8 Empty 

9 MON 87705-produced CP4 EPSPS protein 1 

10 MON 87705-produced CP4 EPSPS protein 1 

11 MON 87705-produced CP4 EPSPS protein 2 

12 MON 87705-produced CP4 EPSPS protein 2 

13 MON 87705-produced CP4 EPSPS protein 3 

14 MON 87705-produced CP4 EPSPS protein 3 

 

250

148

98

64

50

34

17

16

7

4

*

Lane # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

kDa

CP4 EPSPS 
Monsanto Company 09-SY-201U Page 219 of 471

Table C-2. Comparison of Immunoreactive Signal between MON 87705-produced 

and E. coli -produced CP4 EPSPS Proteins 

Sample Gel 

lane 

Amount 

(ng) 

Density 

(OD x 

mm2

) 

Average 

Density1

Percent 

Difference2 

(%) 

Average 

Difference3

(%) 

E. coli CP4 EPSPS 2 1 0.994 0.957 9.8 10.5 ± 1.8 

E. coli CP4 EPSPS 3 1 0.920 

MON 87705 CP4 EPSPS 9 1 1.027 

1.062 

MON 87705 CP4 EPSPS 10 1 1.096 

E. coli CP4 EPSPS 4 2 1.904 

2.163 13.0 

E. coli CP4 EPSPS 5 2 2.421 

MON 87705 CP4 EPSPS 11 2 2.584 

2.485 

MON 87705 CP4 EPSPS 12 2 2.386 

E. coli CP4 EPSPS 6 3 3.296 

3.766 8.7 

E. coli CP4 EPSPS 7 3 4.236 

MON 87705 CP4 EPSPS 13 3 4.039 

4.124 

MON 87705 CP4 EPSPS 14 3 4.208 

1

Average Density =∑〖Density〗/2 

2

Percent Difference ሺ%ሻ ൌ |A୴ୣ୰ୟ୥ୣ Dୣ୬ୱ୧୲୷ ୮୪ୟ୬୲ିA୴ୣ୰ୟ୥ୣ Dୣ୬ୱ୧୲୷ E.ୡ୭୪୧|

A୴ୣ୰ୟ୥ୣ Dୣ୬ୱ୧୲୷ ୮୪ୟ୬୲  X 100%                      

3

Average difference (%) =∑〖% difference〗/3. The standard deviation was calculated using 

Microsoft Office Excel 2007 (12.0.6324.5001) SP1 MSO (12.0.6320.5000). 

 
Monsanto Company 09-SY-201U Page 220 of 471

C.13 Results of MALDI-TOF Tryptic Mass Map Analysis 

The MON 87705-produced, CP4 EPSPS protein was assessed by MALDI-TOF MS. 

Prior to analysis, the protein sample was chemically reduced, alkylated, and digested with 

trypsin. The ability to identify a protein using this method is dependent on matching a 

sufficient number of observed mass segments to expected (theoretical) mass segments. 

In general, protein identification made by peptide mapping is considered to be reliable if 

the measured coverage of the sequence is 15% or higher with a minimum of five matched 

peptides. Observed tryptic peptides were considered a match to the expected tryptic mass 

when differences in molecular weight of less than one dalton (Da) were found between 

the observed and predicted segment masses. Such matches were made without 

consideration for potential natural amino acid modifications such as glycosylation. The 

protein sample was digested with trypsin and the masses of the tryptic peptides were 

measured. 

There were 30 unique peptide segments (Table C-3), identified that corresponded to the 

expected masses of the CP4 EPSPS trypsin-digested peptides. The identified masses 

were used to assemble a coverage map indicating the matched peptide sequences for the 

entire CP4 EPSPS protein (Figure C-3), resulting in ~80% (362 out of 455 amino acids) 

coverage of the total protein. This analysis confirmed the identity of the MON 87705-

produced CP4 EPSPS protein. 

 
Monsanto Company 09-SY-201U Page 221 of 471

Table C-3. Summary of the Tryptic Masses Identified for the MON 87705-Produced CP4 EPSPS Using M

Matrix 

Expected 

Mass1

Difference2 AA 

position3

α-Cyano DHB Sinapinic Segment 

acid 

599.31 616.32 629.32 629.32 711.45 835.39 863.46 872.45 872.45 948.52 991.56 

1115.58 1357.73 1359.67 1359.67 1646.86 1705.82 1994.03 2183.24 2367.43 

389.18 416.23 474.20 506.17 599.27 616.29 629.28 629.28 711.42 835.37 863.44 872.43 872.43 948.50 

1115.58 1357.73 1359.69 1359.69 1558.90 1646.89 1705.88 1994.07 2183.30 2367.50 3186.35 3249.77 

616.10 835.29 

1359.54 1359.54 1558.73 1993.82 2183.05 2367.21 2450.13 2450.13 3186.30 3249.46 4188.82 

389.25 0.07 225-227 TIR 

416.30 474.27 506.22 599.33 616.34 629.29 629.34 711.45 835.39 863.46 872.45 872.52 948.52 991.55 

1115.57 1357.71 1359.72 1359.64 1558.83 1646.84 1705.81 

0.07 0.07 0.05 0.02 0.02 0.03 0.02 

0 0 0 0 

0.07 

0 

0.01 0.01 0.02 0.05 0.03 0.07 0.02 0.01 

70-72 

228-231 354-357 

29-33 

128-132 201-205 383-388 133-138 

62-69 15-23 

313-320 358-366 161-168 

14-23 

295-305 146-157 354-366 

34-46 47-61 

389-405 367-382 

IRK 

LEGR 

ESDR 

SISHR 

RPMGR 

DHTEK 

GRPDGK 

VLNPLR 

AMQAMGAR 

SSGLSGTVR 

GVTVPEDR 

LSAVANGLK 

TPTPITYR 

KSSGLSGTVR 

LAGGEDVADLR 

SEDGDRLPVTLR 

ESDRLSAVANGLK 

SFMFGGLASGETR 

ITGLLEGEDVINTGK 

GLGNASGAAVATHLDHR 

LNGVDCDEGETSLVVR 

1993.97 2183.17 2367.33 2450.23 2450.22 3186.52 3249.62 4188.26 

0.06 0.07 0.10 0.10 0.09 0.17 0.15 0.56 

206-224 275-294 178-200 

24-46 

105-127 

73-104 

321-351 234-274 

MLQGFGANLTVETDADGVR 

TGLILTLQEMGADIEVINPR 

SAVLLAGLNTPGITTVIEPIMT

IPGDKSISHRSFMFGGLASGET

LTMGLVGVYDFDSTFIGDASL

EGDTWIIDGVGNGGLLAPEAPLDFGN

APSMIDEYPILAVAAAFAEGATVMN

LTGQVIDVPGDPSSTAFPLVAALLVPGSDV

1Only experimental masses that matched expected masses are listed in the table. 2The numbers represent the difference between the expected mass and the first column which has the corresponding numbers. 3AA position refers to amino acid position within the predicted CP4 EPSPS sequence as depicted in Figure C-3. 
Monsanto Company 09-SY-201U Page 222 of 471

. 

001 MLHGASSRPA TARKSSGLSG TVRIPGDKSI SHRSFMFGGL ASGETRITGL 

051 LEGEDVINTG KAMQAMGARI RKEGDTWIID GVGNGGLLAP EAPLDFGNAA 

101 TGCRLTMGLV GVYDFDSTFI GDASLTKRPM GRVLNPLREM GVQVKSEDGD 

151 RLPVTLRGPK TPTPITYRVP MASAQVKSAV LLAGLNTPGI TTVIEPIMTR 

201 DHTEKMLQGF GANLTVETDA DGVRTIRLEG RGKLTGQVID VPGDPSSTAF 

251 PLVAALLVPG SDVTILNVLM NPTRTGLILT LQEMGADIEV INPRLAGGED 

301 VADLRVRSST LKGVTVPEDR APSMIDEYPI LAVAAAFAEG ATVMNGLEEL 

351 RVKESDRLSA VANGLKLNGV DCDEGETSLV VRGRPDGKGL GNASGAAVAT 

401 HLDHRIAMSF LVMGLVSENP VTVDDATMIA TSFPEFMDLM AGLGAKIELS 

451 DTKAA 

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87705-produced CP4 

EPSPS. 

The amino acid sequence of the CP4 EPSPS protein was deduced from the cp4 epsps

gene present in MON 87705. Boxed regions correspond to tryptic peptides that were 

identified from the MON 87705-produced CP4 EPSPS protein sample using MALDITOF MS. In total, 79.6% (362 of 455 total amino acids) of the expected protein sequence 

was identified. 

 
Monsanto Company 09-SY-201U Page 223 of 471

C.14 Results of N-terminal Sequence Analysis 

N-terminal sequencing of the first 10 amino acids performed on MON 87705-produced 

CP4 EPSPS protein resulted in the sequence expected for the CP4 EPSPS protein (Table 

C-4) with the exception of the N-terminal methionine, which was not detected. This 

result is expected as removal of the N-terminal methionine, catalyzed by methionine 

aminopeptidase, is a common modification that occurs co-translationally before 

completion of the nascent protein chain and has no effect on protein structure or activity. 

The N-terminal sequence information, therefore, confirms the identity of the CP4 EPSPS 

protein isolated from MON 87705 and the intactness of its N-terminus. 

Table C-4. N-Terminal Sequence of the MON 87705-produced CP4 EPSPS. 

Amino acid 

residue # from 

the N-terminus → 1 2 3 4 5 6 7 8 9 10 11 

Expected 

Sequence1 → M L H G A S S R P A T 

│ │ │ │ │ │ │ │ │ │ │

Experimental 

Sequence → - L H G A S S R P A T 

The expected amino acid sequence of the N-terminus of the CP4 EPSPS protein was 

deduced from the cp4 epsps gene present in soybean MON 87705. The experimental 

sequence obtained from the MON 87705-produced CP4 EPSPS was compared to the 

expected sequence. 1 The single letter IUPAC-IUB amino acid code is M, methionine; L, 

Leucine; H, histidine; G, glycine; A, alanine, S, serine; R, Arginine; P, proline; T, threonine; 

and (-) Indicates the M residue not observed.

C.15 Results of Glycosylation Analysis 

Many eukaryotic proteins are post-translationally modified with carbohydrate moieties 

(Rademacher et al., 1988). These carbohydrate moieties may be complex, branched 

polysaccharide structures or simple monosaccharides. In contrast, strains of E. coli used 

for recombinant protein expression lack the necessary biochemical pathways required for 

protein glycosylation. To assess whether potential post-translational glycosylation of the 

MON 87705-produced CP4 EPSPS protein occurred, the purified protein sample was 

subjected to glycosylation analysis. The E. coli-produced CP4 EPSPS reference standard 

represented a negative control. The positive controls were the transferrin and horseradish 

peroxidase (HRP) proteins which are known to have multiple covalently linked 

carbohydrate modifications. The transferrin protein and HRP, as well as the purified CP4 

EPSPS protein isolated from MON 87705 and E. coli were separated on SDS-PAGE, 
Monsanto Company 09-SY-201U Page 224 of 471

transferred to a PVDF membrane, and glycosylation analysis was performed to detect 

carbohydrate moieties on the proteins. The results of this analysis are shown in Figure C4. The positive controls, transferrin and HRP, were detected at the expected molecular 

weights of ~75 and ~50 kDa, respectively, in a concentration-dependent manner (Figure 

C-4, Panel A, Lanes 4-5 and 2-3). A very faint signal, slightly above background, was 

observed for the MON 87705-produced and E. coli-produced CP4 EPSPS proteins

(Figure C-4, Panel A, Lanes 6-7 and 8-9). This low level signal could be due to low level 

oxidation of amino acid residues of the protein and/or nonspecific binding of florescent 

reagents. Further evidence that the signals observed for MON 87705-produced and 

E. coli-produced CP4 EPSPS proteins are nonspecific are the following: (1) The faint 

signal was also associated with the E. coli-produced CP4 EPSPS could not be the product 

of a glycan moiety because E. coli-produced proteins are not glycosylated. (2) Mass 

spectrometry data demonstrated the absence of glycosylation of the MON 87705-

produced CP4 EPSPS. Glycosylation would result in an increase in the protein mass 

relative to the theoretically calculated mass. No increase in protein mass was observed 

for the MON 87705-produced CP4 EPSPS protein determined by MALDI-TOF MS 

(47396 Da) as compared to its theoretical mass (47481.48 Da) (3) Four potential 

glycosylation sites can be identified in the amino acid sequence of the CP4 EPSPS 

protein: one O linked at T248 and three N-linked at N213, N271 and N392 (see Figure C3 for amino acid positions). The tryptic segments containing these amino acids were 

identified for the MON 87705-produced CP4 EPSPS protein by MALDI-TOF mass 

spectrometry. All identified masses matched the expected non-modified peptide masses, 

indicating that no glycosylation had occurred. 

To confirm that sufficient MON 87705-produced and E. coli-produced CP4 EPSPS 

proteins were present for carbohydrate detection and glycosylation analysis, the 

membrane was stained with Coomassie Blue R250 stain to detect proteins (Figure C-4, 

Panel B). Both MON 87705-produced and E. coli-produced CP4 EPSPS were clearly 

detected on the membrane (Figure C-4, Panel B, Lanes 6-9). 

These results indicate that the MON 87705-produced CP4 EPSPS protein is not 

glycosylated and, thus is equivalent to the E. coli-produced CP4 EPSPS reference 

standard. 
Monsanto Company 09-SY-201U Page 225 of 471

Figure C- 4. Glycosylation Analysis of the MON 87705-Produced CP4 EPSPS 

Protein 

Aliquots of the MON 87705-produced CP4 EPSPS protein, E. coli-produced CP4 EPSPS 

reference standard (negative control), horseradish peroxidase (positive control) and 

transferrin (positive control) were separated by SDS-PAGE (4-20% gradient) and 

electrotransferred to a PVDF membrane. (A) Where present, periodate-oxidized proteinbound carbohydrate moieties reacted with Pro-Q Emerald 488 glycoprotein stain and 

emitted a fluorescent signal at 488 nm (Lanes 1-5). (B) The same blot was stained with 

Coomassie Blue R250. The signal was captured using a Bio-Rad GS-800 scanner. 

Approximate molecular weights (kDa) correspond to the CandyCane glycosylated 

markers loaded in Lane 1 

Lane Sample Amount (ng) 

 1 CandyCane Glycoprotein molecular weight standards ⎯

 2 Horseradish Peroxidase (positive control) 50 

 3 Horseradish Peroxidase (positive control) 100 

 4 Transferrin (positive control) 50 

 5 Transferrin (positive control) 100 

 6 MON 87705-produced CP4 EPSPS 50 

 7 MON 87705-produced CP4 EPSPS 100 

 8 E. coli-produced CP4 EPSPS (negative control) 50 

 9 E. coli-produced CP4 EPSPS (negative control) 100 

 10 See Blue® Plus2 Pre-Stained molecular weight markers ⎯

 

Lane # 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

A B

180

42

82

18

CP4 EPSPS
Monsanto Company 09-SY-201U Page 226 of 471

C.16 Results of Functional Activity 

The functional activities of the MON 87705-produced and E. coli-produced CP4 EPSPS 

proteins were estimated using an assay that measures the EPSPS-catalyzed formation of 

inorganic phosphate (Pi) and 5-enolpyruvylshikimate-3-phosphate (EPSP) from 

Shikimate-3-phosphate (S3P) and Phosphoenolpyruvate (PEP). In this assay protein 

specific activity is expressed as units per milligram of protein (U/mg), where a unit is 

defined as one μmole of inorganic phosphate released from PEP per minute at 25 °C. 

The MON 87705-produced and E. coli-produced CP4 EPSPS proteins were considered 

functionally equivalent if the specific activity of one protein was within 50% (assay 

variability) of the other. 

Results indicated that the specific activity was 4.10 U/mg protein for the MON 87705-

produced CP4 EPSPS, and 4.38 U/mg protein for the E. coli-produced reference standard. 

The difference in specific activities was 6.4% (Table C-5) which is within assay 

variability. These results clearly demonstrate that the CP4 EPSPS proteins derived from 

MON 87705 and E. coli have equivalent functional activities. 

Table C-5. CP4 EPSPS Functional Assay 

The specific activity of the plant-produced CP4 EPSPS protein was determined using a 

phosphate release assay. This end-point type colorimetric assay measures the release of 

inorganic phosphate from one of the substrates, PEP, by the action of the CP4 EPSPS 

enzyme. 

MON 87705-produced

CP4 EPSPS1 

(U/mg) 

E. coli-produced

CP4 EPSPS1 

(U/mg) 

Difference 

(%, Plant vs E. coli)2

4.10 ± 0.1 4.38 ± 0.33 6.4 

1 Value refers to mean and standard deviation calculated based on n= 6 

2

% Difference ൌ ሺAୡ୲୧୴୧୲୷ ா.௖௢௟௜ିAୡ୲୧୴୧୲୷ MON ଼଻଻଴ହሻ

Aୡ୲୧୴୧୲୷ ா.௖௢௟௜

×  100%
Monsanto Company 09-SY-201U Page 227 of 471

Appendix C References 

Aebersold, R. 1993. Mass spectrometry of proteins and peptides in biotechnology. 

Current Opininons in Biotechnology 4:412-419. 

Billeci, T.M., and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrixassisted laser desorption/ionization mass spectrometry. Analytical Chemistry 65:1709-

1716. 

Gorisch, H. 1988. Drop Dialysis: Time Course of Salt and Protein Exchange. Analytical 

Biochemistry 173:393-398. 

Hunkapillar, M.W., R.M. Hewick, W.J. Dreyer, and L.E. Hood. 1983. High-sensitivity 

sequencing with gas-phase sequenator. Methods in Enzymology. 91:399-413. 

Rademacher, T.W., R.B. Parekh, and R.A. Dwek. 1988. Glycobiology. Annual Review in 

Biochemistry 57:785-838. 

 
Monsanto Company 09-SY-201U Page 228 of 471

Appendix D. Materials and Methods Used for the Analysis of the 

Levels of CP4 EPSPS Protein in MON 87705 

D.1 Materials 

Tissue samples analyzed in this study were produced from five field sites in Chile during 

the 2007-2008 planting season from seed lot GLP-0702-18254-S for MON 87705 and 

GLP-0702-18252-S for the conventional control. The conventional control, A3525, does 

not contain the cp4 epsps coding region. Samples were stored in a -80 °C freezer 

throughout the study. An E. coli-produced CP4 EPSPS protein (Monsanto APS lot 20-

100015) was used as a reference standard for the assay. 

D.2 Characterization of the Materials 

The identities of the MON 87705 and conventional control substances were confirmed by 

analysis of the harvested seed DNA by an event-specific polymerase chain reaction (PCR) 

method and the resulting Verifications of Identity (VOIs) were archived in the Monsanto 

Regulatory Archives. The copies of VOIs were also included in the study file. 

D.3 Field Design and Tissue Collection 

Production Plan REG-07-170 was initiated during the 2007-2008 planting season to 

generate MON 87705 and conventional control substances at various soybean-growing 

locations in Chile. The field sites were as follows: Quilapilum, Chacabuco; Melipilla, 

Melipilla; Calera de Tango, Maipo; Rancagua, Cachapoal; and San Fernando, Colchagua. 

These field sites were representative of soybean producing regions suitable for 

commercial soybean production. At each site, three replicated plots of soybean plants 

containing MON 87705 and the conventional control were planted using a randomized 

complete block field design. Throughout the field production sample identity was 

maintained by using unique sample identifiers and proper chain-of-custody 

documentation. All tissue samples, except seed, were stored in a -80 oC freezer and 

shipped on dry ice to the Monsanto processing facility in St. Louis, Missouri. Seed 

samples were stored and shipped at ambient temperature. 

Over-season leaf tissue samples were collected from the youngest set of fully expanded 

trifoliate leaves at the following growth stages: OSL1 at V2-V3 growth stage; OSL2 at 

V7; OSL3 at V10; and OSL4 at V14. The root and forage tissues were collected at 

approximately the R5-R6 growth stage, and the above-ground portion of the plant was 

labelled as the forage, and the below ground portion was washed and labelled as root 

tissue. Harvested seed samples were collected at the R8 growth stage. 

 
Monsanto Company 09-SY-201U Page 229 of 471

D.4 Tissue Processing and Protein Extraction 

All tissue samples produced at the field sites were shipped to Monsanto’s processing 

facility. During the processing step, dry ice was combined with the individual samples, 

and vertical cutters or mixers were used to thoroughly grind and mix the tissues. 

Processed samples were transferred into capped 50 ml tubes and stored in a -80°C freezer 

until use. 

The CP4 EPSPS protein was extracted from soybean tissues as described in Table D-1. 

The CP4 EPSPS protein was extracted from all leaf, forage, root and seed tissues using a 

Harbil Mixer with the appropriate amount of Tris-borate buffer with L-ascorbic acid 

(TBA) [0.1 M Tris, 0.1 M Na2B4O7 · 10H2O, 0.01 M MgCl2, 0.05% (v:v) Tween-20 at 

pH 7.8, and 0.2% (w:v) L-ascorbic acid]. Insoluble material was removed from all tissue 

extracts using a serum filter (Fisher Scientific, Pittsburgh, PA). The extracts were 

aliquotted and stored frozen in a -80°C freezer until ELISA analysis. 

Table D-1. Protein Extraction Methods for Tissue Samples 

Tissue Extraction 

Buffer 

T:B 

Ratio 

No. of 1⁄4 in 

Chrome-steel 

beads 

Shake 

time 

(minutes) 

Sample 

Clarification 

method 

Leaf1 TBA 1:50 8 3.5 Serum Filter 

Root TBA 1:50 8 3.5 Serum Filter 

Forage TBA 1:50 8 3.5 Serum Filter 

Seed TBA 1:50 8 3.5 Serum Filter 

1

Over-season leaf (OSL-1, OSL-2, OSL-3, and OSL-4). 

D.5 CP4 EPSPS Antibodies 

The capture monoclonal antibody (Lot 10002190) was specific for the CP4 EPSPS 

protein. The concentration of the purified antibody was determined to be 2.3 mg/ml by 

spectrophotometric methods. The detection reagent was goat anti-CP4 EPSPS polyclonal 

antibodies (Sigma, St. Louis, MO) conjugated to horseradish peroxidase (HRP). 

D.6 CP4 EPSPS ELISA Method 

The CP4 EPSPS ELISA was performed manually according to an in-house SOP. Mouse 

anti-CP4 EPSPS antibody was diluted in coating buffer (15 mM Na2CO3, 35 mM 

NaHCO3, and 150 mM NaCl, pH 9.6) and immobilized onto 96-well microtiter plates at 

2.0 μg/ml by dispensing 100 μl per well, followed by incubation in a 4°C refrigerator for 

greater than 12 hours. Plates were washed three times with 1X phosphate buffered saline 

(PBS) with 0.05% (v:v) Tween-20 (1X PBST) followed by the addition of 100 μl per 

well of CP4 EPSPS protein standard or sample extract and incubated at 37 °C for one 

hour. Plates were washed three times with 1X PBST, followed by the addition of 100 μl 

per well of goat anti-CP4 EPSPS peroxidase conjugate and incubated at 37 °C for one 

hour. Plates were washed 3 times with 1X PBST, and developed by adding 100 μl per 

well of HRP substrate, 3,3',5,5'- tetramethylbenzidine (TMB, Kirkegaard & Perry, 
Monsanto Company 09-SY-201U Page 230 of 471

Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100 μl per 

well of 6 M H3PO4 after a 10 minute development. Quantitation of CP4 EPSPS protein 

was accomplished by interpolation from a CP4 EPSPS protein standard curve that ranged 

in concentration from 0.456 – 14.6 ng/ml. 

D.7 Moisture Analysis 

All tissues were analyzed for moisture content using an IR-200 Moisture Analyzer 

(Denver Instrument Company, Arvada, CO). A homogeneous tissue-specific site pool 

(TSSP) was prepared using the test and control samples of a given tissue type grown at a 

given site. These pools were prepared for all tissues in this study. The average percent 

moisture for each TSSP was calculated from triplicate analyses. A TSSP Dry Weight 

Conversion Factor (DWCF) was calculated as follows: 

 DWCF = 1 – [Mean % TSSP Moisture / 100] 

The DWCF was used to convert protein levels assessed on a μg/g fresh weight (fwt) basis 

into levels reported on a μg/g dry weight (dwt) basis using the following calculation: 

 

 ( )

( ) DWCF

Protein Level Fresh Weight Protein Level in Dry Weight =

The protein levels that were reported to be less than or equal to the LOD or less than the 

LOQ on a fresh weight basis were not reported on a dry weight basis. 

D.8 Data Analyses 

The CP4 EPSPS ELISA plates were analyzed on a SPECTRAmax Plus (Molecular 

Devices, Sunnyvale, CA) microplate spectrophotometer, using a dual wavelength 

detection method. All protein concentrations were determined by optical absorbance at a 

wavelength of 450 nm with a simultaneous reference reading of 620 nm. Data reduction 

analyses were performed using Molecular Devices SOFTmax PRO GxP version 5.0.1 

software. Absorbance readings and protein standard concentrations were fitted with a 

four-parameter logistic curve fit. Following the interpolation from the standard curve, the 

amount of protein (ng/ml) in the tissue was converted to a “μg/g fwt” basis. For all 

proteins, this conversion utilized a sample dilution factor and a tissue-to-buffer ratio. The 

protein values in “μg/g fwt” were also converted to “μg/g dwt” by applying the DWCF. 

Microsoft Excel 2007 (12.0.6324.5001) SP1 MSO (12.0.6320.5000) (Microsoft, 

Redmond, WA) was used to calculate the CP4 EPSPS protein levels in soybean tissues. 
Monsanto Company 09-SY-201U Page 231 of 471

Appendix E. Materials and Methods Used for Compositional Analysis of 

MON 87705 Soybean Seed, Forage and Processed Fractions 

E.1 Materials 

MON 87705, a conventional soybean control (A3525), and conventional commercial 

reference soybean varieties were grown at five locations in Chile in the 2007-2008 

growing season. MON 87705 and the conventional control were grown from seed lots 

GLP-0702-18254-S and GLP-0702-18252-S, respectively. The conventional control 

material, A3525, has background genetics representative of MON 87705 but does not 

contain FATB1-A and FAD2-1A gene segments. Samples from all three replicates of 

test, control, and reference plots were analyzed with the exception of a single replicate of 

the control substance from site QUI, which was lost. In addition, 20 commercial 

conventional soybean varieties were produced alongside of MON 87705 and 19 of these 

were analyzed for generation of a 99% tolerance interval. One commercial soybean 

variety had all replicates damaged by an early frost and was excluded from the study (see 

Section E.3 below). The 19 varieties, locations, and seed lot numbers are listed below: 

Material Name Seed Lot Number Site Code 

Asgrow A2869 GLP-0707-18806-S SFR 

Stine 2788 GLP-0707-18832-S SFR 

Asgrow A3244 GLP-0707-18807-S SFR 

Hoegemeyer 333 GLP-0707-18823-S SFR 

NK 32Z3 GLP-0707-18827-S CdT 

Stine 3300-0 GLP-0707-18833-S CdT 

Channel Bio 3461 GLP-0707-18816-S CdT 

Stewart 3454 GLP-0707-18831-S CdT 

Croplan 3596STS GLP-0707-18818-S MEL 

Garst 3585N GLP-0707-18820-S MEL 

Pioneer 93B52 GLP-0707-18829-S MEL 

Quality Plus 365C GLP-0707-18830-S MEL 

Stine 3600-0 GLP-0707-18834-S QUI 

Channel Bio 37002 GLP-0707-18817-S QUI 

Lewis 372 GLP-0707-18825-S QUI 

Pioneer 93B82 GLP-0707-18828-S QUI 

Lewis 391 GLP-0707-18826-S RAN 

Stine 3870-0 GLP-0707-18835-S RAN 

Asgrow A4324 GLP-0707-18809-S RAN 

E.2. Characterization of the Materials 

The identities of the forage and seed samples from MON 87705, conventional control, 

and reference soybean varieties were verified prior to their use in the study by confirming 
Monsanto Company 09-SY-201U Page 232 of 471

the chain-of-custody documentation supplied with the forage and seed collected from the 

field plots. The presence or absence of FATB1-A and FAD2-1A gene segments in the 

seed of MON 87705, conventional control and reference soybean varieties was verified 

by event-specific polymerase chain reaction (PCR). As expected, the results confirm that 

MON 87705 contains these gene segments and the conventional control and reference 

varieties do not. 

E.3. Field Production of the Samples 

A total of 20 different conventional commercial soybean reference varieties were planted 

at five field locations with four different varieties grown at each site. Seeds were planted 

in a randomized complete block design with three replicates per block for each test, 

control, and reference variety. Samples from all three replicates of test, control, and 

reference plots were analyzed, with the exception of a single replicate of the control 

variety from site QUI, which was lost in transit from the field location to its destination in 

the U.S. All replicates of the reference variety UA 4805 from site RAN were damaged 

by an early frost and were excluded from this study. All the samples were grown under 

normal agronomic field conditions for their respective geographic regions. 

E.4. Summary of Analytical Methods 

Harvested soybean seed and forage samples from MON 87705, the conventional control 

and conventional reference soybean varieties were shipped on dry ice to Covance 

Laboratories Inc. (Madison, WI) for compositional analyses. Analyses were performed 

using methods that are currently used to evaluate the nutritional quality of food and feed. 

E.4.1. Acid Detergent Fiber 

The sample was placed in a fritted vessel and washed with an acidic boiling detergent 

solution that dissolved the protein, carbohydrate, and ash. An acetone wash removed the 

fats and pigments. The lignocellulose fraction was collected on the frit and determined 

gravimetrically. The limit of quantitation was 0.100%. 

Literature Reference 

USDA. 1970. Forage and Fiber Analyses. Agriculture Handbook No. 379. United States 

Department of Agriculture, Washington, D.C. 

E.4.2. Amino Acid Composition 

The following 18 amino acids were analyzed: 

• Total aspartic acid (including asparagine) 

• Total threonine 

• Total serine 

• Total glutamic acid (including glutamine) 

• Total proline 

• Total glycine 

• Total alanine 
Monsanto Company 09-SY-201U Page 233 of 471

• Total valine 

• Total isoleucine 

• Total leucine 

• Total tyrosine 

• Total phenylalanine 

• Total histidine 

• Total lysine 

• Total arginine 

• Total tryptophan 

• Total methionine - (sulfur containing) 

• Total cystine (including cysteine) - (sulfur containing) 

The sample was assayed by three methods to obtain the full profile. Tryptophan required 

a base hydrolysis with sodium hydroxide. The sulfur-containing amino acids required an 

oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the 

samples for the remaining amino acids was accomplished through direct acid hydrolysis 

with hydrochloric acid. Once hydrolyzed, the individual amino acids were then 

quantitated using an automated amino acid analyzer. The limit of quantitation was 0.100 

mg/g. 

Reference Standards 

• ThermoScientific K18, 2.5 μmol/mL per constituent except cystine (1.25 

μmol/mL), Lot Number JC120602 

• Sigma, L-Tryptophan, 100%, Lot Number 076K50075 

• Sigma/BioChemika, L-Cysteic Acid Monohydrate, >99% (used as 100%), Lot 

Number 1305674 

• Sigma, L-Methionine Sulfone, 100%, Lot Number 012H3349 

Literature Reference 

AOAC. 2005. Method 982.30 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD.

E.4.3. Ash 

The sample was placed in an electric furnace at 550° C and ignited to drive off all volatile 

organic matter. The nonvolatile matter remaining was quantitated gravimetrically and 

calculated to determine percent ash. The limit of quantitation was 0.100%. 

Literature Reference 

AOAC. 2005. Method 923.03 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD.

E.4.4. Carbohydrates 

The total carbohydrate level was calculated by difference using the fresh weight-derived 

data and the following equation: 
Monsanto Company 09-SY-201U Page 234 of 471

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash) 

The limit of quantitation was 0.100%. 

Literature Reference 

USDA. 1973. Energy value of foods. Agriculture Handbook No. 74. United States 

Department of Agriculture, Washington, D.C. 

E.4.5. Fat by Acid Hydrolysis 

The sample was hydrolyzed with hydrochloric acid at an elevated temperature. The fat 

was extracted with ether and hexane. The extract was evaporated on a steambath, redissolved in hexane and filtered through a sodium sulfate column. The hexane extract 

was then evaporated again on a steambath under nitrogen, dried, and weighed. The limit 

of quantitation was 0.100%. 

Literature Reference 

AOAC. 2005. Methods 922.06 and 954.02 in Official Methods of Analysis of AOAC 

International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.6. Fat by Soxhlet Extraction 

The sample was weighed into a cellulose thimble containing sodium sulfate and dried to 

remove excess moisture. Pentane was dripped through the sample to remove the fat. The 

extract was then evaporated, dried, and weighed. The limit of quantitation was 0.100%. 

Literature Reference 

AOAC. 2005. Method 960.39 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD.

E.4.7. Fatty Acid Profile with Trans Fat by GC 

The lipid was extracted, saponified with 0.5 N methanolic sodium hydroxide, and 

methylated with 14% BF3-methanol. The resulting methyl esters of the fatty acids were 

extracted with heptane. An internal standard was added prior to the lipid extraction. The 

methyl esters of the fatty acids were analyzed by gas chromatography using external 

standards for quantitation. The limit of quantitation was 0.0200%. 

Reference Standards

• Nu Chek Prep GLC Reference Standard Hazelton No. 1*, Lot Number AU18-S 

• Nu Chek Prep GLC Reference Standard Hazelton No. 2*, Lot Number M13-O 

• Nu Chek Prep GLC Reference Standard Hazelton No. 3*, Lot Number MA18-S 

• Nu Chek Prep GLC Reference Standard Hazelton No. 4*, Lot Number AU18-S 

 (*Overall purity of the sum of the mixture of components is 100%) 

• Nu Chek Prep Methyl Gamma Linolenate, used as 100%, Lot Number U-63M-JY12-

R 
Monsanto Company 09-SY-201U Page 235 of 471

• Nu Chek Prep Methyl Tridecanoate, used as 100%, Lot Number N-13M-A2-S 

• Nu Chek Prep Methyl Butyrate, used as 100%, Lot Number N-4M-J20-R 

• Nu Chek Prep Methyl Hexanoate, used as 100%, Lot Number N-6M-A25-R 

• Nu Chek Prep Methyl Erucate, used as 100%, Lot Number U-79M-AU3-Q 

• Nu Chek Prep Methyl Lignocerate, used as 100%, Lot Number N-24M-AU18-S 

• Nu Chek Prep Methyl Docosapentaenoate, used as 100%, Lot Number U-101MJY14-S 

• Nu Chek Prep Methyl Docosahexaenoate, used as 100%, Lot Number U-84M-M30-S 

• Nu Chek Prep Methyl Eicosapentaenoate, used as 100%, Lot Number U-99M-D14-R 

• Cayman Chemicals Steariodonic Acid Methyl Ester, used as 100%, Lot Number 

182102-1 

• Nu Chek Prep Methyl Elaidate, used as 100%, Lot Number U-47M-JA18-R 

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-F27-R 

• Nu Chek Prep Methyl Nervonate, used as 100%, Lot Number U-88M-AU4-S 

• Nu Chek Prep Methyl Palmitelaidate, used as as 100%, Lot Number U-41M-M21-S 

• Monsanto Mono Trans SDA, used as 99%, Lot Number GLP-0804-19309-A 

• Monsanto Mono Trans Alpha Linolenic Acid, used as 100%, Lot Number GLP-0804-

19308-A 

• Monsanto 9c, 15c Octadecadienoate (Omnisoy), used as 100%, Lot Number GLP0802-19168-A 

• Larodan Methyl 6(z), 9(z)-Octadecadienoate, used as 99.3%, Lot Number LX-017 

• Monsanto Omnisoy C17:1 Methyl 9-cis-Heptadecenoate, used as 99%, Lot Number 

GLP-0806-19436-A 

Literature References

AOAC. 2005. Method 996.06 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD. 

AOCS. 1997. Method Ce 2c-66 and Ce 1C-89 in Official Methods and Recommended 

Practices of the AOCS, 5th ed. American Oil Chemists' Society, Champaign, IL. 

AOAC. 2005. Method 983.23 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD 

E.4.8. Isoflavone Analysis 

The sample was extracted using a solution of hydrochloric acid and reagent alcohol 

heated on steam baths or hot plates. The extract was brought to volume, diluted, and 

centrifuged. An aliquot of the supernatant was placed onto a C18 solid-phase extraction 

column. Unwanted components of the matrix were rinsed off with 20% methanol and 

then the isoflavones were eluted with 80% methanol. The sample was analyzed on a 

high-performance liquid chromatography (HPLC) system with ultraviolet detection and 

was compared to an external standard curve of known standards for quantitation. The 

limit of quantitation was 10.0 ppm for each component. 
Monsanto Company 09-SY-201U Page 236 of 471

Reference Standards 

Chromadex, Daidzein, 96.5%, Lot Number 04007-120 

Chromadex, Glycitein, 96.3%, Lot Number 07344-571 

Indofine, Genistein, >99%1

, Lot Number 0309074 

1

Used as 100% in calculations. 

Literature References 

Seo, A. and C.V. Morr. 1984. Improved high-performance liquid chromatographic 

analysis of phenolic acids and isoflavonoids from soybean protein products. Journal of 

Agricultural and Food Chemistry, 32(3):530-533. 

Pettersson, H., and K.H. Kiessling. 1984. Liquid Chromatographic Determination of the 

Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official 

Analytical Chemists Journal, 67(3):503-506. 

 E.4.9. Lectin 

The sample was suspended in phosphate buffered saline (PBS), shaken, and filtered. An 

aliquot of the resulting extract was serially diluted in 10 cuvettes containing PBS. A 10% 

hematocrit of lyophilized rabbit blood in PBS was added to each dilution. After 2.5 

hours, the absorbance of each dilution of the sample and lectin control was measured on a 

spectrophotometer at 620 nm, using PBS to zero the instrument. One hemagglutinating 

unit (H.U.) was defined as the level that caused 50% of the standard cell suspension to 

sediment in 2.5 hours. The limit of quantitation was 0.10 H.U./mg. 

Reference Standard 

Sigma-Aldrich, Red Blood Cells, Rabbit, Product #R1629, Lot Number 105K6042. 

Literature References 

Klurfeld, D. M. and Kritchevsky, D. 1987. Isolation and quantitation of lectins from 

vegetable oils. Lipids 22:667-668. 

Klurfeld, D. M., Personal communication. 

Liener, I. E., 1955. The photometric determination of the hemagglutinating activity of 

soyin and crude soybean extracts. Archives of Biochemistry and Biophysics 54:223-231, 

E.4.10. Moisture 

The sample was dried in a vacuum oven at approximately 100° C to a constant weight. 

The moisture weight loss was determined and converted to percent moisture. The limit 

of quantitation was 0.100%. 

 
Monsanto Company 09-SY-201U Page 237 of 471

Literature Reference 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods 926.08 

and 925.09, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005). 

E.4.11. Neutral Detergent Fiber, Enzyme Method 

The sample was placed in a fritted vessel and washed with a neutral boiling detergent 

solution that dissolved the protein, carbohydrate, enzyme, and ash. An acetone wash 

removed the fats and pigments. Hemicellulose, cellulose, and lignin fractions were 

collected on the frit and determined gravimetrically. The limit of quantitation was 

0.100%. 

Literature References 

Approved Methods of the American Association of Cereal Chemists, 9th Ed., 

Method 32.20, (1998). 

USDA. 1970. Forage and Fiber Analyses. Agriculture Handbook No. 379. United States 

Department of Agriculture, Washington, D.C. 

E.4.12. Phytic Acid

The sample was extracted using 0.5 M HCl with ultrasonication. Purification and 

concentration were accomplished on a silica-based anion-exchange column. The sample 

was analyzed on a polymer HPLC column PRP-1, 5 μm (150 x 4.1 mm) with a refractive 

index detector. The limit of quantitation was 0.100%. 

Reference Standard 

Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 95%, Lot Number 077K0693. 

Literature References 

Lehrfeld, Jacob, “HPLC Separation and Quantitation of Phytic Acid and Some Inositol 

Phosphates in Foods: Problem and Solutions,” Journal of Agricultural and Food 

Chemistry, 42:2726-2731, (1994). 

Lehrfeld, Jacob, “High-Performance Liquid Chromatography Analysis of Phytic Acid on 

a pH-Stable, Macroporous Polymer Column,” Cereal Chemistry, 66(6):510-515, (1989). 

E.4.13. Protein 

Nitrogenous compounds in the sample were reduced in the presence of boiling sulfuric 

acid and a mercury catalyst mixture to form ammonia. The acid digest was made alkaline. 

The ammonia was distilled and then titrated with a previously standardized acid. The 

percent nitrogen was calculated and converted to equivalent protein using the factor 6.25. 

The limit of quantitation was 0.100%. 
Monsanto Company 09-SY-201U Page 238 of 471

Literature References 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods 955.04 

and 979.09, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005). 

Bradstreet, R. B., The Kjeldahl Method for Organic Nitrogen, Academic Press: New 

York, New York, (1965). 

Kolthoff, I. M., and Sandell, E. B., Quantitative Inorganic Analysis, MacMillan: New 

York, (1948). 

E.4.14. Raffinose and Stachyose 

The sample was extracted with deionized water and the extract treated with a 

hydroxylamine hydrochloride solution in pyridine, containing phenyl-β-D-glucoside as 

an internal standard. The resulting oximes were converted to silyl derivatives by 

treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas 

chromatography using a flame ionization detector. The quantitation limit was 0.0500%. 

Reference Standards 

• Sigma, Raffinose Pentahydrate, 99% (84% after correction for degree of hydration), 

Lot Number 037K1059 

• Sigma, Stachyose, 98% (96.8% after correction for degree of hydration), Lot 

Number 038K3775 

Literature References 

Brobst, K. M. 1972. Gas-Liquid Chromatography of Trimethylsilyl Derivatives in 

Methods in Carbohydrate Chemistry, Vol. 6. Academic Press, New York. 

Mason, B.S., and H.T. Slover. 1971. A Gas Chromatographic Method for the 

Determination of Sugars in Foods. Journal of Agricultural and Food Chemistry, 

19(3):551-554. 

E.4.15. Trypsin Inhibitor 

The sample was ground and defatted with petroleum ether. A sample of matrix was 

extracted with 0.01 N sodium hydroxide. Varying aliquots of the sample suspension 

were exposed to a known amount of trypsin and benzoy1-DL-arginine~p~nitroanilide 

hydrochloride. The sample was allowed to react for 10 minutes at 37°C. After 10 

minutes, the reaction was halted by the addition of acetic acid. The solution was 

centrifuged, then the absorbance was determined at 410 nm. Trypsin inhibitor activity 

was determined by photometrically measuring the inhibition of trypsin’s reaction with 

benzoyl-DL-arginine~p~nitroanilide hydrochloride. The limit of quantitation was 1.00 

Trypsin Inhibitor Units (TIU)/mg. 

Literature Reference 

AOCS. 1997. Method Ba 12-75 in Official Methods and Recommended Practices of the 

American Oil Chemists’ Society. AOCS Press, Champaign, IL. 
Monsanto Company 09-SY-201U Page 239 of 471

E.4.16. Vitamin E 

The sample was saponified to break down any fat and release vitamin E. The saponified 

mixture was extracted with ethyl ether and then quantitated by high-performance liquid 

chromatography using a silica column. The limit of quantitation was 0.500 mg/100 g. 

Reference Standard 

USP, Alpha Tocopherol, 100%, Lot Number M. 

Literature References 

Cort, W.M., T.S. Vincente, E.H. Waysek, and B.D. Williams. 1983. Vitamin E content of 

feedstuffs determined by high-performance liquid chromatographic Fluorescence. Journal 

of Agricultural Food Chemistry, 31:1330-1333. 

McMurray, C.H., W.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction 

techniques on determination of α-tocopherol in animal feedstuffs. Journal of the 

Association of Official Analytical Chemists, 63(6):1258-1261. 

Speek, A.J., J. Schijver, and W.H.P. Schreurs. 1985. Vitamin E composition of some seed 

oils as determined by high-performance liquid chromatography with fluorometric 

quantitation. Journal of Food Science, 50(1):121-124. 

 
Monsanto Company 09-SY-201U Page 240 of 471

E.5. Data Processing and Statistical Analysis 

After compositional analyses were performed at Covance Laboratories, Inc., data 

spreadsheets were forwarded to Monsanto Company. The data were reviewed, formatted, 

and sent to Certus International, Inc. for statistical analysis. 

The following formulas were used for re-expression of composition data for statistical 

analysis: 

Component From (X) To Formula1

Proximates (excluding Moisture), 

Fiber, Phytic Acid, Raffinose, 

Stachyose 

% FW % DW X/d 

Amino Acids (AA) mg/g FW % DW X/(10d) 

Isoflavones μg/g FW μg/g DW X/d 

Trypsin Inhibitor TIU/mg FW TIU/mg DW X/d 

Vitamin E mg/100g FW mg/100g DW X/d 

Lectin H.U./mg FW H.U./mg DW X/d 

Fatty Acids (FA) % FW % Total FA 

(100)Xj/ΣX, 

for each FAj

where ΣX is 

over all the FA 

1

‘X’ is the individual sample value; ‘d’ is the fraction of the sample that is dry matter. 

In order to complete a statistical analysis for a compositional component, at least 50% of 

the values for a component had to be greater than the assay limit of quantitation (LOQ). 

Components with more than 50% of observations below the assay LOQ were excluded 

from summaries and analysis as there was insufficient data to conduct the statistical 

analysis. The following 17 components with more than 50% of the observations below 

the assay LOQ were excluded: 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 

myristic acid, 14:1 myristoleic acid, 15:0 pentadecylic acid, 15:1 10c pentadecenoic acid, 

16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 9c heptadecenoic acid, 18:1 9t 

octadecenoic acid, 18:2 9c,15c octadecadienoic acid, 18:2 6c,9c octadecadienoic acid, 

18:3 gamma linolenic acid, 20:2 11c,14c eicosadienoic acid, 20:3 11c,14c,17c 

eicosatrienoic acid, and 20:4 arachidonic acid. 

Otherwise, results below the LOQ were assigned a value equal to one half of the 

quantitation limit. Thirteen observations for 24:0 lignoceric acid were assigned a value, 

equal to one half of the quantitation limit (<0.0200%). 

PRESS residuals were used to identify outliers. A PRESS residual is the difference 

between any value and its predicted value from a statistical model that excludes the data 

point. The studentized version scales these residuals so that the values tend to have a 

standard normal distribution when outliers are absent. Thus, most values are expected to 

be between ±3. Extreme data points that were outside the ±6 studentized PRESS residual 

range were considered for exclusion from the final analyses. No results had PRESS 

residual values outside of the ±6 range. 
Monsanto Company 09-SY-201U Page 241 of 471

Soybean components were statistically analyzed using a mixed model analysis of 

variance. The five replicated sites were analyzed both separately and combined. 

Individual replicated site analyses used model (1). 

(1) Yij = U + Ti + Bj + eij, 

where Yij = unique individual observation, U = overall mean, Ti = substance effect, Bj = 

random block effect, and eij = residual error. 

Combined-site analyses used model (2). 

(2) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk, 

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, Lj 

= random location effect, B(L)jk = random block within location effect, LTij = random 

location by substance interaction effect, and eijk = residual error. 

For each compositional component, 99% tolerance intervals were calculated. A tolerance 

interval is an interval that one can claim, with a specified degree of confidence, contains 

at least a specified proportion, p, of an entire sampled population for the parameter 

measured. The calculated tolerance intervals in this study are expected to contain, with 

95% confidence, 99% of the quantities expressed in the population of conventional 

soybean. Each tolerance interval estimate was based upon the average of three 

observations per unique reference substance. Because negative quantities are not 

possible, negative calculated lower tolerance bounds were set to zero. 

SAS software was used to generate all summary statistics and perform all analyses. 

Report tables present p-values from SAS as either <0.001 or the actual value truncated to 

three decimal places. 
Monsanto Company 09-SY-201U Page 242 of 471

Table E-1. Statistical Summary of Site CdT Soybean Forage Fiber and Proximate Content for MON 87705 Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fiber and Proximate (% DW)Acid Detergent Fiber 32.08 (3.03) 27.25 (3.03) 4.83 (3.78) -11.44, 21.09 0.3

[30.53 - 34.11] [19.21 - 33.03] [1.02 - 12.39] 

Ash 8.74 (0.36) 8.53 (0.36) 0.22 (0.51) -1.97, 2.40 0.7

[8.40 - 9.19] [7.76 - 9.32] [-0.68 - 1.43] 

Carbohydrates 72.60 (0.75) 73.03 (0.75) -0.43 (1.05) -4.96, 4.11 0.7

[72.28 - 72.79] [70.97 - 74.33] [-1.61 - 1.82] 

Moisture (% FW) 71.53 (0.58) 71.17 (0.58) 0.37 (0.83) -3.19, 3.92 0.7

[70.60 - 72.50] [70.00 - 72.10] [-1.50 - 2.50] 

Neutral Detergent Fiber 37.26 (1.43) 32.98 (1.43) 4.28 (1.89) -3.86, 12.42 0.1

[34.35 - 41.05] [32.26 - 33.67] [2.10 - 8.05] 

Protein 13.34 (0.51) 12.97 (0.51) 0.37 (0.72) -2.74, 3.47 0.6

[13.05 - 13.89] [11.85 - 14.16] [-1.10 - 2.04] 
Monsanto Company 09-SY-201U Page 243 of 471

Table E-1. Statistical Summary of Site CdT Soybean Forage Fiber and Proximate Content for MON 8770Conventional Control (A3525) 

 Difference (MON 87705 minus control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fiber and Proximate (%DW)Total Fat 5.32 (0.47) 5.52 (0.47) -0.20 (0.67) -3.07, 2.66 0.7

[4.77 - 5.71] [4.40 - 6.47] [-1.70 - 1.31] 

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Nto zero.
Monsanto Company 09-SY-201U Page 244 of 471

Table E-2. Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitaand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Amino Acid (% DW)Alanine 1.53 (0.017) 1.56 (0.017) -0.023 (0.012) -0.077, 0.030 0.2

[1.51 - 1.54] [1.51 - 1.58] [-0.041 - 0.00038] 

Arginine 2.55 (0.036) 2.55 (0.036) -0.0053 (0.040) -0.18, 0.16 0.9

[2.48 - 2.63] [2.51 - 2.58] [-0.062 - 0.071] 

Aspartic Acid 3.84 (0.045) 3.94 (0.045) -0.10 (0.035) -0.25, 0.051 0.1

[3.76 - 3.93] [3.86 - 4.00] [-0.16 - -0.039] 

Cystine 0.62 (0.0081) 0.62 (0.0081) 0.0014 (0.011) -0.048, 0.051 0.9

[0.60 - 0.63] [0.60 - 0.63] [-0.027 - 0.028] 

Glutamic Acid 5.99 (0.083) 6.17 (0.083) -0.19 (0.070) -0.49, 0.11 0.1

[5.86 - 6.13] [6.00 - 6.29] [-0.32 - -0.094] 

Glycine 1.52 (0.016) 1.55 (0.016) -0.027 (0.0087) -0.064, 0.010 0.0

[1.50 - 1.54] [1.51 - 1.57] [-0.040 - -0.011] 
Monsanto Company 09-SY-201U Page 245 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Amino Acid (% DW)Histidine 0.94 (0.012) 0.94 (0.012) 0.0024 (0.014) -0.056, 0.061 0.8

[0.92 - 0.97] [0.92 - 0.96] [-0.021 - 0.025] 

Isoleucine 1.61 (0.039) 1.64 (0.039) -0.034 (0.038) -0.20, 0.13 0.4

[1.57 - 1.64] [1.55 - 1.71] [-0.11 - 0.022] 

Leucine 2.56 (0.031) 2.63 (0.031) -0.073 (0.030) -0.20, 0.057 0.1

[2.53 - 2.59] [2.55 - 2.69] [-0.13 - -0.026] 

Lysine 2.31 (0.026) 2.32 (0.026) -0.013 (0.019) -0.096, 0.071 0.5

[2.27 - 2.34] [2.26 - 2.36] [-0.051 - 0.012] 

Methionine 0.53 (0.0091) 0.54 (0.0091) -0.0089 (0.013) -0.064, 0.046 0.5

[0.51 - 0.54] [0.52 - 0.55] [-0.035 - 0.024] 

Phenylalanine 1.71 (0.024) 1.76 (0.024) -0.050 (0.024) -0.15, 0.053 0.1

[1.68 - 1.74] [1.70 - 1.80] [-0.097 - -0.025] 
Monsanto Company 09-SY-201U Page 246 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Proline 1.65 (0.026) 1.68 (0.026) -0.027 (0.011) -0.075, 0.021 0.1

[1.60 - 1.70] [1.65 - 1.73] [-0.046 - -0.0075] 

Serine 1.69 (0.069) 1.71 (0.069) -0.013 (0.097) -0.43, 0.40 0.9

[1.59 - 1.80] [1.56 - 1.81] [-0.22 - 0.24] 

Threonine 1.28 (0.034) 1.33 (0.034) -0.050 (0.048) -0.26, 0.16 0.4

[1.25 - 1.34] [1.26 - 1.39] [-0.13 - 0.080] 

Tryptophan 0.40 (0.0088) 0.42 (0.0088) -0.017 (0.0061) -0.043, 0.0094 0.1

[0.38 - 0.41] [0.41 - 0.43] [-0.028 - -0.0070] 

Tyrosine 1.22 (0.021) 1.17 (0.021) 0.048 (0.029) -0.079, 0.17 0.2

[1.18 - 1.27] [1.15 - 1.19] [-0.0086 - 0.12] 

Valine 1.72 (0.042) 1.74 (0.042) -0.023 (0.049) -0.24, 0.19 0.6

[1.69 - 1.75] [1.63 - 1.82] [-0.11 - 0.065] 
Monsanto Company 09-SY-201U Page 247 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 16:0 Palmitic 2.31 (0.040) 10.80 (0.040) -8.49 (0.047) -8.70, -8.29 <0.

[2.29 - 2.32] [10.72 - 10.91] [-8.59 - -8.43] 

18:0 Stearic 3.17 (0.040) 4.58 (0.040) -1.42 (0.038) -1.58, -1.25 <0.

[3.09 - 3.23] [4.53 - 4.66] [-1.47 - -1.34] 

18:1 Oleic 76.44 (0.19) 23.02 (0.19) 53.42 (0.21) 52.50, 54.34 <0.

[76.35 - 76.60] [22.51 - 23.37] [53.19 - 53.85] 

18:2 Linoleic 10.09 (0.15) 52.43 (0.15) -42.34 (0.11) -42.83, -41.84 <0.

[9.94 - 10.22] [52.13 - 52.78] [-42.56 - -42.19] 

18:3 Linolenic 6.90 (0.047) 8.15 (0.047) -1.25 (0.044) -1.44, -1.06 0.0

[6.85 - 6.94] [8.08 - 8.28] [-1.33 - -1.18] 

20:0 Arachidic 0.29 (0.0058) 0.35 (0.0058) -0.063 (0.0082) -0.099, -0.028 0.0

[0.28 - 0.29] [0.34 - 0.36] [-0.082 - -0.046] 
Monsanto Company 09-SY-201U Page 248 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fatty Acid (% Total FA)20:1 Eicosenoic 0.36 (0.0056) 0.21 (0.0056) 0.16 (0.0045) 0.14, 0.18 <0.

[0.36 - 0.38] [0.19 - 0.21] [0.15 - 0.16] 

22:0 Behenic 0.28 (0.0046) 0.30 (0.0046) -0.016 (0.0066) -0.045, 0.012 0.1

[0.28 - 0.29] [0.29 - 0.31] [-0.029 - -0.0044] 

24:0 Lignoceric 0.15 (0.0029) 0.15 (0.0029) -0.0049 (0.00046) -0.0069, -0.0030 0.0

[0.14 - 0.15] [0.15 - 0.16] [-0.0055 - -0.0040] 

Fiber (% DW)Acid Detergent Fiber 18.23 (0.32) 16.27 (0.32) 1.97 (0.45) 0.014, 3.92 0.0

[17.57 - 18.58] [15.69 - 16.74] [0.83 - 2.89] 

Neutral Detergent Fiber 21.04 (0.45) 17.99 (0.45) 3.05 (0.30) 1.78, 4.33 0.0

[20.47 - 22.18] [17.71 - 18.54] [2.75 - 3.65] 

Proximate (% DW)Ash 6.37 (0.093) 6.22 (0.093) 0.15 (0.099) -0.28, 0.57 0.2

[6.13 - 6.54] [6.18 - 6.31] [-0.050 - 0.26] 
Monsanto Company 09-SY-201U Page 249 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Proximate (% DW)Carbohydrates 41.82 (0.20) 40.05 (0.20) 1.76 (0.23) 0.78, 2.75 0.0

[41.62 - 42.00] [39.64 - 40.52] [1.31 - 2.00] 

Moisture (% FW) 10.80 (0.35) 10.87 (0.35) -0.067 (0.50) -2.21, 2.08 0.9

[10.10 - 11.20] [10.20 - 11.40] [-1.30 - 0.90] 

Protein 33.75 (0.33) 34.22 (0.33) -0.47 (0.36) -2.02, 1.08 0.3

[33.26 - 34.65] [34.04 - 34.41] [-0.94 - 0.24] 

Total Fat 18.09 (0.25) 19.52 (0.25) -1.43 (0.35) -2.95, 0.083 0.0

[17.66 - 18.35] [18.96 - 19.82] [-2.16 - -0.61] 

Vitamin (mg/100g DW)Vitamin E 3.34 (0.13) 4.03 (0.13) -0.69 (0.18) -1.46, 0.068 0.0

[3.17 - 3.50] [3.74 - 4.19] [-1.01 - -0.39] 

AntinutrientLectin (H.U./mg DW) 1.93 (0.74) 3.12 (0.74) -1.19 (1.01) -5.54, 3.15 0.3

[1.01 - 2.77] [2.20 - 4.93] [-2.93 - 0.57] 
Monsanto Company 09-SY-201U Page 250 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Antinutrient (% DW)Phytic Acid 1.81 (0.10) 1.79 (0.10) 0.017 (0.14) -0.59, 0.62 0.9

[1.52 - 1.97] [1.78 - 1.81] [-0.26 - 0.16] 

Raffinose 0.51 (0.018) 0.52 (0.018) -0.0095 (0.025) -0.12, 0.099 0.7

[0.50 - 0.54] [0.50 - 0.56] [-0.069 - 0.030] 

Stachyose 3.76 (0.15) 3.10 (0.15) 0.66 (0.15) 0.027, 1.29 0.0

[3.55 - 4.16] [3.04 - 3.21] [0.51 - 0.95] 

Trypsin Inhibitor (TIU/mg DW) 42.86 (3.21) 43.65 (3.21) -0.79 (2.92) -13.37, 11.80 0.8

[38.51 - 48.03] [37.36 - 49.78] [-5.31 - 4.69] 

Isoflavone (μg/g DW)Daidzein 1192.24 (52.19) 1182.58 (52.19) 9.66 (73.81) -307.91, 327.23 0.9

[1145.72 - 1259.84] [1092.43 - 1309.26] [-163.54 - 167.41] 

Genistein 813.17 (33.64) 825.33 (33.64) -12.17 (47.57) -216.84, 192.50 0.8

[809.79 - 815.32] [751.67 - 914.22] [-104.43 - 62.73] 
Monsanto Company 09-SY-201U Page 251 of 471

Table E-2 (continued). Statistical Summary of Site CdT Soybean Seed Amino Acid, Fatty Acid, Fiber, ProAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Isoflavone (μg/g DW)Glycitein 140.40 (18.28) 146.58 (18.28) -6.18 (25.85) -117.41, 105.05 0.8

[90.89 - 176.80] [141.57 - 152.56] [-61.67 - 35.23] 

1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Nto zero.
Monsanto Company 09-SY-201U Page 252 of 471

Table E-3. Statistical Summary of Site MEL Soybean Forage Fiber and Proximate Content for MON 8770Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fiber and Proximate (% DW)Acid Detergent Fiber 27.77 (1.88) 27.46 (1.88) 0.31 (1.52) -6.24, 6.86 0.8

[24.18 - 29.83] [25.10 - 31.32] [-1.49 - 3.34] 

Ash 8.58 (0.33) 8.20 (0.33) 0.38 (0.46) -1.61, 2.38 0.4

[8.32 - 8.86] [7.66 - 9.08] [-0.52 - 1.01] 

Carbohydrates 71.69 (1.41) 71.00 (1.41) 0.69 (2.00) -7.91, 9.28 0.7

[69.52 - 73.15] [67.88 - 73.56] [-2.05 - 5.27] 

Moisture (% FW) 70.43 (0.26) 69.90 (0.26) 0.53 (0.37) -1.06, 2.13 0.2

[70.20 - 70.80] [69.40 - 70.50] [-0.20 - 1.40] 

Neutral Detergent Fiber 30.89 (1.70) 32.58 (1.70) -1.69 (2.21) -11.22, 7.84 0.5

[27.64 - 35.57] [32.19 - 33.01] [-4.90 - 2.56] 

Protein 13.95 (1.60) 14.11 (1.60) -0.16 (2.27) -9.90, 9.59 0.9

[12.65 - 16.40] [11.63 - 17.85] [-5.20 - 3.56] 
Monsanto Company 09-SY-201U Page 253 of 471

Table E-3 (continued). Statistical Summary of Site MEL Soybean Forage Fiber and Proximate Conten

the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fiber and Proximate (% DW)Total Fat 5.79 (0.31) 6.76 (0.31) -0.97 (0.17) -1.71, -0.22 0.0

[5.37 - 6.57] [6.49 - 7.19] [-1.16 - -0.62] 

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Nto zero.
Monsanto Company 09-SY-201U Page 254 of 471

Table E-4. Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vit

and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Amino Acid (% DW)Alanine 1.62 (0.032) 1.56 (0.032) 0.052 (0.040) -0.12, 0.23 0.3

[1.54 - 1.68] [1.54 - 1.61] [-0.00088 - 0.13] 

Arginine 2.78 (0.097) 2.63 (0.097) 0.15 (0.12) -0.37, 0.66 0.3

[2.57 - 2.94] [2.54 - 2.79] [0.025 - 0.39] 

Aspartic Acid 4.14 (0.12) 3.94 (0.12) 0.19 (0.16) -0.51, 0.90 0.3

[3.87 - 4.36] [3.84 - 4.15] [0.026 - 0.52] 

Cystine 0.61 (0.013) 0.58 (0.013) 0.030 (0.018) -0.048, 0.11 0.2

[0.60 - 0.62] [0.55 - 0.61] [-0.0014 - 0.062] 

Glutamic Acid 6.59 (0.23) 6.18 (0.23) 0.41 (0.31) -0.90, 1.73 0.3

[6.09 - 7.02] [5.99 - 6.55] [0.098 - 1.02] 

Glycine 1.61 (0.042) 1.55 (0.042) 0.060 (0.053) -0.17, 0.29 0.3

[1.52 - 1.69] [1.52 - 1.62] [-0.00070 - 0.17] 
Monsanto Company 09-SY-201U Page 255 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Amino Acid (% DW)Histidine 0.96 (0.025) 0.91 (0.025) 0.047 (0.033) -0.096, 0.19 0.2

[0.91 - 1.01] [0.89 - 0.95] [0.011 - 0.11] 

Isoleucine 1.74 (0.052) 1.62 (0.052) 0.12 (0.073) -0.19, 0.44 0.2

[1.61 - 1.84] [1.58 - 1.66] [-0.0014 - 0.26] 

Leucine 2.78 (0.072) 2.65 (0.072) 0.13 (0.096) -0.28, 0.54 0.3

[2.62 - 2.92] [2.60 - 2.76] [0.024 - 0.32] 

Lysine 2.41 (0.049) 2.31 (0.049) 0.10 (0.069) -0.19, 0.40 0.2

[2.31 - 2.50] [2.25 - 2.39] [0.016 - 0.25] 

Methionine 0.56 (0.0097) 0.52 (0.0097) 0.038 (0.013) -0.018, 0.094 0.1

[0.53 - 0.57] [0.51 - 0.53] [0.013 - 0.056] 

Phenylalanine 1.85 (0.053) 1.78 (0.053) 0.070 (0.074) -0.25, 0.39 0.4

[1.74 - 1.95] [1.73 - 1.87] [-0.0084 - 0.22] 
Monsanto Company 09-SY-201U Page 256 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, 

Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Amino Acid (% DW)Proline 1.81 (0.056) 1.68 (0.056) 0.13 (0.070) -0.18, 0.43 0.2

[1.69 - 1.89] [1.63 - 1.78] [0.043 - 0.27] 

Serine 1.79 (0.046) 1.79 (0.046) -0.0013 (0.065) -0.28, 0.28 0.9

[1.72 - 1.86] [1.73 - 1.89] [-0.17 - 0.12] 

Threonine 1.34 (0.042) 1.37 (0.042) -0.025 (0.053) -0.25, 0.20 0.6

[1.31 - 1.39] [1.28 - 1.47] [-0.13 - 0.031] 

Tryptophan 0.41 (0.0088) 0.41 (0.0088) -0.0023 (0.012) -0.056, 0.051 0.8

[0.39 - 0.43] [0.40 - 0.42] [-0.028 - 0.019] 

Tyrosine 1.25 (0.023) 1.24 (0.023) 0.0014 (0.027) -0.12, 0.12 0.9

[1.23 - 1.26] [1.20 - 1.30] [-0.040 - 0.054] 

Valine 1.86 (0.054) 1.73 (0.054) 0.13 (0.077) -0.20, 0.46 0.2

[1.72 - 1.96] [1.68 - 1.76] [-0.014 - 0.28] 
Monsanto Company 09-SY-201U Page 257 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fatty Acid (% Total FA)16:0 Palmitic 2.39 (0.021) 10.83 (0.021) -8.44 (0.029) -8.56, -8.31 <0.

[2.35 - 2.42] [10.80 - 10.87] [-8.47 - -8.38] 

18:0 Stearic 3.33 (0.10) 4.39 (0.10) -1.06 (0.15) -1.69, -0.42 0.0

[3.20 - 3.47] [4.26 - 4.64] [-1.32 - -0.79] 

18:1 Oleic 76.10 (0.22) 22.31 (0.22) 53.79 (0.31) 52.45, 55.14 <0.

[75.68 - 76.33] [21.86 - 22.61] [53.06 - 54.47] 

18:2 Linoleic 10.50 (0.22) 53.48 (0.22) -42.98 (0.31) -44.31, -41.66 <0.

[10.16 - 10.92] [53.22 - 53.89] [-43.74 - -42.40] 

18:3 Linolenic 6.58 (0.055) 8.00 (0.055) -1.42 (0.076) -1.74, -1.09 0.0

[6.53 - 6.65] [7.86 - 8.10] [-1.56 - -1.31] 

20:0 Arachidic 0.30 (0.0058) 0.34 (0.0058) -0.040 (0.0066) -0.068, -0.012 0.0

[0.29 - 0.30] [0.32 - 0.34] [-0.052 - -0.030] 
Monsanto Company 09-SY-201U Page 258 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Fatty Acid (% Total FA)20:1 Eicosenoic 0.35 (0.0050) 0.20 (0.0050) 0.14 (0.0063) 0.12, 0.17 0.0

[0.34 - 0.36] [0.20 - 0.21] [0.14 - 0.16] 

22:0 Behenic 0.30 (0.0063) 0.30 (0.0063) 0.0014 (0.0089) -0.037, 0.040 0.8

[0.29 - 0.30] [0.28 - 0.31] [-0.016 - 0.020] 

24:0 Lignoceric 0.15 (0.0080) 0.16 (0.0080) -0.0026 (0.0096) -0.044, 0.039 0.8

[0.14 - 0.17] [0.15 - 0.16] [-0.019 - 0.014] 

Fiber (% DW)Acid Detergent Fiber 16.35 (0.29) 16.74 (0.29) -0.38 (0.29) -1.61, 0.85 0.3

[16.22 - 16.63] [16.03 - 17.32] [-0.69 - 0.19] 

Neutral Detergent Fiber 20.24 (0.63) 17.83 (0.63) 2.42 (0.89) -1.40, 6.23 0.1

[18.92 - 21.65] [17.32 - 18.62] [0.30 - 4.33] 

Proximate (% DW)Ash 6.11 (0.11) 6.41 (0.11) -0.30 (0.14) -0.90, 0.30 0.1

[5.99 - 6.26] [6.14 - 6.55] [-0.56 - -0.073] 
Monsanto Company 09-SY-201U Page 259 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Proximate (% DW)Carbohydrates 39.70 (0.55) 39.60 (0.55) 0.10 (0.59) -2.43, 2.64 0.8

[38.85 - 41.07] [38.91 - 40.04] [-0.99 - 1.03] 

Moisture (% FW) 10.93 (0.21) 11.37 (0.21) -0.43 (0.15) -1.06, 0.19 0.0

[10.40 - 11.20] [11.10 - 11.60] [-0.70 - -0.20] 

Protein 35.56 (0.92) 34.38 (0.92) 1.18 (0.95) -2.89, 5.26 0.3

[33.59 - 36.94] [33.30 - 35.97] [0.18 - 3.08] 

Total Fat 18.64 (0.29) 19.63 (0.29) -0.99 (0.31) -2.35, 0.36 0.0

[18.24 - 19.08] [19.00 - 20.13] [-1.51 - -0.42] 

Vitamin (mg/100g DW)Vitamin E 3.26 (0.11) 3.83 (0.11) -0.58 (0.043) -0.76, -0.39 0.0

[3.15 - 3.45] [3.66 - 4.05] [-0.64 - -0.49] 

AntinutrientLectin (H.U./mg DW) 1.84 (0.30) 1.69 (0.30) 0.15 (0.42) -1.68, 1.98 0.7

[1.61 - 2.14] [1.04 - 2.40] [-0.63 - 1.11] 
Monsanto Company 09-SY-201U Page 260 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control 

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Antinutrient (% DW)Phytic Acid 1.89 (0.099) 1.90 (0.099) -0.0094 (0.14) -0.60, 0.58 0.9

[1.80 - 2.02] [1.66 - 2.08] [-0.27 - 0.18] 

Raffinose 0.57 (0.019) 0.56 (0.019) 0.018 (0.0085) -0.019, 0.054 0.1

[0.54 - 0.61] [0.53 - 0.58] [0.0046 - 0.034] 

Stachyose 3.68 (0.13) 3.78 (0.13) -0.10 (0.18) -0.87, 0.66 0.6

[3.56 - 3.77] [3.51 - 4.08] [-0.53 - 0.26] 

Trypsin Inhibitor (TIU/mg DW) 35.12 (2.90) 36.06 (2.90) -0.93 (1.36) -6.80, 4.93 0.5

[31.64 - 41.07] [31.67 - 41.28] [-3.57 - 0.98] 

Isoflavone (μg/g DW)Daidzein 2095.83 (90.09) 2012.16 (90.09) 83.67 (127.41) -464.54, 631.88 0.5

[2060.81 - 2139.64] [1844.77 - 2257.34] [-196.53 - 242.28] 

Genistein 1362.28 (58.14) 1286.17 (58.14) 76.11 (82.22) -277.67, 429.88 0.4

[1340.09 - 1385.14] [1176.47 - 1444.70] [-104.61 - 208.66] 
Monsanto Company 09-SY-201U Page 261 of 471

Table E-4 (continued). Statistical Summary of Site MEL Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Isoflavone (μg/g DW) Glycitein 144.06 (9.70) 142.17 (9.70) 1.90 (13.72) -57.13, 60.93 0.9

[132.88 - 151.79] [128.23 - 167.04] [-34.16 - 23.55] 

1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 262 of 471

Table E-5. Statistical Summary of Site QUI Soybean Forage Fiber and Proximate Content for MON 87705 Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Acid Detergent Fiber 24.62 (1.87) 22.74 (2.29) 1.88 (2.96) -35.75, 39.51 0.6

[20.04 - 26.98] [22.72 - 22.76] [4.10 - 4.25] 

Ash 8.26 (0.32) 7.87 (0.39) 0.39 (0.50) -5.97, 6.76 0.5

[8.02 - 8.51] [7.24 - 8.49] [-0.24 - 1.27] 

Carbohydrates 71.07 (0.43) 72.76 (0.53) -1.69 (0.68) -10.36, 6.98 0.2

[70.11 - 71.67] [72.40 - 73.12] [-3.01 - -0.73] 

Moisture (% FW) 71.77 (0.47) 72.10 (0.58) -0.33 (0.75) -9.87, 9.20 0.7

[70.70 - 72.70] [72.10 - 72.10] [-1.40 - -0.20] 

Neutral Detergent Fiber 30.94 (2.43) 32.99 (2.51) -2.05 (1.09) -15.95, 11.85 0.3

[26.12 - 33.59] [33.91 - 36.56] [-2.96 - -0.80] 

Protein 16.36 (0.79) 15.09 (0.97) 1.27 (1.26) -14.70, 17.24 0.4

[15.22 - 18.10] [14.37 - 15.81] [-0.58 - 1.39] 
Monsanto Company 09-SY-201U Page 263 of 471

Table E-5 (continued). Statistical Summary of Site QUI Soybean Forage Fiber and Proximate Content foConventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Total Fat 4.35 (1.10) 3.42 (1.15) 0.93 (0.57) -6.30, 8.16 0.3

[2.29 - 5.80] [3.36 - 5.34] [0.46 - 1.59] 

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 264 of 471

Table E-6. Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitaand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Alanine 1.64 (0.031) 1.68 (0.031) -0.036 (0.0029) -0.072, 0.00038 0.0

[1.60 - 1.70] [1.63 - 1.66] [-0.039 - -0.033] 

Arginine 2.96 (0.090) 2.98 (0.091) -0.022 (0.028) -0.38, 0.33 0.5

[2.82 - 3.12] [2.82 - 2.99] [-0.052 - 0.0034] 

Aspartic Acid 4.31 (0.10) 4.40 (0.11) -0.095 (0.053) -0.77, 0.58 0.3

[4.16 - 4.49] [4.21 - 4.43] [-0.16 - -0.051] 

Cystine 0.63 (0.0098) 0.62 (0.011) 0.0059 (0.010) -0.12, 0.14 0.6

[0.60 - 0.64] [0.61 - 0.63] [-0.0061 - 0.017] 

Glutamic Acid 6.85 (0.20) 7.06 (0.20) -0.21 (0.058) -0.94, 0.52 0.1

[6.54 - 7.19] [6.70 - 7.09] [-0.27 - -0.16] 

Glycine 1.64 (0.029) 1.65 (0.035) -0.012 (0.046) -0.59, 0.57 0.8

[1.60 - 1.71] [1.65 - 1.65] [-0.050 - -0.045] 
Monsanto Company 09-SY-201U Page 265 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Histidine 0.99 (0.024) 1.01 (0.024) -0.018 (0.0035) -0.062, 0.027 0.1

[0.95 - 1.04] [0.97 - 1.01] [-0.021 - -0.014] 

Isoleucine 1.76 (0.054) 1.87 (0.058) -0.10 (0.036) -0.56, 0.36 0.2

[1.65 - 1.86] [1.80 - 1.86] [-0.15 - -0.074] 

Leucine 2.88 (0.073) 2.94 (0.074) -0.061 (0.022) -0.34, 0.22 0.2

[2.77 - 3.01] [2.81 - 2.94] [-0.085 - -0.041] 

Lysine 2.48 (0.043) 2.50 (0.044) -0.019 (0.016) -0.22, 0.18 0.4

[2.41 - 2.55] [2.42 - 2.51] [-0.036 - -0.0050] 

Methionine 0.55 (0.0071) 0.56 (0.0087) -0.0063 (0.011) -0.15, 0.14 0.6

[0.54 - 0.57] [0.55 - 0.57] [-0.022 - -0.0066] 

Phenylalanine 1.89 (0.053) 1.93 (0.065) -0.044 (0.084) -1.11, 1.03 0.6

[1.83 - 2.00] [1.88 - 1.99] [-0.16 - -0.035] 
Monsanto Company 09-SY-201U Page 266 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Proline 1.85 (0.045) 1.88 (0.045) -0.032 (0.0091) -0.15, 0.083 0.1

[1.77 - 1.92] [1.80 - 1.90] [-0.041 - -0.023] 

Serine 1.92 (0.051) 1.83 (0.062) 0.083 (0.080) -0.93, 1.10 0.4

[1.87 - 1.98] [1.74 - 1.92] [-0.053 - 0.16] 

Threonine 1.42 (0.023) 1.37 (0.028) 0.046 (0.037) -0.42, 0.51 0.4

[1.38 - 1.45] [1.34 - 1.41] [-0.025 - 0.11] 

Tryptophan 0.43 (0.0092) 0.44 (0.011) -0.0041 (0.015) -0.19, 0.18 0.8

[0.41 - 0.45] [0.43 - 0.44] [-0.025 - -0.0021] 

Tyrosine 1.31 (0.018) 1.28 (0.021) 0.024 (0.028) -0.33, 0.38 0.5

[1.29 - 1.32] [1.25 - 1.32] [-0.024 - 0.059] 

Valine 1.86 (0.059) 1.96 (0.066) -0.10 (0.053) -0.77, 0.56 0.3

[1.73 - 1.96] [1.90 - 1.96] [-0.17 - -0.064] 
Monsanto Company 09-SY-201U Page 267 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 16:0 Palmitic 2.30 (0.042) 10.56 (0.051) -8.26 (0.066) -9.10, -7.43 0.0

[2.25 - 2.37] [10.51 - 10.62] [-8.37 - -8.13] 

18:0 Stearic 3.51 (0.19) 4.82 (0.19) -1.31 (0.0088) -1.42, -1.20 0.0

[3.15 - 3.82] [4.47 - 4.85] [-1.32 - -1.30] 

18:1 Oleic 78.61 (0.41) 24.95 (0.44) 53.66 (0.26) 50.35, 56.96 0.0

[77.70 - 79.17] [24.34 - 25.08] [53.36 - 53.88] 

18:2 Linoleic 8.75 (0.58) 51.70 (0.63) -42.95 (0.44) -48.50, -37.39 0.0

[7.85 - 10.02] [51.68 - 52.44] [-43.30 - -42.42] 

18:3 Linolenic 5.64 (0.074) 7.02 (0.082) -1.38 (0.063) -2.19, -0.58 0.0

[5.55 - 5.71] [6.86 - 7.16] [-1.45 - -1.32] 

20:0 Arachidic 0.33 (0.017) 0.36 (0.017) -0.032 (0.0021) -0.058, -0.0055 0.0

[0.30 - 0.36] [0.33 - 0.36] [-0.034 - -0.030] 
Monsanto Company 09-SY-201U Page 268 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 20:1 Eicosenoic 0.38 (0.0089) 0.20 (0.011) 0.18 (0.014) 0.0019, 0.36 0.0

[0.37 - 0.40] [0.20 - 0.20] [0.18 - 0.20] 

22:0 Behenic 0.31 (0.0094) 0.31 (0.0095) 0.0051 (0.0029) -0.032, 0.042 0.3

[0.30 - 0.33] [0.29 - 0.31] [0.0019 - 0.0078] 

24:0 Lignoceric 0.17 (0.0027) 0.15 (0.0033) 0.021 (0.0042) -0.032, 0.075 0.1

[0.17 - 0.17] [0.15 - 0.16] [0.015 - 0.027] 

Fiber (% DW) Acid Detergent Fiber 18.15 (0.59) 15.96 (0.73) 2.19 (0.94) -9.75, 14.13 0.2

[17.23 - 19.31] [15.28 - 16.65] [1.28 - 4.03] 

Neutral Detergent Fiber 18.01 (0.71) 16.53 (0.87) 1.48 (1.13) -12.86, 15.82 0.4

[16.93 - 19.69] [16.18 - 16.87] [0.054 - 1.23] 

Proximate (% DW) Ash 6.22 (0.089) 6.10 (0.10) 0.12 (0.091) -1.04, 1.28 0.4

[6.06 - 6.42] [6.13 - 6.18] [0.061 - 0.24] 
Monsanto Company 09-SY-201U Page 269 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Proximate (% DW) Carbohydrates 37.54 (0.51) 37.41 (0.55) 0.12 (0.34) -4.20, 4.44 0.7

[36.69 - 38.62] [37.46 - 38.09] [-0.15 - 0.53] 

Moisture (% FW) 10.40 (0.097) 11.05 (0.12) -0.65 (0.15) -2.60, 1.30 0.1

[10.20 - 10.60] [11.00 - 11.10] [-0.90 - -0.60] 

Protein 37.05 (0.83) 37.11 (0.84) -0.055 (0.27) -3.50, 3.39 0.8

[35.60 - 38.59] [35.96 - 36.78] [-0.35 - 0.19] 

Total Fat 19.20 (0.21) 19.73 (0.26) -0.53 (0.34) -4.80, 3.73 0.3

[18.68 - 19.49] [19.69 - 19.78] [-0.36 - -0.20] 

Vitamin (mg/100g DW) Vitamin E 3.93 (0.19) 3.72 (0.23) 0.21 (0.29) -3.51, 3.93 0.5

[3.70 - 4.36] [3.61 - 3.82] [-0.13 - 0.76] 

Antinutrient Lectin (H.U./mg DW) 1.45 (0.82) 3.92 (1.00) -2.47 (1.30) -18.93, 13.99 0.3

[0.72 - 2.03] [2.32 - 5.53] [-4.80 - -0.29] 
Monsanto Company 09-SY-201U Page 270 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Antinutrient (% DW)Phytic Acid 1.89 (0.12) 1.97 (0.12) -0.085 (0.024) -0.39, 0.22 0.1

[1.73 - 2.13] [1.90 - 2.19] [-0.11 - -0.059] 

Raffinose 0.60 (0.023) 0.55 (0.029) 0.049 (0.037) -0.42, 0.52 0.4

[0.56 - 0.64] [0.52 - 0.57] [-0.015 - 0.12] 

Stachyose 4.08 (0.31) 3.84 (0.38) 0.24 (0.49) -5.93, 6.42 0.7

[3.43 - 4.48] [3.52 - 4.16] [0.18 - 0.96] 

Trypsin Inhibitor (TIU/mg DW) 42.52 (5.28) 40.77 (5.31) 1.75 (0.99) -10.88, 14.38 0.3

[33.22 - 52.01] [41.51 - 49.21] [0.81 - 2.80] 

Isoflavone (μg/g DW)Daidzein 1346.63 (67.43) 1411.91 (69.07) -65.28 (26.14) -397.44, 266.88 0.2

[1230.43 - 1450.89] [1394.83 - 1539.33] [-88.43 - -36.25] 

Genistein 952.69 (44.70) 932.29 (52.06) 20.40 (50.70) -623.77, 664.56 0.7

[882.55 - 1005.58] [881.89 - 1020.22] [-14.64 - 88.04] 
Monsanto Company 09-SY-201U Page 271 of 471

Table E-6 (continued). Statistical Summary of Site QUI Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Isoflavone (μg/g DW) Glycitein 87.69 (17.12) 98.58 (18.14) -10.89 (10.64) -146.05, 124.28 0.4

[49.11 - 108.04] [72.10 - 109.21] [-22.99 - -1.18] 

1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 272 of 471

Table E-7. Statistical Summary of Site RAN Soybean Forage Fiber and Proximate Content for MON 8770Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Acid Detergent Fiber 32.11 (2.53) 29.37 (2.53) 2.73 (3.57) -12.65, 18.11 0.5

[31.02 - 33.39] [24.37 - 36.13] [-4.22 - 9.02] 

Ash 9.64 (0.30) 8.56 (0.30) 1.07 (0.42) -0.75, 2.90 0.1

[8.81 - 10.11] [8.40 - 8.67] [0.14 - 1.60] 

Carbohydrates 69.77 (0.62) 72.09 (0.62) -2.32 (0.40) -4.02, -0.62 0.0

[68.94 - 71.06] [70.97 - 72.89] [-3.10 - -1.83] 

Moisture (% FW) 78.03 (1.32) 75.53 (1.32) 2.50 (1.86) -5.50, 10.50 0.3

[76.50 - 81.10] [73.90 - 77.50] [-1.00 - 7.20] 

Neutral Detergent Fiber 41.60 (1.83) 36.03 (1.83) 5.58 (1.75) -1.97, 13.12 0.0

[39.19 - 46.30] [33.91 - 37.55] [2.70 - 8.75] 

Protein 16.73 (0.63) 14.71 (0.63) 2.02 (0.89) -1.82, 5.87 0.1

[15.19 - 17.78] [14.02 - 15.48] [0.57 - 3.75] 
Monsanto Company 09-SY-201U Page 273 of 471

Table E-7 (continued). Statistical Summary of Site RAN Soybean Forage Fiber and Proximate Contenthe Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Total Fat 3.87 (0.48) 4.66 (0.48) -0.80 (0.67) -3.69, 2.10 0.3

[2.83 - 4.89] [4.05 - 5.06] [-2.23 - 0.015] 

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 274 of 471

Table E-8. Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitaand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Alanine 1.66 (0.021) 1.62 (0.021) 0.036 (0.027) -0.080, 0.15 0.3

[1.62 - 1.70] [1.59 - 1.66] [-0.0069 - 0.085] 

Arginine 3.08 (0.032) 2.95 (0.032) 0.13 (0.045) -0.062, 0.33 0.0

[3.01 - 3.16] [2.94 - 2.97] [0.043 - 0.22] 

Aspartic Acid 4.35 (0.036) 4.24 (0.036) 0.11 (0.051) -0.11, 0.33 0.1

[4.30 - 4.44] [4.20 - 4.29] [0.026 - 0.23] 

Cystine 0.60 (0.011) 0.58 (0.011) 0.022 (0.011) -0.024, 0.069 0.1

[0.57 - 0.62] [0.57 - 0.59] [0.00097 - 0.033] 

Glutamic Acid 7.00 (0.060) 6.82 (0.060) 0.17 (0.085) -0.19, 0.54 0.1

[6.92 - 7.14] [6.73 - 6.89] [0.039 - 0.41] 

Glycine 1.68 (0.030) 1.63 (0.030) 0.046 (0.020) -0.041, 0.13 0.1

[1.62 - 1.74] [1.58 - 1.67] [0.015 - 0.084] 
Monsanto Company 09-SY-201U Page 275 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Histidine 1.02 (0.0088) 0.99 (0.0088) 0.031 (0.012) -0.022, 0.085 0.1

[1.01 - 1.04] [0.98 - 1.01] [0.0086 - 0.060] 

Isoleucine 1.83 (0.022) 1.76 (0.022) 0.069 (0.031) -0.063, 0.20 0.1

[1.78 - 1.88] [1.74 - 1.77] [0.0045 - 0.14] 

Leucine 2.91 (0.022) 2.86 (0.022) 0.049 (0.031) -0.085, 0.18 0.2

[2.89 - 2.97] [2.85 - 2.90] [-0.0094 - 0.12] 

Lysine 2.49 (0.021) 2.43 (0.021) 0.065 (0.030) -0.065, 0.19 0.1

[2.46 - 2.55] [2.41 - 2.45] [0.018 - 0.13] 

Methionine 0.57 (0.0092) 0.53 (0.0092) 0.035 (0.010) -0.0098, 0.079 0.0

[0.55 - 0.58] [0.52 - 0.54] [0.015 - 0.049] 

Phenylalanine 1.95 (0.013) 1.92 (0.013) 0.028 (0.018) -0.050, 0.11 0.2

[1.93 - 1.98] [1.90 - 1.94] [-0.0066 - 0.075] 
Monsanto Company 09-SY-201U Page 276 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Proline 1.92 (0.018) 1.83 (0.018) 0.093 (0.025) -0.014, 0.20 0.0

[1.88 - 1.95] [1.80 - 1.85] [0.026 - 0.14] 

Serine 1.79 (0.041) 1.91 (0.041) -0.11 (0.058) -0.37, 0.14 0.1

[1.74 - 1.90] [1.88 - 1.94] [-0.20 - 0.0033] 

Threonine 1.36 (0.014) 1.36 (0.014) -0.00098 (0.020) -0.088, 0.086 0.9

[1.33 - 1.39] [1.34 - 1.38] [-0.052 - 0.042] 

Tryptophan 0.45 (0.0040) 0.44 (0.0040) 0.0065 (0.0057) -0.018, 0.031 0.3

[0.44 - 0.46] [0.44 - 0.44] [-0.0022 - 0.022] 

Tyrosine 1.29 (0.014) 1.30 (0.014) -0.0067 (0.014) -0.065, 0.052 0.6

[1.26 - 1.33] [1.29 - 1.31] [-0.026 - 0.019] 

Valine 1.97 (0.023) 1.89 (0.023) 0.080 (0.033) -0.063, 0.22 0.1

[1.91 - 2.02] [1.87 - 1.91] [0.026 - 0.15] 
Monsanto Company 09-SY-201U Page 277 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 16:0 Palmitic 2.40 (0.0088) 10.96 (0.0088) -8.56 (0.012) -8.61, -8.51 <0.

[2.39 - 2.40] [10.94 - 10.98] [-8.58 - -8.54] 

18:0 Stearic 3.34 (0.032) 4.50 (0.032) -1.16 (0.045) -1.35, -0.96 0.0

[3.28 - 3.41] [4.47 - 4.55] [-1.22 - -1.07] 

18:1 Oleic 74.69 (0.59) 21.53 (0.59) 53.15 (0.79) 49.76, 56.55 <0.

[73.13 - 75.98] [21.41 - 21.63] [51.71 - 54.43] 

18:2 Linoleic 11.32 (0.42) 53.73 (0.42) -42.41 (0.57) -44.84, -39.97 <0.

[10.37 - 12.42] [53.67 - 53.81] [-43.33 - -41.38] 

18:3 Linolenic 7.32 (0.19) 8.43 (0.19) -1.11 (0.27) -2.28, 0.066 0.0

[6.87 - 7.81] [8.40 - 8.47] [-1.60 - -0.59] 

20:0 Arachidic 0.28 (0.0036) 0.33 (0.0036) -0.043 (0.0051) -0.065, -0.021 0.0

[0.28 - 0.29] [0.32 - 0.33] [-0.053 - -0.031] 
Monsanto Company 09-SY-201U Page 278 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 20:1 Eicosenoic 0.29 (0.0087) 0.16 (0.0087) 0.13 (0.0081) 0.096, 0.17 0.0

[0.27 - 0.31] [0.15 - 0.16] [0.12 - 0.15] 

22:0 Behenic 0.28 (0.0041) 0.30 (0.0041) -0.016 (0.0047) -0.036, 0.0048 0.0

[0.28 - 0.29] [0.30 - 0.30] [-0.021 - -0.0062] 

24:0 Lignoceric 0.069 (0.0011) 0.067 (0.0011) 0.0017 (0.0012) -0.0034, 0.0069 0.2

[0.066 - 0.071] [0.067 - 0.068] [-0.00065 - 

0.0032] 

Fiber(% DW) Acid Detergent Fiber 16.32 (0.34) 13.94 (0.34) 2.38 (0.11) 1.91, 2.85 0.0

[15.71 - 16.78] [13.36 - 14.58] [2.20 - 2.58] 

Neutral Detergent Fiber 15.14 (0.91) 16.40 (0.91) -1.26 (0.39) -2.95, 0.43 0.0

[13.41 - 16.48] [14.61 - 17.51] [-1.97 - -0.61] 

Proximate (% DW) Ash 5.53 (0.088) 5.68 (0.088) -0.14 (0.12) -0.68, 0.39 0.3

[5.46 - 5.63] [5.48 - 5.88] [-0.42 - 0.15] 
Monsanto Company 09-SY-201U Page 279 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Proximate (% DW) Carbohydrates 40.12 (0.24) 39.60 (0.24) 0.52 (0.34) -0.93, 1.97 0.2

[39.56 - 40.64] [39.41 - 39.84] [-0.28 - 1.23] 

Moisture (% FW) 9.52 (0.43) 12.03 (0.43) -2.51 (0.61) -5.12, 0.090 0.0

[8.96 - 10.60] [11.50 - 12.40] [-3.40 - -0.90] 

Protein 37.54 (0.20) 36.87 (0.20) 0.67 (0.19) -0.16, 1.51 0.0

[37.35 - 37.70] [36.56 - 37.40] [0.29 - 0.94] 

Total Fat 16.83 (0.21) 17.85 (0.21) -1.01 (0.30) -2.30, 0.27 0.0

[16.55 - 17.36] [17.63 - 18.11] [-1.52 - -0.45] 

Vitamin (mg/100g DW) Vitamin E 1.44 (0.094) 1.81 (0.094) -0.38 (0.094) -0.78, 0.026 0.0

[1.23 - 1.64] [1.69 - 1.91] [-0.47 - -0.19] 

Antinutrient Lectin (H.U./mg DW) 3.57 (0.50) 1.36 (0.50) 2.21 (0.61) -0.39, 4.82 0.0

[3.30 - 3.77] [0.61 - 2.73] [1.04 - 3.04] 
Monsanto Company 09-SY-201U Page 280 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Antinutrient (% DW)Phytic Acid 1.62 (0.032) 1.72 (0.032) -0.10 (0.042) -0.28, 0.079 0.1

[1.61 - 1.63] [1.63 - 1.77] [-0.14 - -0.016] 

Raffinose 0.52 (0.017) 0.56 (0.017) -0.044 (0.023) -0.14, 0.056 0.1

[0.48 - 0.56] [0.55 - 0.57] [-0.088 - -0.011] 

Stachyose 3.79 (0.21) 3.70 (0.21) 0.084 (0.30) -1.20, 1.37 0.8

[3.39 - 4.35] [3.56 - 3.80] [-0.35 - 0.79] 

Trypsin Inhibitor (TIU/mg DW) 29.38 (1.10) 28.54 (1.10) 0.85 (0.95) -3.23, 4.92 0.4

[26.73 - 31.10] [27.23 - 29.95] [-0.50 - 2.67] 

Isoflavone (μg/g DW)Daidzein 1915.89 (53.73) 1947.43 (53.73) -31.54 (48.74) -241.26, 178.18 0.5

[1868.13 - 1946.31] [1803.65 - 2027.33] [-94.12 - 64.48] 

Genistein 1186.26 (34.98) 1149.45 (34.98) 36.81 (39.06) -131.24, 204.87 0.4

[1164.84 - 1208.26] [1054.79 - 1209.04] [-23.36 - 110.04] 
Monsanto Company 09-SY-201U Page 281 of 471

Table E-8 (continued). Statistical Summary of Site RAN Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Isoflavone (μg/g DW) Glycitein 117.22 (15.47) 130.37 (15.47) -13.15 (21.88) -107.29, 80.99 0.6

[79.19 - 140.66] [110.84 - 146.92] [-54.14 - 29.81] 

1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 282 of 471

Table E-9. Statistical Summary of Site SFR Soybean Forage Fiber and Proximate Content for MON 87705 Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Acid Detergent Fiber 34.33 (3.08) 38.52 (3.08) -4.19 (4.36) -22.93, 14.55 0.4

[30.23 - 42.14] [34.68 - 40.67] [-10.04 - 7.47] 

Ash 8.54 (0.51) 7.73 (0.51) 0.81 (0.73) -2.31, 3.94 0.3

[7.39 - 9.74] [7.21 - 8.02] [-0.63 - 1.79] 

Carbohydrates 76.37 (1.00) 78.35 (1.00) -1.98 (1.42) -8.09, 4.13 0.2

[74.24 - 78.93] [77.70 - 78.99] [-4.74 - 1.23] 

Moisture (% FW) 72.87 (0.39) 72.60 (0.39) 0.27 (0.47) -1.74, 2.27 0.6

[72.00 - 73.60] [72.20 - 73.20] [-0.20 - 1.20] 

Neutral Detergent Fiber 38.24 (1.13) 43.34 (1.13) -5.10 (1.53) -11.67, 1.48 0.0

[37.27 - 38.89] [40.65 - 45.90] [-7.01 - -2.08] 

Protein 9.84 (0.24) 9.77 (0.24) 0.070 (0.32) -1.29, 1.43 0.8

[9.25 - 10.42] [9.71 - 9.81] [-0.46 - 0.63] 
Monsanto Company 09-SY-201U Page 283 of 471

Table E-9 (continued). Statistical Summary of Site SFR Soybean Forage Fiber and Proximate Content foConventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Total Fat 5.25 (0.64) 4.31 (0.64) 0.93 (0.91) -2.98, 4.84 0.4

[4.36 - 7.01] [3.99 - 4.75] [-0.39 - 2.80] 

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 284 of 471

Table E-10. Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Proximate, Vitand Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Alanine 1.51 (0.011) 1.44 (0.011) 0.067 (0.011) 0.021, 0.11 0.0

[1.49 - 1.54] [1.43 - 1.45] [0.055 - 0.088] 

Arginine 2.52 (0.048) 2.34 (0.048) 0.18 (0.040) 0.0051, 0.35 0.0

[2.43 - 2.64] [2.31 - 2.39] [0.11 - 0.24] 

Aspartic Acid 3.76 (0.054) 3.56 (0.054) 0.20 (0.019) 0.11, 0.28 0.0

[3.67 - 3.88] [3.51 - 3.65] [0.17 - 0.23] 

Cystine 0.61 (0.010) 0.57 (0.010) 0.043 (0.015) -0.020, 0.11 0.0

[0.60 - 0.62] [0.55 - 0.59] [0.0062 - 0.073] 

Glutamic Acid 5.90 (0.11) 5.53 (0.11) 0.37 (0.034) 0.22, 0.51 0.0

[5.72 - 6.12] [5.42 - 5.71] [0.30 - 0.41] 

Glycine 1.49 (0.015) 1.42 (0.015) 0.078 (0.022) -0.016, 0.17 0.0

[1.47 - 1.54] [1.41 - 1.43] [0.055 - 0.12] 
Monsanto Company 09-SY-201U Page 285 of 471

Table-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Histidine 0.90 (0.014) 0.85 (0.014) 0.050 (0.0069) 0.021, 0.080 0.0

[0.88 - 0.94] [0.84 - 0.87] [0.043 - 0.064] 

Isoleucine 1.60 (0.029) 1.51 (0.029) 0.093 (0.030) -0.037, 0.22 0.0

[1.56 - 1.66] [1.45 - 1.54] [0.032 - 0.12] 

Leucine 2.54 (0.036) 2.41 (0.036) 0.12 (0.015) 0.059, 0.19 0.0

[2.47 - 2.61] [2.37 - 2.47] [0.095 - 0.15] 

Lysine 2.25 (0.026) 2.13 (0.026) 0.11 (0.010) 0.070, 0.16 0.0

[2.19 - 2.30] [2.10 - 2.17] [0.097 - 0.13] 

Methionine 0.53 (0.0080) 0.50 (0.0080) 0.037 (0.011) -0.011, 0.086 0.0

[0.52 - 0.55] [0.49 - 0.51] [0.0074 - 0.056] 

Phenylalanine 1.68 (0.023) 1.60 (0.023) 0.077 (0.011) 0.030, 0.12 0.0

[1.64 - 1.73] [1.58 - 1.64] [0.056 - 0.089] 
Monsanto Company 09-SY-201U Page 286 of 471

Table E-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Proline 1.62 (0.023) 1.55 (0.023) 0.078 (0.012) 0.028, 0.13 0.0

[1.59 - 1.66] [1.52 - 1.60] [0.065 - 0.10] 

Serine 1.56 (0.063) 1.58 (0.063) -0.017 (0.089) -0.40, 0.36 0.8

[1.49 - 1.68] [1.45 - 1.68] [-0.15 - 0.23] 

Threonine 1.24 (0.022) 1.22 (0.022) 0.022 (0.032) -0.11, 0.16 0.5

[1.20 - 1.28] [1.18 - 1.25] [-0.044 - 0.10] 

Tryptophan 0.39 (0.011) 0.37 (0.011) 0.024 (0.016) -0.045, 0.092 0.2

[0.37 - 0.41] [0.35 - 0.38] [-0.0073 - 0.046] 

Tyrosine 1.18 (0.0097) 1.12 (0.0097) 0.064 (0.014) 0.0052, 0.12 0.0

[1.17 - 1.20] [1.10 - 1.13] [0.039 - 0.10] 

Valine 1.72 (0.031) 1.61 (0.031) 0.12 (0.036) -0.039, 0.27 0.0

[1.69 - 1.79] [1.55 - 1.64] [0.044 - 0.16] 
Monsanto Company 09-SY-201U Page 287 of 471

Table E-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, 

Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 16:0 Palmitic 2.42 (0.029) 11.00 (0.029) -8.58 (0.042) -8.76, -8.40 <0.

[2.40 - 2.44] [10.95 - 11.08] [-8.68 - -8.53] 

18:0 Stearic 3.22 (0.084) 4.31 (0.084) -1.09 (0.044) -1.28, -0.90 0.0

[3.07 - 3.41] [4.24 - 4.44] [-1.17 - -1.03] 

18:1 Oleic 76.49 (0.43) 22.42 (0.43) 54.07 (0.60) 51.47, 56.67 <0.

[75.33 - 77.21] [22.16 - 22.65] [52.87 - 55.05] 

18:2 Linoleic 9.82 (0.27) 52.84 (0.27) -43.02 (0.38) -44.64, -41.39 <0.

[9.33 - 10.55] [52.75 - 52.98] [-43.65 - -42.21] 

18:3 Linolenic 6.98 (0.11) 8.49 (0.11) -1.50 (0.15) -2.16, -0.85 0.0

[6.79 - 7.26] [8.41 - 8.60] [-1.80 - -1.20] 

20:0 Arachidic 0.29 (0.0035) 0.32 (0.0035) -0.026 (0.0021) -0.035, -0.017 0.0

[0.29 - 0.29] [0.31 - 0.32] [-0.029 - -0.022] 
Monsanto Company 09-SY-201U Page 288 of 471

Table E-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, 

Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 20:1 Eicosenoic 0.33 (0.0077) 0.18 (0.0077) 0.15 (0.011) 0.10, 0.19 0.0

[0.32 - 0.35] [0.17 - 0.19] [0.13 - 0.17] 

22:0 Behenic 0.29 (0.0025) 0.30 (0.0025) -0.0071 (0.0033) -0.021, 0.0069 0.1

[0.29 - 0.29] [0.29 - 0.30] [-0.012 - -0.0012] 

24:0 Lignoceric 0.15 (0.0045) 0.14 (0.0045) 0.0090 (0.0039) -0.0077, 0.026 0.1

[0.14 - 0.16] [0.13 - 0.14] [0.0013 - 0.014] 

Fiber (% DW) Acid Detergent Fiber 16.62 (0.28) 17.78 (0.28) -1.17 (0.36) -2.74, 0.40 0.0

[15.85 - 17.05] [17.64 - 18.02] [-1.84 - -0.59] 

Neutral Detergent Fiber 17.75 (0.93) 19.99 (0.93) -2.24 (1.01) -6.57, 2.09 0.1

[15.62 - 19.68] [19.01 - 21.09] [-4.24 - -1.06] 

Proximate (% DW) Ash 6.05 (0.10) 6.17 (0.10) -0.12 (0.14) -0.73, 0.49 0.4

[5.84 - 6.32] [6.15 - 6.22] [-0.38 - 0.16] 
Monsanto Company 09-SY-201U Page 289 of 471

Table E-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Proximate (% DW) Carbohydrates 42.56 (0.37) 42.80 (0.37) -0.24 (0.47) -2.28, 1.80 0.6

[41.73 - 43.52] [42.65 - 42.96] [-1.08 - 0.57] 

Moisture (% FW) 12.13 (0.11) 12.47 (0.11) -0.33 (0.15) -0.97, 0.31 0.1

[12.00 - 12.30] [12.30 - 12.70] [-0.70 - -0.10] 

Protein 32.70 (0.60) 31.15 (0.60) 1.55 (0.52) -0.70, 3.81 0.0

[31.48 - 34.13] [30.71 - 31.81] [0.55 - 2.32] 

Total Fat 18.70 (0.50) 19.88 (0.50) -1.18 (0.40) -2.91, 0.56 0.0

[17.41 - 19.50] [19.38 - 20.32] [-1.98 - -0.73] 

Vitamin (mg/100g DW) Vitamin E 2.20 (0.22) 2.91 (0.22) -0.72 (0.31) -2.06, 0.63 0.1

[2.07 - 2.42] [2.58 - 3.49] [-1.40 - -0.16] 

Antinutrient Lectin (H.U./mg DW) 2.28 (0.63) 2.46 (0.63) -0.18 (0.90) -4.04, 3.67 0.8

[0.82 - 3.38] [1.92 - 3.41] [-2.59 - 1.31] 
Monsanto Company 09-SY-201U Page 290 of 471

Table E-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Antinutrient (% DW)Phytic Acid 1.90 (0.072) 1.88 (0.072) 0.023 (0.076) -0.30, 0.35 0.7

[1.83 - 2.03] [1.72 - 1.97] [-0.13 - 0.11] 

Raffinose 0.68 (0.021) 0.68 (0.021) -0.0041 (0.029) -0.13, 0.12 0.9

[0.62 - 0.71] [0.66 - 0.70] [-0.064 - 0.051] 

Stachyose 4.06 (0.24) 4.10 (0.24) -0.038 (0.35) -1.53, 1.45 0.9

[3.88 - 4.41] [3.50 - 4.43] [-0.56 - 0.91] 

Trypsin Inhibitor (TIU/mg DW) 40.79 (3.57) 36.63 (3.57) 4.16 (1.82) -3.67, 11.98 0.1

[33.98 - 47.66] [33.22 - 42.92] [0.76 - 6.98] 

Isoflavone (μg/g DW)Daidzein 2481.08 (83.81) 2406.95 (83.81) 74.13 (58.64) -178.17, 326.42 0.3

[2354.95 - 2565.56] [2223.49 - 2565.86] [-43.14 - 134.06] 

Genistein 1487.13 (52.05) 1390.09 (52.05) 97.04 (43.02) -88.04, 282.12 0.1

[1433.45 - 1527.94] [1254.28 - 1466.21] [33.79 - 179.17] 
Monsanto Company 09-SY-201U Page 291 of 471

Table E-10 (continued). Statistical Summary of Site SFR Soybean Seed Amino Acid, Fatty Acid, Fiber, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Isoflavone (μg/g DW) Glycitein 174.86 (13.28) 122.44 (13.28) 52.42 (18.78) -28.36, 133.21 0.1

[152.79 - 196.59] [98.05 - 146.12] [6.67 - 98.54] 

1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 292 of 471

Table E-11. Statistical Summary of Combined Site Soybean Forage Fiber and Proximate Content for MON Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Acid Detergent Fiber 30.18 (2.20) 29.17 (2.23) 1.01 (1.64) -2.38, 4.39 0.5

[20.04 - 42.14] [19.21 - 40.67] [-10.04 - 12.39] 

Ash 8.75 (0.22) 8.18 (0.22) 0.57 (0.23) 0.096, 1.05 0.0

[7.39 - 10.11] [7.21 - 9.32] [-0.68 - 1.79] 

Carbohydrates 72.30 (1.20) 73.43 (1.21) -1.13 (0.59) -2.36, 0.097 0.0

[68.94 - 78.93] [67.88 - 78.99] [-4.74 - 5.27] 

Moisture (% FW) 72.93 (1.16) 72.22 (1.16) 0.70 (0.48) -0.64, 2.04 0.2

[70.20 - 81.10] [69.40 - 77.50] [-1.50 - 7.20] 

Neutral Detergent Fiber 35.79 (2.05) 35.85 (2.06) -0.062 (2.14) -6.00, 5.88 0.9

[26.12 - 46.30] [32.19 - 45.90] [-7.01 - 8.75] 

Protein 14.04 (1.13) 13.34 (1.13) 0.70 (0.55) -0.43, 1.84 0.2

[9.25 - 18.10] [9.71 - 17.85] [-5.20 - 3.75] 
Monsanto Company 09-SY-201U Page 293 of 471

Table E-11 (continued). Statistical Summary of Combined Site Soybean Forage Fiber and Proximate Convs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fiber and Proximate (% DW) Total Fat 4.91 (0.42) 5.14 (0.42) -0.23 (0.38) -1.01, 0.55 0.5

[2.29 - 7.01] [3.36 - 7.19] [-2.23 - 2.80] 

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 294 of 471

Table E-12. Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty Acid, Fiber, ProximaAntinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Alanine 1.59 (0.033) 1.57 (0.033) 0.025 (0.018) -0.026, 0.075 0.2

[1.49 - 1.70] [1.43 - 1.66] [-0.041 - 0.13] 

Arginine 2.78 (0.11) 2.68 (0.11) 0.10 (0.036) 0.0012, 0.20 0.0

[2.43 - 3.16] [2.31 - 2.99] [-0.062 - 0.39] 

Aspartic Acid 4.08 (0.13) 4.00 (0.13) 0.075 (0.060) -0.092, 0.24 0.2

[3.67 - 4.49] [3.51 - 4.43] [-0.16 - 0.52] 

Cystine 0.61 (0.0075) 0.59 (0.0076) 0.022 (0.0075) 0.0010, 0.042 0.0

[0.57 - 0.64] [0.55 - 0.63] [-0.027 - 0.073] 

Glutamic Acid 6.46 (0.24) 6.32 (0.24) 0.14 (0.12) -0.19, 0.47 0.3

[5.72 - 7.19] [5.42 - 7.09] [-0.32 - 1.02] 

Glycine 1.59 (0.039) 1.56 (0.039) 0.028 (0.022) -0.033, 0.089 0.2

[1.47 - 1.74] [1.41 - 1.67] [-0.050 - 0.17] 
Monsanto Company 09-SY-201U Page 295 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Alanine 1.59 (0.033) 1.57 (0.033) 0.025 (0.018) -0.026, 0.075 0.2

[1.49 - 1.70] [1.43 - 1.66] [-0.041 - 0.13] 

Arginine 2.78 (0.11) 2.68 (0.11) 0.10 (0.036) 0.0012, 0.20 0.0

[2.43 - 3.16] [2.31 - 2.99] [-0.062 - 0.39] 

Aspartic Acid 4.08 (0.13) 4.00 (0.13) 0.075 (0.060) -0.092, 0.24 0.2

[3.67 - 4.49] [3.51 - 4.43] [-0.16 - 0.52] 

Cystine 0.61 (0.0075) 0.59 (0.0076) 0.022 (0.0075) 0.0010, 0.042 0.0

[0.57 - 0.64] [0.55 - 0.63] [-0.027 - 0.073] 

Glutamic Acid 6.46 (0.24) 6.32 (0.24) 0.14 (0.12) -0.19, 0.47 0.3

[5.72 - 7.19] [5.42 - 7.09] [-0.32 - 1.02] 

Glycine 1.59 (0.039) 1.56 (0.039) 0.028 (0.022) -0.033, 0.089 0.2

[1.47 - 1.74] [1.41 - 1.67] [-0.050 - 0.17] 
Monsanto Company 09-SY-201U Page 296 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Histidine 0.96 (0.023) 0.94 (0.023) 0.027 (0.010) -0.0029, 0.057 0.0

[0.88 - 1.04] [0.84 - 1.01] [-0.021 - 0.11] 

Isoleucine 1.71 (0.050) 1.67 (0.051) 0.039 (0.036) -0.064, 0.14 0.3

[1.56 - 1.88] [1.45 - 1.86] [-0.15 - 0.26] 

Leucine 2.73 (0.083) 2.69 (0.083) 0.044 (0.040) -0.065, 0.15 0.3

[2.47 - 3.01] [2.37 - 2.94] [-0.13 - 0.32] 

Lysine 2.39 (0.053) 2.33 (0.053) 0.057 (0.025) -0.011, 0.13 0.0

[2.19 - 2.55] [2.10 - 2.51] [-0.051 - 0.25] 

Methionine 0.55 (0.0088) 0.53 (0.0089) 0.020 (0.011) -0.010, 0.049 0.1

[0.51 - 0.58] [0.49 - 0.57] [-0.035 - 0.056] 

Phenylalanine 1.82 (0.056) 1.80 (0.056) 0.019 (0.027) -0.056, 0.093 0.5

[1.64 - 2.00] [1.58 - 1.99] [-0.16 - 0.22] 
Monsanto Company 09-SY-201U Page 297 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Amino Acid (% DW) Proline 1.77 (0.057) 1.72 (0.057) 0.054 (0.030) -0.029, 0.14 0.1

[1.59 - 1.95] [1.52 - 1.90] [-0.046 - 0.27] 

Serine 1.75 (0.059) 1.77 (0.059) -0.016 (0.035) -0.087, 0.056 0.6

[1.49 - 1.98] [1.45 - 1.94] [-0.22 - 0.24] 

Threonine 1.33 (0.030) 1.33 (0.030) -0.0031 (0.018) -0.041, 0.035 0.8

[1.20 - 1.45] [1.18 - 1.47] [-0.13 - 0.11] 

Tryptophan 0.42 (0.011) 0.41 (0.012) 0.0016 (0.0069) -0.017, 0.020 0.8

[0.37 - 0.46] [0.35 - 0.44] [-0.028 - 0.046] 

Tyrosine 1.25 (0.029) 1.22 (0.029) 0.026 (0.014) -0.011, 0.064 0.1

[1.17 - 1.33] [1.10 - 1.32] [-0.040 - 0.12] 

Valine 1.83 (0.052) 1.77 (0.053) 0.051 (0.038) -0.059, 0.16 0.2

[1.69 - 2.02] [1.55 - 1.96] [-0.17 - 0.28] 
Monsanto Company 09-SY-201U Page 298 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 16:0 Palmitic 2.36 (0.056) 10.83 (0.056) -8.47 (0.055) -8.62, -8.31 <0.

[2.25 - 2.44] [10.51 - 11.08] [-8.68 - -8.13] 

18:0 Stearic 3.31 (0.067) 4.50 (0.067) -1.19 (0.065) -1.37, -1.01 <0.

[3.07 - 3.82] [4.24 - 4.85] [-1.47 - -0.79] 

18:1 Oleic 76.47 (0.59) 22.81 (0.59) 53.65 (0.22) 53.17, 54.13 <0.

[73.13 - 79.17] [21.41 - 25.08] [51.71 - 55.05] 

18:2 Linoleic 10.10 (0.39) 52.86 (0.39) -42.77 (0.18) -43.17, -42.37 <0.

[7.85 - 12.42] [51.68 - 53.89] [-43.74 - -41.38] 

18:3 Linolenic 6.69 (0.28) 8.02 (0.28) -1.33 (0.072) -1.53, -1.13 <0.

[5.55 - 7.81] [6.86 - 8.60] [-1.80 - -0.59] 

20:0 Arachidic 0.30 (0.0076) 0.34 (0.0077) -0.039 (0.0071) -0.059, -0.019 0.0

[0.28 - 0.36] [0.31 - 0.36] [-0.082 - -0.022] 
Monsanto Company 09-SY-201U Page 299 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Fatty Acid (% Total FA) 20:1 Eicosenoic 0.34 (0.013) 0.19 (0.013) 0.15 (0.0086) 0.13, 0.18 <0.

[0.27 - 0.40] [0.15 - 0.21] [0.12 - 0.20] 

22:0 Behenic 0.29 (0.0037) 0.30 (0.0038) -0.0052 (0.0051) -0.017, 0.0070 0.3

[0.28 - 0.33] [0.28 - 0.31] [-0.029 - 0.020] 

24:0 Lignoceric 0.14 (0.017) 0.13 (0.017) 0.0046 (0.0046) -0.0084, 0.018 0.3

[0.066 - 0.17] [0.067 - 0.16] [-0.019 - 0.027] 

Fiber (% DW) Acid Detergent Fiber 17.14 (0.54) 16.14 (0.54) 1.00 (0.74) -1.06, 3.05 0.2

[15.71 - 19.31] [13.36 - 18.02] [-1.84 - 4.03] 

Neutral Detergent Fiber 18.44 (0.85) 17.83 (0.86) 0.60 (1.03) -2.25, 3.46 0.5

[13.41 - 22.18] [14.61 - 21.09] [-4.24 - 4.33] 

Proximate (% DW) Ash 6.06 (0.13) 6.13 (0.13) -0.072 (0.081) -0.30, 0.15 0.4

[5.46 - 6.54] [5.48 - 6.55] [-0.56 - 0.26] 
Monsanto Company 09-SY-201U Page 300 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Proximate (% DW) Carbohydrates 40.35 (0.86) 39.93 (0.86) 0.42 (0.37) -0.59, 1.43 0.3

[36.69 - 43.52] [37.46 - 42.96] [-1.08 - 2.00] 

Moisture (% FW) 10.76 (0.37) 11.56 (0.37) -0.80 (0.44) -2.02, 0.42 0.1

[8.96 - 12.30] [10.20 - 12.70] [-3.40 - 0.90] 

Protein 35.32 (0.99) 34.66 (0.99) 0.66 (0.36) -0.33, 1.65 0.1

[31.48 - 38.59] [30.71 - 37.40] [-0.94 - 3.08] 

Total Fat 18.29 (0.39) 19.33 (0.39) -1.04 (0.16) -1.39, -0.69 <0.

[16.55 - 19.50] [17.63 - 20.32] [-2.16 - -0.20] 

Vitamin (mg/100g DW) Vitamin E 2.83 (0.43) 3.27 (0.43) -0.44 (0.17) -0.92, 0.037 0.0

[1.23 - 4.36] [1.69 - 4.19] [-1.40 - 0.76] 

Antinutrient Lectin (H.U./mg DW) 2.21 (0.40) 2.45 (0.41) -0.24 (0.57) -1.57, 1.09 0.6

[0.72 - 3.77] [0.61 - 5.53] [-4.80 - 3.04] 
Monsanto Company 09-SY-201U Page 301 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Control)

Component (Units)1

MON 87705 

Mean (S.E.) 

[Range]

 Control

Mean (S.E.) 

[Range]

Mean (S.E.) 

[Range]

95% CI 

(Lower, Upper) p-V

Antinutrient (% DW)Phytic Acid 1.82 (0.052) 1.85 (0.053) -0.031 (0.041) -0.12, 0.057 0.4

[1.52 - 2.13] [1.63 - 2.19] [-0.27 - 0.18] 

Raffinose 0.58 (0.029) 0.58 (0.029) 0.00036 (0.015) -0.042, 0.043 0.9

[0.48 - 0.71] [0.50 - 0.70] [-0.088 - 0.12] 

Stachyose 3.87 (0.13) 3.70 (0.13) 0.17 (0.14) -0.22, 0.56 0.2

[3.39 - 4.48] [3.04 - 4.43] [-0.56 - 0.96] 

Trypsin Inhibitor (TIU/mg DW) 38.14 (2.60) 37.25 (2.61) 0.89 (0.97) -1.74, 3.51 0.4

[26.73 - 52.01] [27.23 - 49.78] [-5.31 - 6.98] 

Isoflavone (μg/g DW)Daidzein 1806.33 (229.35) 1794.07 (229.50) 12.26 (37.50) -68.88, 93.40 0.7

[1145.72 - 2565.56] [1092.43 - 2565.86] [-196.53 - 242.28] 

Genistein 1160.30 (115.82) 1117.27 (115.95) 43.04 (24.92) -10.74, 96.81 0.1

[809.79 - 1527.94] [751.67 - 1466.21] [-104.61 - 208.66] 
Monsanto Company 09-SY-201U Page 302 of 471

Table E-12 (continued). Statistical Summary of Combined Site Soybean Seed Amino Acid, Fatty AciVitamin, Antinutrient and Isoflavone Content for MON 87705 vs. the Conventional Control (A3525) 

 Difference (MON 87705 minus Contro

Component (Units)1 

MON 87705 

Mean (S.E.) 

[Range] 

 Control 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

p

Va

Isoflavone (μg/g DW) Glycitein 132.85 (12.38) 126.86 (12.53) 5.98 (12.18) -27.41, 39.38 0.6

[49.11 - 196.59] [72.10 - 167.04] [-61.67 - 98.54] 

1DW = dry weight; FW = fresh weight; FA = fatty acid; S.E. = standard error; CI = Confidence Interval. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean vlimits were set to zero. 
Monsanto Company 09-SY-201U Page 303 of 471

Table E-13. Literature and ILSI-CCD Ranges for Components in Soybean Forage 

and Seed 

 

Forage Tissue/Component1 Literature Range2 ILSI-CCD Range3

Proximate (% dw) 

Ash 5.36 – 8.91 6.72 – 10.78 

Carbohydrates 62.25 – 72.28 59.8 – 74.7 

Moisture (% fw) 68.50 – 78.40 73.5 – 81.6 

Protein 16.48 – 24.29 14.38 – 24.71 

Total Fat 2.65 – 9.87 1.302 – 5.132 

 

Fiber (% dw) 

Acid Detergent Fiber (ADF) 23.86 – 50.69 not available 

Neutral Detergent Fiber (NDF) 19.61 – 43.70 not available 

 

Seed Tissue Component1 Literature Range2

 ILSI Range3

Proximates (% dw) 

Ash 4.61 – 6.32 3.89 – 6.99 

Carbohydrates 32.75 – 40.98 29.6 – 50.2 

Moisture (% fw) 6.24 – 11.10 4.7 – 34.4 

Protein 34.78 – 43.35 33.19 – 45.48 

Total Fat 14.62 – 20.68 8.10 – 23.56 

 

Fiber (% dw) 

Acid Detergent Fiber (ADF) 9.22 – 26.26 7.81 – 18.61 

Neutral Detergent Fiber (NDF) 10.79 – 23.90 8.53 – 21.25 

 

Amino Acids (% dw) 

Alanine 1.62 – 1.89 1.51-2.10 

Arginine 2.57 – 3.27 2.29-3.40 

Aspartic acid 4.16 – 5.02 3.81-5.12 

Cystine/Cysteine 0.52 – 0.69 0.37-0.81 

Glutamic acid 6.52 – 8.19 5.84-8.20 

Glycine 1.59 – 1.90 1.46-2.00 

Histidine 0.96 – 1.13 0.88-1.18 

Isoleucine 1.59 – 2.00 1.54-2.08 

Leucine 2.79 – 3.42 2.59-3.62 

Lysine 2.36 – 2.77 2.29-2.84 

Methionine 0.45 – 0.63 0.43-0.68 

Phenylalanine 1.82 – 2.29 1.63-2.35 

Proline 1.83 – 2.23 1.69-2.28 

Serine 1.95 – 2.42 1.11-2.48 

Threonine 1.44 – 1.71 1.14-1.86 

Tryptophan 0.30 – 0.48 0.36-0.50 

Tyrosine 1.27 – 1.53 1.02-1.61 

Valine 1.68 – 2.09 1.60-2.20 

 
Monsanto Company 09-SY-201U Page 304 of 471

Table E-13 (continued). Literature and ILSI-CCD Ranges for Components in 

Soybean Forage and Seed 

1

fw=fresh weight; dw=dry weight; H.U. = hemagglutinating unit; TIU = trypsin inhibitor unit. 

2

Lundry et al. (2008). 3ILSI Crop Composition Database at: http://www.cropcomposition.org. 4Padgette et al. (1996). 

Conversions: % dw x 104

 = μg/g dw; mg/g dw x 103 = mg/kg dw; mg/100g dw x 10 = mg/kg dw; g/100g dw x 10 = 

mg/g dw 

 

 

Seed Tissue Component1 Literature Range2

 ILSI-CCD Range3

Fatty Acids (% dw) (% total)

8:0 Caprylic not available 0.148 – 0.148 

10:0 Capric not available not available 

12:0 Lauric not available 0.082 – 0.132 

14:0 Myristic not available 0.071 – 0.238 

14:1 Myristoleic not available 0.121 – 0.125 

15:0 Pentadecanoic not available not available 

15:1 Pentadecenoic not available not available 

16:0 Palmitic 1.44 – 2.35 9.55 – 15.77 

16:1 Palmitoleic not available 0.086 – 0.194 

17:0 Heptadecanoic not available 0.085 – 0.146 

17:1 Heptadecenoic not available 0.073 – 0.087 

18:0 Stearic 0.54 – 1.12 2.70 – 5.88 

18:1 Oleic 2.87 – 8.82 14.3 – 32.2 

18:2 Linoleic 6.48 – 11.6 42.3 – 58.8 

18:3 Gamma Linolenic not available not available 

18:3 Linolenic 0.72 – 2.16 3.00 – 12.52 

20:0 Arachidic 0.04 – 0.7 0.163 – 0.482 

20:1 Eicosenoic 0.026 – 0.057 0.140 – 0.350 

20:2 Eicosadienoic not available 0.077 – 0.245 

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.044 – 0.073 0.277 – 0.595 

22:1 Erucic not available not available 

24:0 Lignoceric 0.13 – 0.244

 not available 

 

Vitamins (mg/100g dw) 

Vitamin E 1.29 – 4.80 0.19-6.17 

 

Antinutrients 

Lectin (H.U./mg fw) 0.45 – 9.95 0.09 – 8.46 

Trypsin Inhibitor (TIU/mg dw) 20.79 – 59.03 19.59 – 118.68 

Phytic Acid (% dw) 0.41 – 1.92 0.63 – 1.96 

 

Isoflavones (μg/g dw) (mg/kg dw)

Daidzein 224.03 – 1485.52 60.0 – 2453.5 

Genistein 338.24 – 1488.89 144.3 – 2837.2 

Glycitein 52.72 – 298.57 15.3 – 310.4 

 

Bio-Actives (% dw) 

Raffinose 0.26 – 0.84 0.21 – 0.66 

Stachyose 1.53 – 2.98 1.21 – 3.50 

 
Monsanto Company 09-SY-201U Page 305 of 471

E.6. Compositional Comparison of Processed Fractions from Soybean Seed of 

MON 87705 and the Conventional Control 

To prepare soybean processed fractions, seed samples were collected from field trials 

conducted with MON 87705 and the conventional control at two field sites (Jefferson 

County, IA and Clinton County, IL) in the U.S. during the 2007 growing season. In 

addition, 12 commercial conventional soybean reference varieties were grown separately 

at three field sites in the U.S. to determine a 99% tolerance interval for each component 

analyzed. The seed samples were processed into defatted toasted soybean meal (TD 

soybean meal); refined, bleached, and deodorized soybean oil (RBD oil); protein isolate; 

and crude lecithin fractions. The processed fractions were analyzed according to the 

principles outlined in the OECD consensus document for soybean composition (OECD, 

2001). Samples from all three replicates of MON 87705, the control, and the 12 

references were analyzed from all plots. The TD soybean meal was analyzed for 

proximates (moisture, protein, fat, ash and carbohydrates by calculation), ADF, NDF, 

amino acids, trypsin inhibitors and phytic acid. The RBD oil was analyzed for fatty acids 

and vitamin E (α-tocopherol). The protein isolate fraction was analyzed for amino acids 

and moisture. The crude lecithin fraction was analyzed for phosphatides (α-phosphatidic 

acid, α phosphatidylcholine, α phosphatidylethanolamine, and α-phosphatidylinositiol). 

Compositional analyses were conducted to assess whether the processed fractions 

prepared from MON 87705 are comparable to those of the conventional control, A3525, 

which has background genetics similar to MON 87705, but lacks the intentionally 

modified fatty acid profile and glyphosate tolerance traits. Statistically significant 

differences were determined at the 5% level of significance (p<0.05) using established 

statistical methods. In addition, 12 commercial conventional soybean varieties were 

grown and analyzed separately 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. The methods used for composition analysis of the processed fractions are 

summarized in Section E.7. The statistical analysis compared MON 87705 and the 

conventional control across the two sites (combined-site). A summary of the significant 

differences observed between the processed fractions prepared from the seed of 

MON 87705 and the conventional control is included in Table E-15. A statistical 

summary of the composition of each processed fraction is included in Tables E-16 to E19. Literature ranges are provided in Table E-20 for soybean meal, Table E-21 for 

protein isolate, Table E-22 for soybean lecithin, and Table E-23 for soybean oil. 

Results show that except for the intended changes in fatty acid composition, minor 

differences in the levels of less abundant fatty acids in RBD oil and occurrence of low 

levels of minor fatty acids due to spontaneous isomerisation during the oil refining 

process, the processed fractions produced from MON 87705 are compositionally 

equivalent to those of conventional soybean. Further details are provided below. 

E.6.1. Composition of TD Soybean Meal 

Comparison of the composition of TD soybean meal prepared from MON 87705 and the 

conventional soybean control showed no differences (p>0.05) for 21 of the 27 

components analyzed. Significant differences (p<0.05) were observed for six 
Monsanto Company 09-SY-201U Page 306 of 471

components (Table E-15): alanine, glycine, isoleucine, lysine, valine, and NDF. For the 

statistically significant amino acid differences, the absolute magnitude of the mean 

differences from the control were small (<0.1% dw) and the MON 87705 mean values 

fell within the 99% tolerance interval for the conventional soybean varieties and also 

within the range of published values for conventional soybean. For the statistically 

significant difference observed for NDF, the absolute magnitude of the mean difference 

from the control was small (1.7% dw) and the MON 87705 mean value was within the 

99% tolerance interval for the conventional soybean varieties and also within the range of 

published values for conventional soybean (Table E-20). Therefore, these differences 

were not considered biologically relevant from a food and feed safety or nutritional 

perspective. 

As expected, the TD soybean meal from MON 87705 contained a small amount of total 

fat or oil (0.78% dw), which was also present at similar levels (0.86% dw) in the meal 

from the conventional control (Table E-16). TD soybean meal is expected to contain oil, 

and accordingly, the National Oil Processors Association has established a minimum oil 

content (0.5% dw) for defatted soybean meal that meets quality standards and guidelines 

for soybean meal for domestic and international shipping (NOPA, 2006). Although not 

analyzed for, the composition of the residual oil present in TD soybean meal is expected 

be consistent with the intended fatty acid changes observed in seed and in RBD oil (see 

discussion in Section E.6.2 below). Based on these results, apart from the intended fatty 

acid changes in residual oil, the meal from MON 87705 soybean is considered 

compositionally equivalent to the meal from conventional soybean. 

E.6.2. Composition of RBD Oil 

Of the 38 fatty acids analyzed, 21 were excluded from statistical analysis since more than 

50% of the observations were below the assay limit of quantitation. Of the 17 fatty acids 

that could be statistically analyzed, significant differences (p<0.05) between MON 87705 

and control RBD oil were observed for 13 fatty acids (Table E-15). Four of the 13 

differences were expected as they were due to the intended changes in fatty acid levels. 

Thus, 16:0 palmitic acid levels were significantly lower in MON 87705 (2.49% total FA) 

than the control (11.59% total FA), 18:0 stearic acid levels were significantly lower in 

MON 87705 (3.22% total FA) than the control (4.47% total FA); 18:1 oleic acid levels 

were significantly higher in MON 87705 (71.51% total FA) than the control (23.16% 

total FA), and 18:2 linoleic acid levels were significantly lower in MON 87705 (14.41% 

total FA) than the control (51.08% total FA) (Table E-15). 

In addition to the intended changes, six fatty acids were detected in RBD oil that were not 

detected in seed: 14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric (heptadecanoic) 

acid, 17:1 9c heptadecenoic acid, 18:2 other trans isomer fatty acids (excluding 9t,12t 

linolelaidic), and 18:2 6c,9c, octadecadienoic acid. As observed in seed, levels of several 

less abundant fatty acids were significantly different (p<0.05) between the RBD oil from 

MON 87705 and the conventional control. For six of the nine remaining fatty acids 

where a difference was observed (14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric 

[heptadecanoic] acid, 20:0 arachidic, 20:1 eicosenoic and 22:0 behenic acids), the 

absolute magnitude of the differences was small (<0.15% total FA), and the MON 87705 
Monsanto Company 09-SY-201U Page 307 of 471

mean values fell within the 99% tolerance intervals for the reference soybean varieties 

and/or within published ranges for conventional soybean oil (Codex, 2005). 

A significant increase in the level of the minor fatty acid 17:1 9c heptadecenoic acid was 

observed in MON 87705 RBD oil (0.12% total FA) compared to its level in control RBD 

oil (0.031% total FA). This is not unexpected given the intended shift in fatty acid levels 

in MON 87705. The mean level of 17:1 9c heptadecenoic acid in MON 87705 RBD oil 

was higher than the level in the commercial reference varieties (<0.06% total FA) and 

slightly outside reported literature values for soybean oil (ND-0.1% total FA; Codex, 

2005). However, 17:1 9c heptadecenoic acid is known to be present in other vegetables 

oils such as canola (0.3% total FA), corn (0.1% total FA), peanut (0.1% total FA), high 

oleic safflower (0.1% total FA), and high oleic sunflower (0.1% total FA) (Codex, 2005). 

The presence of 17:1 9c heptadecenoic acid has also been documented in a variety of 

foods, with levels highest in meats and oils. As shown in the Table E-14, comparable or 

higher intakes of 17:1 9c heptadecenoic acid can be achieved in a single serving of tofu, 

ground beef or soft-spread margarine compared to a serving of MON 87705 RBD oil. 

Therefore, it is concluded that there are no adverse food and feed safety or nutrition 

effects associated with the levels of 17:1 9c heptadecenoic acid observed in MON 87705 

RBD oil. 
Monsanto Company 09-SY-201U Page 308 of 471

Table E-14. Estimation of 17:1 9c Heptadecenoic Acid Intake in Selected Foods and 

MON 87705 

Food Level of 17:1 

(g/100 g food) 

 RACC1

 (g) 

Amount of 17:1 

consumed per 

serving (mg/RACC) 

Extra firm nigari tofu 

(16159)2 1.085 85 922.0 

Ground beef, 80% lean 

(23572)2 0.135 85 115.0 

Soft-spread margarine, 

80% fat, canola based 

(04684)2

0.053 14 7.0 

MON 87705 0.12 14 17.0 

1

Reference Amount Customarily Consumed, which is the U.S. serving size for labeling purposes (21CFR 

101.12) 

2

Number in parentheses refers to Nutrient Database Number used to identify a particular food within the 

USDA-ARS food composition database. 

The 18:2 other trans fatty acids and 18:2 6c,9c, octadecadienoic acid observed in both 

MON 87705 and the conventional control RBD oil were not observed in seed fatty acid 

analyses and are believed to arise from the spontaneous isomerization of unsaturated fatty 

acids during the oil refining process, particularly during the heat-requiring deodorization 

step (Chardigny et al. 1996). The primary source of industrially produced trans fatty 

acids (TFAs) in the human diet is the consumption of hydrogenated vegetable oils in 

liquid or solid form resulting in food products that may contain in excess of 30% TFA 

(Chardigny et al. 1996; Ledoux et al, 2007). Naturally occurring trans fats also arise as a 

result of bacterial reduction of unsaturated fatty acids in the gut of ruminant animals, with 

intake resulting from consumption of meat and dairy products (Chardigny et al. 1996; 

Ledoux et al., 2007). 

The mean level of 18:2 other trans fatty acids in MON 87705 RBD oil was significantly 

lower than in control RBD oil, and both values were outside the range of the commercial 

reference varieties. This is likely due to minor differences in processing methods as the 

commercial reference soybean varieties were grown and processed separately from MON 

87705 and the conventional control. However, the levels of 18:2 other trans fatty acids 

in MON 87705 RBD oil were within the range of total TFA content in samples of 

unhydrogenated commercial soybean oil (<3.5%) (Chardigny et al., 1996; Ledoux et al.,

2007; Wolff, 1993). The contribution of MON 87705 RBD oil to overall dietary TFA 

intake will be minimal relative to commonly experienced dietary intakes, and therefore, 

are not biologically relevant from a food and feed safety or nutritional perspective. 
Monsanto Company 09-SY-201U Page 309 of 471

The mean level of 18:2 6c,9c, octadecadienoic acid in MON 87705 RBD oil was 

significantly lower than in conventional control RBD oil, and both values were outside 

the range of the commercial references. This is likely due to minor differences in 

processing methods as the commercial reference soybean varieties were grown and 

processed separately from MON 87705 and the conventional control. It has been 

reported that although most fatty acid double bonds are in the cis configuration, some 

processes (such as heat treatment) may lead to the migration of double bonds from their 

naturally occurring positions in the carbon chain, leading to an increase in the levels of 

other cis isomers (Ledoux et al., 2007). 

The RBD oil was also analyzed for vitamin E levels which were not significantly 

different (p>0.05) between MON 87705 and conventional control RBD oil. 

In summary, except for intended changes in fatty acid composition, minor differences in 

the levels of less abundant fatty acids and occurrence of low levels of minor fatty acids 

due to spontaneous isomerisation during the oil refining process, the RBD oil from 

MON 87705 is considered compositionally equivalent to oil from conventional soybean. 

E.6.3. Composition of Soybean Protein Isolate 

There were no statistically significant differences (p<0.05) between MON 87705 and the 

conventional control for components measured in the protein isolate fraction. Statistical 

summary data on the composition of the soybean protein isolate fraction is included in 

Table E-18. Based on these results, the protein isolate prepared from MON 87705 is 

considered compositionally equivalent to protein isolate from conventional soybean. 

E.6.4. Composition of Crude Lecithin 

There were no statistically significant differences (p<0.05) between MON 87705 and the 

conventional control for phosphatides of crude lecithin. Statistical summary data on the 

composition of the crude lecithin fraction is included in Table E-19. Based on these 

results, the MON 87705 soybean lecithin is considered compositionally equivalent to 

conventional soybean lecithin. 

E.6.5. Compositional Equivalence of MON 87705 and Conventional Soybean 

Processed Fractions 

The processed fractions, TD soybean meal, RBD oil, protein isolate and crude lecithin, 

were analyzed according to the principles outlined in the OECD consensus document for 

soybean composition (OECD, 2001). There were no statistically significant differences 

(p<0.05) observed between MON 87705 and the conventional control (A3525) for the 

components measured in protein isolates or crude lecithin. Significant differences 

(p<0.05) were observed for six (alanine, glycine, isoleucine, lysine, valine, and NDF) of 

27 components measured in TD soybean meal; however, the magnitude of the differences 

from the conventional control was small and the MON 87705 mean values were within 

the 99% tolerance interval for the conventional reference soybean varieties and/or within 

the range of published values for conventional soybean meal, indicating that they were 

not biologically meaningful. The low levels of oil (0.78% dw, as total fat) present in TD 

soybean meal are expected to reflect the intended fatty acid changes observed in seed. 
Monsanto Company 09-SY-201U Page 310 of 471

As expected, and consistent with results obtained for seed fatty acid levels, the intended 

fatty acid changes were observed in the RBD oil fraction from MON 87705. As observed 

in seed, levels of several less abundant fatty acids were significantly different (p<0.05) 

between the RBD oil from MON 87705 and the conventional control. Of the 17 fatty 

acids that could be statistically analyzed in RBD oil, significant differences (p<0.05) 

between MON 87705 and the conventional control were observed for 13 fatty acids. 

Four of the 13 differences were expected as they were due to the intended changes in 

fatty acid levels (16:0 palmitic, 18:0 palmitic, 18:1 oleic and 18:2 linoleic). For six of the 

nine remaining differences (14:0 myristic acid, 16:1 palmitoleic acid, 17:0 margaric 

[heptadecanoic] acid, 20:0 arachidic, 20:1 eicosenoic and 22:0 behenic acids), the 

magnitude of the differences was small (<0.15% total FA), and the MON 87705 mean 

values fell within the 99% tolerance intervals for the reference varieties and/or within 

published ranges for conventional RBD soybean oil (Codex, 2005, Table E-23). 

The remaining three differences were for 17:1 9c heptadecenoic acid, 18:2 trans isomer 

fatty acids (excluding linolelaidic), and 18:2 6c,9c, octadecadienoic acid. A significant 

increase in the level of the minor fatty acid 17:1 9c heptadecenoic acid was observed in 

MON 87705 compared to conventional control RBD oil. This is not unexpected given 

the intended shift in fatty acid levels in MON 87705. The mean level of 17:1 9c 

heptadecenoic acid in MON 87705 was outside the range of values obtained for the RBD 

oil from commercial reference soybean varieties. However, 17:1 9c heptadecenoic acid 

is present at similar or higher levels in a variety of oils (canola, corn, peanut, high oleic 

safflower, and high oleic sunflower) and foods (tofu, ground beef, and soft-spread 

margarine). Therefore, it is concluded that there are no adverse food and feed safety or 

nutrition effects associated with the levels of 17:1 9c heptadecenoic acid observed in 

MON 87705 soybean oil. The minor fatty acids, 18:2 other trans (excluding linolelaidic) 

and 18:2 6c,9c, octadecadienoic acid, are believed to arise from the spontaneous 

isomerization of unsaturated fatty acids during the oil refining process. 

Therefore, this compositional assessment supports the conclusion that, except for the 

intended changes in fatty acid composition, minor differences in the levels of less 

abundant fatty acids and occurrence of low levels of minor fatty acids due to spontaneous 

isomerisation during the oil refining process, the processed fractions produced from 

MON 87705 are compositionally equivalent to those of conventional soybean.
Monsanto Company 09-SY-201U Page 311 of 471

Table E-15. Summary of Differences (p<0.05) for the Comparison of Soybean Processed Fraction Compon

MON 87705 vs. the Conventional Control (A3525) and Reference Varieties 

 Mean Difference 

(MON 87705 minus Control) 

Component (Units)1 

MON 87705

Mean 

Control 

Mean 

Mean Difference 

(% of A3525) 

Signif. 

(p-Value) 

Test 

Range 

(99

Meal Amino Acid (% DW) Alanine 2.26 2.33 -3.06 0.019 2.22 - 2.29 

 

Glycine 2.28 2.34 -2.44 0.023 2.26 - 2.33 

 

Isoleucine 2.45 2.53 -3.17 0.006 2.40 - 2.51 

 

Lysine 3.25 3.34 -2.49 0.030 3.22 - 3.29 

 

Valine 2.55 2.64 -3.47 0.003 2.51 - 2.60 

 

Meal Fiber (% DW) Neutral Detergent Fiber 8.55 6.81 25.47 0.016 8.05 - 8.96 

 

RBD Oil Fatty Acid (% Total FA) 

14:0 Myristic 0.031 0.090 -65.15 <0.001 0.031 - 0.032 

 

16:0 Palmitic 2.49 11.59 -78.50 <0.001 2.36 - 2.69 

 

16:1 Palmitoleic 0.13 0.11 22.10 0.012 0.12 - 0.14 

 

17:0 Margaric [Heptadecanoic] 0.036 0.10 -65.27 0.002 0.031 - 0.048 

 

17:1 9c Heptadecenoic 0.12 0.031 279.62 0.006 0.092 - 0.14 

 

18:0 Stearic 3.22 4.47 -28.05 <0.001 3.00 - 3.40 

 
Monsanto Company 09-SY-201U Page 312 of 471

Table E-15 (continued). Summary of Differences (p<0.05) for the Comparison of Soybean Processed FLevels for MON 87705 vs. the Conventional Control (A3525) and Reference Varieties 

 Mean Difference 

(MON 87705 minus Control) 

Component (Units)1 

MON 87705

Mean 

Control 

Mean 

Mean Difference 

(% of A3525) 

Signif. 

(p-Value) 

Test 

Range 

(99

RBD Oil Fatty Acid (% Total FA) 

18:1 Oleic 71.51 23.16 208.71 <0.001 69.30 - 73.01 

 

18:2 6c,9c Octadecadienoic 0.20 0.65 -69.86 <0.001 0.16 - 0.24 

18:2 Linoleic 14.41 51.08 -71.78 <0.001 12.25 - 17.39 

 

18:2 Other Trans 0.18 0.63 -70.92 <0.001 0.14 - 0.23 

 

20:0 Arachidic 0.29 0.36 -19.97 0.001 0.27 - 0.31 

 

20:1 11c Eicosenoic 0.33 0.19 73.17 <0.001 0.29 - 0.37 

 

22:0 Behenic 0.31 0.35 -9.74 0.001 0.30 - 0.35 

 

1 DW = dry weight; FA = fatty acid. 

2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were se
Monsanto Company 09-SY-201U Page 313 of 471

E.7. Methods and Materials for Compositional Analysis of Processed Fractions 

MON 87705 and a conventional soybean control (A3525) were grown at two U.S. 

locations in 2007. MON 87705 was grown from seed lots GLP-0704-18516-S and GLP0705-18715-S, and the conventional soybean control was grown from seed lots GLP0704-18512-S and GLP-0705-18716-S. The conventional soybean control has 

background genetics representative of MON 87705 but does not contain cp4 epsps or the 

FATB1-A and FAD2-1A gene segments. In addition, 12 commercial conventional 

soybean varieties produced at three locations in separate U.S. field trials in 2007 were 

included for the generation of a 99% tolerance interval. The conventional varieties, 

locations, and seed lot numbers are listed below: 

Material Name Starting Seed Lot No. Field Site 

Anand GLP-0705-18678-S AR 

UA4805 GLP-0705-18679-S AR 

Ozark GLP-0705-18680-S AR 

Delta & Pine 5989 GLP-0705-18681-S AR 

H437 GLP-0705-18686-S IL 

NK S38-T8 GLP-0705-18687-S IL 

LG C3540 GLP-0705-18688-S IL 

HS38C60 GLP-0705-18689-S IL 

NuPride 2954 GLP-0705-18682-S NE 

NC+ 2A86 GLP-0705-18683-S NE 

Pioneer 92B72 GLP-0705-18684-S NE 

NK 25-J5 GLP-0705-18685-S NE 

E.7.1. Characterization of the Materials 

The identities of MON 87705, the conventional soybean control, and conventional 

reference soybean varieties were verified prior to use by confirming the chain-of-custody 

documentation of the samples from the field cooperators. The seed samples from 

MON 87705 and the conventional soybean control were further characterized by an 

event-specific PCR analysis of the DNA extracted from the seed to confirm the presence 

or absence of MON 87705. 
Monsanto Company 09-SY-201U Page 314 of 471

E.7.2. Field Production of the Samples 

Seed of the MON 87705 and the conventional soybean control were collected from two 

plots at each of two sites across the U.S. during the 2007 growing season. The field sites 

were in Clinton County, IL and Jefferson County, IA. The reference varieties were 

produced separately from one plot grown at one of three sites in the U.S. in 2007. The 

reference soybean varieties were planted at field sites in Jackson County, AR, Clinton 

County, IL, and York County, NE. All the reference varieties were grown under normal 

agronomic field conditions for their respective geographic regions. Seed was shipped at 

ambient temperature from all production locations to Monsanto Company, St. Louis, MO, 

USA. A subsample for compositional analyses was obtained from each seed sample. The 

seed subsamples were ground and stored in a freezer set to maintain -20° C at Monsanto 

Company (St. Louis, MO) prior to transfer to Covance Laboratories, Inc. (Madison, WI). 

The labels on the samples shipped to Covance laboratories Inc. listed the Monsanto study 

number, crop and sample type, production site and plot number, storage conditions, 

unique sample ID, lot or source number, material name, container type, and contact name. 

Additionally, a subsample of each MON 87705, conventional control and reference seed 

sample was shipped from Monsanto Company (St. Louis, MO) to GLP-Technologies 

(GLP-T) in Navasota, Texas, for processing into TD soybean meal, refined, RBD oil, 

protein isolate and crude lecithin. A subsample for use in compositional analysis was 

obtained from each processed sample generated at GLP-T, and was shipped on dry ice to 

Monsanto Company (St. Louis, MO). The sample containers were relabeled at Monsanto 

with the following information: Monsanto study number, processing site, sample 

identifier, lot number, crop and sample type, material name, container type, contact name, 

and storage conditions, and then shipped overnight on dry ice to Covance Laboratories 

Inc. (Madison, WI) for analyses. 

E.7.3. Compositional Analysis Methods for Processed Fractions 

The compositional analysis methods for ADF, amino acids, ash, carbohydrates, fat, 

isoflavones, lectin, moisture, NDF, phytic acid, protein, and vitamin E were the same as 

described in Section E.6. Methods for analytes that had different standards and/or 

detection limits and the method for phosphatides, are described below. 

E.7.3.1. Fatty Acid Profile with Trans Fat by GC 

The lipid was extracted, saponified with 0.5N methanolic sodium hydroxide, and 

methylated with 14% BF3-methanol. The resulting methyl esters of the fatty acids were 

extracted with heptane. An internal standard was added prior to the lipid extraction. The 

methyl esters of the fatty acids were analyzed by gas chromatography using external 

standards for quantitation. The limit of quantitation was 0.0200% – 0.0600% depending 

on the matrix. 

Reference Standards 
Monsanto Company 09-SY-201U Page 315 of 471

• Nu Chek Prep GLC Reference Standard Hazelton No. 1, Lot Number JY19-R, 

100%* 

• Nu Chek Prep GLC Reference Standard Hazelton No. 2, Lot Number M13-O, 

100%* 

• Nu Chek Prep GLC Reference Standard Hazelton No. 3, Lot Number MA18-S, 

100%* 

• Nu Chek Prep GLC Reference Standard Hazelton No. 4, Lot Number JY19-R, 

100%* 

• Nu Chek Prep Methyl Gamma Linolenate, used as 100% , Lot Number U-63MJY12-R 

• Nu Chek Prep Methyl Tridecanoate, used as 100%, Lot Number N-13M-F5-S 

• Nu Chek Prep Methyl Butyrate, used as 100%, Lot Number N-4M-J20-R 

• Nu Chek Prep Methyl Hexanoate, used as 100%, Lot Number N-6M-A25-R 

• Nu Chek Prep Methyl Erucate, used as 100%, Lot Number U-79M-AU3-Q 

• Nu Chek Prep Methyl Lignocerate, used as 100%, Lot Number N-24M-F5-S 

• Nu Chek Prep Methyl Docosapentaenoate, used as 100%, Lot Number U101M-F18-S 

• Nu Chek Prep Methyl Docosahexaenoate, used as 100%, Lot Number U-84MD24-R 

• Nu Chek Prep Methyl Eicosapentaenoate, used as 100%, Lot Number U-99MD14-R 

• Cayman Chemicals Steariodonic Acid Methyl Ester, used as 100%, Lot 

Number 186208-192001 and 186208-192002 

• Nu Chek Prep Methyl Elaidate, used as 100%, Lot Number U-47M-JA18-R 

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-F27-R 

• Nu Chek Prep Methyl Nervonate, used as 100%, Lot Number U-88M-O19-R 

• Nu Chek Prep Methyl Palmitelaidate, used as as 100%, Lot Number U-41MO26-R 

• Monsanto Mono Trans SDA, 99%, Lot Number GLP-0804-19309-A 

• Monsanto Alpha Linolenic Acid, used as 100%, Lot Number GLP-0804-19308-

A 

• Monsanto 9c, 15c Octadecadienoate (Omnisoy), used as 100%, Lot Number 

GLP-0802-19168-A 

• Larodan Methyl 6(z), 9(z)-Octadecadienoate, used as 99.6%, Lot Number LS113 

• Monsanto Omnisoy C17:1 Methyl 9-cis-Heptadecenoate, used as 99%, Lot 

Number GLP-0806-19436-A 

* Overall purity of the sum of the mixture components 

Literature Reference 
Monsanto Company 09-SY-201U Page 316 of 471

AOCS. 1997. Method Ce 1-62 in Official Methods and Recommended Practices of the 

AOCS, 5th ed. American Oil Chemists' Society, Champaign, IL. 

E.7.3.2. Isoflavones Analysis 

The sample was extracted using a solution of hydrochloric acid and reagent alcohol 

heated on steam baths or hot plates. The extract was brought to volume, diluted, and 

centrifuged. An aliquot of the supernatant was placed onto a C18 solid-phase extraction 

column. Unwanted components of the matrix were rinsed off with 20% methanol and 

then the isoflavones were eluted with 80% methanol. The sample was analyzed on a 

HPLC system with ultraviolet detection and was compared to an external standard curve 

of known standards for quantitation. The limit of quantitation for each component was 

10.0 μg/g. 

Reference Standards 

• Indofine, Daidzein, 99%, Lot Number 020508146

Indofine, Genistein, >99%, (used as 100%), Lot Number 0309074 

Indofine, Glycitein, 99%, Lot Number 0704034 

Literature References 

Seo, A. and C.V. Morr. 1984. Improved high-performance liquid chromatographic 

analysis of phenolic acids and isoflavonoids from soybean protein products. Journal of 

Agricultural and Food Chemistry, 32(3):530-533. 

Pettersson, H., and K.H. Kiessling. 1984. Liquid Chromatographic Determination of the 

Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official 

Analytical Chemists Journal, 67(3):503-506. 

E.7.3.3. Phosphatides 

The sample was extracted with a 98% CHCl3 2% MeOH solvent. The extract was then 

analyzed on an HPLC system equipped with an evaporative light-scattering detector 

(ELSD). A calibration curve was used for quantification. The LOQs for these assays 

were as follows: L-alpha-Phosphatidic Acid 0.70%, L-alpha-Phosphatidylcholine 1.30%, 

L-alpha-Phosphatidylethanolamine 1.30%, and L-alpha-Phosphatidylinositol 0.70%. 

Reference Standards 

• (PA) – Avanti Polar Lipids, L-alpha-Phosphatidic Acid (sodium salt), 100%, 

Lot Numbers SPA-19 and SPA-20. 

• (PC) – Avanti Polar Lipids, L-alpha-Phosphatidylcholine, 100%, 

Lot Numbers PPC-116f and PPC-117 
Monsanto Company 09-SY-201U Page 317 of 471

• (PE) – Avanti Polar Lipids, L-alpha-Phosphatidylethanolamine, 100%, 

Lot Numbers PPE-133 and PPE-133c. 

• (PI) – Avanti Polar Lipids, L-alpha-Phosphatidylinositol (Sodium salt), 100%, 

Lot Numbers PPI-151 and PPI-154. 

Literature Reference 

 AOCS Official Method Ja 7b-91. 1997. Determination of Lecithin Phospholipids by 

HPLC. 

E.7.3.4. Raffinose and Stachyose 

The sample was extracted with deionized water and the extract treated with a 

hydroxylamine hydrochloride solution in pyridine, containing phenyl-β-D-glucoside as 

an internal standard. The resulting oximes were converted to silyl derivatives by 

treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas 

chromatography using a flame ionization detector. The limit of quantitation was 

0.0500%. 

Reference standards 

• Sigma, Raffinose Pentahydrate, 99% / 84.0% after correction for degree of 

hydration, Lot Number 035K1371 

• Sigma, Stachyose, 98% / 96.4% after correction for degree of hydration, Lot 

Number 065K3775 

Literature References 

Brobst, K. M. 1972. Gas-Liquid Chromatography of Trimethylsilyl Derivatives in 

Methods in Carbohydrate Chemistry, Vol. 6. Academic Press, New York. 

Mason, B.S., and H.T. Slover. 1971. A Gas Chromatographic Method for the 

Determination of Sugars in Foods. Journal of Agricultural and Food Chemistry, 

19(3):551-554. 

 
Monsanto Company 09-SY-201U Page 318 of 471

E.7.3.5. Trypsin Inhibitor 

The sample was ground and defatted with petroleum ether. A sample of matrix was 

extracted for three hours with 0.01 N sodium hydroxide. Varying aliquots of the sample 

suspension were exposed to a known amount of trypsin and benzoy1-DLarginine~p~nitroanilide hydrochloride. The sample was allowed to react for 10 minutes 

at 37°C. After 10 minutes, the reaction was halted by the addition of acetic acid. The 

solution was centrifuged, then the absorbance was determined at 410 nm. Trypsin 

inhibitor activity was determined by photometrically measuring the inhibition of trypsin’s 

reaction with benzoyl-DL-arginine~p~nitroanilide hydrochloride. The limit of 

quantitation was 1.00 Trypsin Inhibitor Units (TIU)/mg. 

Literature Reference 

AOCS. 1997. Method Ba 12-75 in Official Methods and Recommended Practices of the 

American Oil Chemists’ Society. AOCS Press, Champaign, IL. 

E.8 Data Processing 

After compositional analyses were performed at Covance Laboratories Inc., data 

spreadsheets were forwarded to Monsanto Company. The data were reviewed, formatted, 

and sent to Certus International, Inc. for statistical analysis. A statistical sub-report was 

generated by Certus International, Inc. and sent to Monsanto Company. In all, 27 

components were analyzed in meal, 39 in RBD oil, 19 in protein isolates, and four in 

lecithin. The following RBD oil components were excluded from statistical analysis: 8:0 

caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:1 myristoleic acid, 15:0 pentadecanoic 

acid, 15:1 10c pentadecenoic acid, 16:1 palmitelaidic, 18:1 elaidic acid, 18:2 9c,15c 

octadecadienoic acid, 18:2 linolelaidic acid, 18:3 gamma linolenic acid, 18:4 

6c,9c,12c,15t trans-SDA, 18:4 stearidonic acid, 20:2 11c, 14c eicosadienoic acid, 20:3 

11c,14,17c eicosatrienoic acid, 20:4 arachidonic acid, 20:5 5c,8c,11c,14c,17c 

eicosapentaenoic acid, 22:1 erucic acid, 22:5 7c,10c,13c,16c,19c docosapentaenoic acid, 

22:6 4c,7c,10c,13c,16c,19c docosehexaenoic acid, and 24:1 nervonic acid. The LOQ for 

the oil fatty acid method was 0.0600% fresh weight. 

The following observations were below the LOQ: five observations for trypsin inhibitor 

in meal; eight observations for 14:0 myristic acid, seven observations for 17:0 

heptadecenoic acid, and eight observations for 17:1 9c heptadecenoic acid in RBD oil; 

four observations for L-alpha-phosphatidic acid and two observations for L-alphaphosphatidylethanolamine in lecithin. These observations were assigned a value equal to 

half the LOQ for the respective assay. 

The data were assessed for potential outliers using a studentized PRESS residuals 

calculation. No values in the data set were removed as outliers. 
Monsanto Company 09-SY-201U Page 319 of 471

E.9. Statistical Methodology 

The compositional components for MON 87705 and the conventional soybean control 

were statistically analyzed across all sites using a mixed-model analysis of variance. The 

combined-site analyses used the model: 

(1) Yij = U + Ti + Bj + eij,

where Yij = unique individual observation, U = overall mean, Ti = substance effect, Bj = 

random replication block effect, and eij = residual error. For the processed fractions 

compositional components, MON 87705 was compared to the conventional soybean 

control. 

A range of observed values from the reference soybean varieties was determined for each 

analytical component. Additionally, the reference soybean varieties’ data were used to 

develop population tolerance intervals. A tolerance interval is an interval that one can 

claim, with a specified degree of confidence, contains at least a specified proportion, p, of 

an entire sampled population for the parameter measured. For each compositional 

component, 99% tolerance intervals were calculated that are expected to contain, with 

95% confidence, 99% of the quantities expressed in the population of conventional 

reference soybean varieties (Ridley et al., 2002; George et al., 2004). Each tolerance 

interval estimate was based upon one observation per unique reference variety. A single 

replicate from each unique reference variety was analyzed for inclusion in tolerance 

interval calculations. Because negative quantities are not possible, calculated negative 

lower tolerance bounds were set to zero. SAS programming was used to generate all 

summary statistics and perform all analyses (SAS Institute 2002-2003). Report tables 

present p-values from SAS as either <0.001 or the actual value truncated to three 

decimals.
Monsanto Company 09-SY-201U Page 320 of 471

Table E-16. Comparison of Amino Acid, Fiber, Proximate, and Antinutrient Content from MON 877Conventional Control (A3525) Soybean Meal Processed from a 2007 U.S. Field Production 

Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E.

[Range] 

95% CI1

(Lower, Upper) p-Value 

[99

Amino Acid (% DW) 

Alanine 2.26 (0.021) 2.33 (0.021) -0.071 (0.016) -0.12, -0.022 0.019  [2.22 - 2.29] [2.26 - 2.38] Arginine 4.22 (0.074) 4.31 (0.074) -0.091 (0.075) -0.33, 0.15 0.312  [4.01 - 4.37] [4.17 - 4.49] Aspartic Acid 5.95 (0.068) 6.16 (0.068) -0.21 (0.066) -0.42, 0.0058 0.053  [5.83 - 6.14] [5.99 - 6.32] Cystine 0.82 (0.028) 0.81 (0.028) 0.0016 (0.019) -0.059, 0.062 0.938  [0.75 - 0.90] [0.78 - 0.87] Glutamic Acid 9.43 (0.11) 9.78 (0.11) -0.35 (0.14) -0.79, 0.091 0.086  [9.17 - 9.70] [9.48 - 9.98] Glycine 2.28 (0.018) 2.34 (0.018) -0.057 (0.013) -0.099, -0.015 0.023  [2.26 - 2.33] [2.29 - 2.38] Histidine 1.40 (0.019) 1.44 (0.019) -0.041 (0.016) -0.093, 0.012 0.089  [1.38 - 1.43] [1.39 - 1.50] Isoleucine 2.45 (0.021) 2.53 (0.021) -0.080 (0.012) -0.12, -0.043 0.006  [2.40 - 2.51] [2.50 - 2.58] 
Monsanto Company 09-SY-201U Page 321 of 471

Table E-16 (continued). Comparison of Amino Acid, Fiber, Proximate, and Antinutrient Content frand Conventional Control (A3525) Soybean Meal Processed from a 2007 U.S. Field Production 

 Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[9

Amino Acid (% DW)

Leucine 4.02 (0.038) 4.14 (0.038) -0.11 (0.038) -0.23, 0.0060 0.056  [3.94 - 4.13] [4.04 - 4.22] Lysine 3.25 (0.019) 3.34 (0.019) -0.083 (0.022) -0.15, -0.015 0.030  [3.22 - 3.29] [3.28 - 3.39] Methionine 0.82 (0.024) 0.84 (0.024) -0.021 (0.025) -0.10, 0.057 0.452  [0.76 - 0.89] [0.79 - 0.89] Phenylalanine 2.70 (0.030) 2.79 (0.030) -0.093 (0.032) -0.19, 0.0074 0.060  [2.65 - 2.78] [2.71 - 2.86] Proline 2.65 (0.040) 2.76 (0.040) -0.11 (0.037) -0.23, 0.0099 0.061  [2.60 - 2.76] [2.65 - 2.85] Serine 2.64 (0.037) 2.73 (0.037) -0.093 (0.050) -0.25, 0.065 0.157  [2.58 - 2.69] [2.61 - 2.81] Threonine 2.06 (0.027) 2.13 (0.027) -0.070 (0.031) -0.17, 0.028 0.106  [1.99 - 2.09] [2.05 - 2.18] Tryptophan 0.60 (0.013) 0.60 (0.013) -0.0030 (0.013) -0.045, 0.039 0.835  [0.57 - 0.62] [0.57 - 0.64] 
Monsanto Company 09-SY-201U Page 322 of 471

Table E-16 (continued). Comparison of Amino Acid, Fiber, Proximate, and Antinutrient Content frand Conventional Control (A3525) Soybean Meal Processed from a 2007 U.S. Field Production

 Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[9

Amino Acid (% DW)

Tyrosine 1.83 (0.023) 1.85 (0.023) -0.027 (0.021) -0.093, 0.039 0.279  [1.78 - 1.85] [1.80 - 1.91] Valine 2.55 (0.020) 2.64 (0.020) -0.092 (0.010) -0.13, -0.058 0.003  [2.51 - 2.60] [2.61 - 2.69] Fiber and Proximate (% DW)

Acid Detergent Fiber 6.54 (0.26) 5.94 (0.26) 0.59 (0.37) -0.30, 1.49 0.156  [5.96 - 6.89] [5.34 - 6.70] Neutral Detergent Fiber 8.55 (0.26) 6.81 (0.26) 1.74 (0.36) 0.59, 2.88 0.016  [8.05 - 8.96] [6.26 - 7.50] Ash 6.99 (0.17) 7.29 (0.17) -0.29 (0.097) -0.60, 0.014 0.056  [6.74 - 7.30] [6.88 - 7.76] Carbohydrates 39.31 (0.71) 37.69 (0.71) 1.62 (0.59) -0.25, 3.49 0.070  [37.73 - 40.04] [35.68 - 39.61] Moisture (% FW) 3.33 (1.41) 6.86 (1.41) -3.53 (2.00) -8.41, 1.35 0.127  [2.72 - 3.91] [3.94 - 12.50] 
Monsanto Company 09-SY-201U Page 323 of 471

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval; ND = not detected; NC = not calculated. 

2With 95% confidence, interval contains 99% of the values expressed in the population of commercial varieties. Negative limits 

Table E-16 (continued). Comparison of Amino Acid, Fiber, Proximate, and Antinutrient Content frand Conventional Control (A3525) Soybean Meal Processed from a 2007 U.S. Field Production

Difference (MON 87705 minus 

Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[9

Protein 52.91 (0.59) 54.16 (0.59) -1.24 (0.63) -3.24, 0.76 0.142  [52.01 - 54.17] [52.52 - 55.85] Total Fat 0.78 (0.084) 0.86 (0.084) -0.079 (0.10) -0.41, 0.25 0.495  [0.61 - 1.04] [0.69 - 1.00] Antinutrient (% DW) Phytic Acid 1.37 (0.033) 1.47 (0.033) -0.10 (0.033) -0.21, 0.0047 0.055  [1.27 - 1.43] [1.44 - 1.54] Trypsin Inhibitor (TIU/mg DW) 2.15 (0.59) 1.41 (0.59) 0.74 (0.83) -1.29, 2.77 0.405  [0.52 - 4.09] [0.54 - 2.07] 
Monsanto Company 09-SY-201U Page 324 of 471

Table E-17. Comparison of Fatty Acids and Vitamin E from MON 87705 and Conventional Control (ASoybean RBD Oil Processed from the 2007 U.S. Field Production 

Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[99

RBD Oil Fatty Acid (% Total FA) 14:0 Myristic 0.031 (0.0017) 0.090 (0.0017) -0.059 (0.0025) -0.065, -0.053 <0.001 

 [0.031 - 0.032] [0.086 - 0.097] 16:0 Palmitic 2.49 (0.087) 11.59 (0.087) -9.10 (0.087) -9.37, -8.82 <0.001  [2.36 - 2.69] [11.36 - 11.83] 16:1 Palmitoleic 0.13 (0.0044) 0.11 (0.0044) 0.023 (0.0044) 0.0094, 0.037 0.012  [0.12 - 0.14] [0.096 - 0.11] 17:0 Margaric 0.036 (0.0070) 0.10 (0.0070) -0.067 (0.0073) -0.091, -0.044 0.002  [0.031 - 0.048] [0.085 - 0.12] 17:1 9c Heptadecenoic 0.12 (0.0090) 0.031 (0.0090) 0.088 (0.013) 0.047, 0.13 0.006 

 [0.092 - 0.14] [0.031 - 0.031] 18:0 Stearic 3.22 (0.072) 4.47 (0.072) -1.25 (0.060) -1.44, -1.06 <0.001 

[3.00 - 3.40] [4.33 - 4.57] 

18:1 Oleic 71.51 (0.85) 23.16 (0.85) 48.35 (1.21) 45.39, 51.30 <0.001 

[69.30 - 73.01] [21.44 - 25.54] 

18:2 6c,9c Octadecadienoic 0.20 (0.017) 0.65 (0.017) -0.46 (0.024) -0.52, -0.40 <0.001 

 [0.16 - 0.24] [0.63 - 0.69] 
Monsanto Company 09-SY-201U Page 325 of 471

Table E-17 (continued). Comparison of Fatty Acids and Vitamin E from MON 87705 and Conven(A3525) Soybean RBD Oil Processed from the 2007 U.S. Field Production

 Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[99% 

RBD Oil Fatty Acid (% Total FA)18:2 Linoleic 14.41 (0.92) 51.08 (0.92) -36.67 (1.18) -40.44, -32.90 <0.001 4 [12.25 - 17.39] [50.02 - 52.06] [18:2 Other Trans 0.18 (0.017) 0.63 (0.017) -0.44 (0.024) -0.50, -0.39 <0.001  [0.14 - 0.23] [0.60 - 0.66] 18:3 9c,12c,15t Octadecatrienoic 

0.92 (0.073) 0.98 (0.073) -0.059 (0.045) -0.20, 0.085 0.284 

 [0.81 - 1.01] [0.80 - 1.19] 18:3 Linolenic 4.76 (0.38) 5.13 (0.38) -0.37 (0.15) -0.87, 0.12 0.094  [4.08 - 5.46] [4.21 - 5.96] 18:3 Other 18:3 Trans 0.92 (0.067) 0.93 (0.067) -0.011 (0.045) -0.15, 0.13 0.815  [0.81 - 1.01] [0.78 - 1.16] 20:0 Arachidic 0.29 (0.0088) 0.36 (0.0088) -0.072 (0.0060) -0.091, -0.053 0.001  [0.27 - 0.31] [0.34 - 0.38] 20:1 Eicosenoic 0.33 (0.016) 0.19 (0.016) 0.14 (0.0086) 0.11, 0.17 <0.001  [0.29 - 0.37] [0.17 - 0.21] 
Monsanto Company 09-SY-201U Page 326 of 471

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval; ND = not detected; NC = not calculated 

2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negativ

Table E-17 (continued). Comparison of Fatty Acids and Vitamin E from MON 87705 and Conv(A3525) Soybean RBD Oil Processed from the 2007 U.S. Field Production 

Difference (MON 87705 minus 

Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[9

RBD Oil Fatty Acid (% Total FA)22:0 Behenic 0.31 (0.011) 0.35 (0.011) -0.034 (0.0033) -0.044, -0.023 0.001 

 [0.30 - 0.35] [0.33 - 0.38] 

24:0 Lignoceric 0.14 (0.018) 0.15 (0.018) -0.0056 (0.0036) -0.017, 0.0059 0.218 

 [0.11 - 0.18] [0.11 - 0.19] 

Vitamin E (mg/100g FW) 11.64 (2.96) 13.09 (2.96) -1.45 (0.67) -3.59, 0.70 0.120  [6.37 - 17.35] [7.72 - 20.35] 
Monsanto Company 09-SY-201U Page 327 of 471

Table E-18. Comparison of Amino Acids and Moisture from MON 87705 and Conventional Control (Soybean Protein Isolate Processed from a 2007 U.S. Field Production 

Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[99

Amino Acid (%DW)Alanine 3.73 (0.029) 3.71 (0.029) 0.017 (0.015) -0.031, 0.064 0.345 

[3.66 - 3.81] [3.64 - 3.76] 

Arginine 7.84 (0.094) 7.83 (0.094) 0.0065 (0.11) -0.34, 0.35 0.955 

[7.57 - 8.08] [7.68 - 8.04] 

Aspartic Acid 10.88 (0.10) 10.87 (0.10) 0.013 (0.14) -0.42, 0.45 0.930 

[10.58 - 11.19] [10.68 - 11.04] 

Cystine 1.21 (0.024) 1.18 (0.024) 0.035 (0.034) -0.050, 0.12 0.351 

[1.17 - 1.28] [1.12 - 1.23] 

Glutamic Acid 17.52 (0.23) 17.66 (0.23) -0.14 (0.20) -0.77, 0.50 0.540 

[16.89 - 18.15] [17.25 - 18.02] 

Glycine 3.93 (0.021) 3.92 (0.021) 0.0027 (0.029) -0.090, 0.095 0.932 

[3.87 - 3.98] [3.90 - 3.97] 

Histidine 2.40 (0.019) 2.39 (0.019) 0.0026 (0.017) -0.052, 0.058 0.888 

[2.35 - 2.44] [2.36 - 2.44] 
Monsanto Company 09-SY-201U Page 328 of 471

Table E-18 (continued). Comparison of Amino Acids and Moisture from MON 87705 and Conve(A3525) Soybean Protein Isolate Processed from a 2007 U.S. Field Production

 Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[9

Amino Acid (% DW) Isoleucine 4.43 (0.039) 4.47 (0.039) -0.032 (0.024) -0.11, 0.046 0.280  [4.35 - 4.48] [4.36 - 4.56] Leucine 7.33 (0.037) 7.33 (0.037) -0.00005 (0.039) -0.12, 0.12 0.999  [7.19 - 7.41] [7.28 - 7.38] Lysine 5.80 (0.017) 5.76 (0.017) 0.034 (0.015) -0.013, 0.081 0.102  [5.75 - 5.83] [5.74 - 5.81] Methionine 1.27 (0.024) 1.24 (0.024) 0.035 (0.030) -0.061, 0.13 0.327  [1.25 - 1.29] [1.15 - 1.29] Phenylalanine 5.05 (0.038) 5.07 (0.038) -0.019 (0.046) -0.17, 0.13 0.713  [4.94 - 5.15] [5.01 - 5.14] Proline 5.02 (0.051) 5.05 (0.051) -0.033 (0.049) -0.19, 0.12 0.555  [4.91 - 5.11] [4.92 - 5.17] Serine 4.96 (0.043) 4.92 (0.043) 0.043 (0.061) -0.11, 0.19 0.513  [4.87 - 5.06] [4.86 - 5.02] Threonine 3.37 (0.037) 3.35 (0.037) 0.018 (0.0085) -0.0086, 0.045 0.118  [3.28 - 3.43] [3.24 - 3.43] 
Monsanto Company 09-SY-201U Page 329 of 471

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 

2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negativ

Table E-18 (continued). Comparison of Amino Acids and Moisture from MON 87705 and Conve(A3525) Soybean Protein Isolate Processed from a 2007 U.S. Field Production

Difference (MON 87705 minus 

Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[99

Amino Acid (% DW)Tryptophan 1.05 (0.016) 1.04 (0.016) 0.0071 (0.011) -0.027, 0.042 0.556 

[1.01 - 1.07] [0.99 - 1.07] 

Tyrosine 3.46 (0.012) 3.45 (0.012) 0.0089 (0.013) -0.032, 0.050 0.536 

[3.43 - 3.49] [3.43 - 3.48] 

Valine 4.42 (0.052) 4.45 (0.052) -0.031 (0.054) -0.20, 0.14 0.610 

[4.34 - 4.49] [4.29 - 4.58] 

Moisture (% FW) 1.91 (0.28) 1.33 (0.28) 0.58 (0.27) -0.27, 1.43 0.117 

[1.30 - 2.96] [0.93 - 1.69] 
Monsanto Company 09-SY-201U Page 330 of 471

1DW = dry weight; FW = fresh weight; S.E. = standard error; CI = confidence interval. 

2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negativ

Table E-19. Comparison of Phosphatides from MON 87705 and Conventional Control (A3525) SoybeProcessed from a 2007 U.S. Field Production 

Difference (MON 87705 minus Control)

Analytical 

Component1

MON 87705 

Mean ± S.E. 1

[Range] 

A3525 

Mean ± S.E. 

[Range] 

Mean ± S.E. 

[Range] 

95% CI1

(Lower, Upper) p-Value 

[99

Phosphatides (% FW)L-alpha-Phosphatidic Acid 1.10 (0.29) 1.24 (0.29) -0.14 (0.41) -1.14, 0.86 0.740 

[0.35 - 1.64] [0.35 - 1.60] 

L-alpha-Phosphatidylcholine 4.38 (1.04) 7.18 (1.04) -2.80 (1.14) -6.44, 0.83 0.091 

[1.93 - 7.85] [5.75 - 9.18] 

L-alpha-Phosphatidylethanolamine 2.45 (0.74) 4.99 (0.74) -2.55 (0.97) -5.62, 0.53 0.077 

[0.65 - 4.79] [4.06 - 6.70] 

L-alpha-Phosphatidylinositol 2.28 (0.66) 4.63 (0.66) -2.36 (0.83) -5.00, 0.29 0.066 

[1.13 - 4.53] [3.99 - 6.06] 
Monsanto Company 09-SY-201U Page 331 of 471

1

Literature range references: aLundry, et al., 2008. bPadgette et al., 1996. 

Table E-20. Literature Ranges for Components in Soybean Meal 

 

Tissue Component Literature Range1

Proximates (% dw)

Ash 5.2 – 9.1a

 

Carbohydrates 32.0 – 38.0b

 

Fat, total 0.5 -3.30a

 

Moisture (% fw) 5.58-11.7a

 

Protein 47.4 – 59.5a

 

 

Fiber (% dw) a

 

Acid detergent fiber (ADF) 5.2 – 6.7 

Neutral detergent fiber (NDF) 7.4 – 12.2 

 

Amino Acids (%dw)a

Alanine 2.18 – 2.59 

Arginine 3.29 – 4.49 

Aspartic acid 5.18 – 6.83 

Cystine/Cysteine 0.6 – 0.92 

Glutamic acid 8.05 – 11.21 

Glycine 2.02 – 2.40 

Histidine 1.32 – 1.63 

Isoleucine 2.11 – 2.74 

Leucine 3.62 – 4.72 

Lysine 2.97 – 3.69 

Methionine 0.5 – 0.9 

Phenylalanine 2.39 – 3.19 

Proline 2.32 – 3.05 

Serine 1.97 – 3.3 

Threonine 0.80 – 2.24 

Tryptophan 0.60 – 2.08 

Tyrosine 1.68 – 2.17 

Valine 2.29 – 2.92 

 

Anti-Nutrients 

Trypsin Inhibitors (TIU/mg dw) 3.8 – 17.9a

 

Phytic Acid (% dw) 1.3 – 4.1 a

 

 
Monsanto Company 09-SY-201U Page 332 of 471

 Table E-21. Literature Ranges for Components in Soybean Protein 

Isolate 

Tissue Component Literature Range or Value1

Proximates 

Moisture (% fw) 3.9 – 7.0 

 

Amino Acids (%dw) 

Alanine NA 

Arginine 6.67 

Aspartic acid NA 

Cystine/Cysteine 1.05 

Glutamic acid NA 

Glycine NA 

Histidine 2.3 

Isoleucine 4.25 

Leucine 6.78 

Lysine 5.33 

Methionine 1.13 

Phenylalanine 4.59 

Proline NA 

Serine NA 

Threonine 3.14 

Tryptophan 1.12 

Tyrosine NA 

Valine 4.1 

 1

Literature range or value reference: Lundry, et. al., 2008. 

Table E-22. Literature Ranges for Components in Soybean Lecithin 

Tissue Component Literature Range1

Phosphatides (%fw) 

α-Phosphatidic Acid 0.2 – 14.0 

α-Phosphatidylcholine 12.0 – 46.0 

α-Phosphatidylethanolamine 8.0 – 34.0 

α-Phosphatidylinositol 1.7 – 21.0 

 

Literature range reference: 1 Lundry, et. al., 2008. 
Monsanto Company 09-SY-201U Page 333 of 471

 

Table E-23. Literature Ranges for Components in Soybean Oil 

Tissue Component Literature Range1

Fatty Acids (FA) 

14:0 Myristic ND – 0.2a

 

16:0 Palmitic 7 - 12b

 

16:1 Palmitoleic ≤ 0.2b

 

17:0 Margaric [Heptadecanoic] ND – 0.1a

 

17:1 9c Heptadecenoic ND – 0.1a

 

18:0 Stearic 2 – 5b

 

18:1 Oleic 19 – 34b

 

18:2 Linoleic 48 – 60b

 

18:3 Linolenic 2 -10b

 

20:0 Arachidic 0.1 – 0.6a

 

20:1 Eicosenoic ND – 0.5a

 

20:2 Eicosadienoic ND – 0.1a

 

22:0 Behenic ND – 0.7a

 

 

Vitamins mg/100g fw

Vitamin E 0.9 – 35.2a

 

 

1

Literature range references: aCodex, 2005 (% Total FA). bLundry, et al., 

2008, (% FW). ND = not detected. 
Monsanto Company 09-SY-201U Page 334 of 471

Appendix E References 

Chardigny J.M., J.L. Sébédio, and O. Berdeux. 1996. Trans Polyunsaturated Fatty Acids: 

Occurrence and Nutritional Implications. Ann. Appl. Lipid Reearch 2:1-33. 

Codex. 2005. Codex Standard for named vegetable oils. Codex Alimentarus. 

George, C., W.P. Ridley, J.C. Obert, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 

2004. Composition of grain and forage from corn rootworm-protected corn event MON 

863 is equivalent to that of conventional corn (Zea mays L.). Journal of Agricultural and 

Food Chemistry 52:4149-4158. 

Ledoux, M., P. Juaneda, and J-L Sebedio. 2007. Tran fatty acids: Definition and 

occurrence in foods. European Journal of Lipid Science Technology 109:891-900. 

Lundry, D.R., Ridley, W. P., Meyer, J.J., Riordan, S.G., Nemeth, M.A., Trujillo, W.A., 

Breeze, M.L., and Sorbet, R. 2008. 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.). J. Agric. Food Chem., 

56:4611-4622. 

NOPA. 2006. Trading rules for the purchase and sale of soybean meal. National Oilseed 

Processors Association, Washington, D.C. 

OECD. 2001. Consensus document on compositional considerations for new varieties of 

soybean: key food and feed nutrients and anti-nutrients. OECD 

ENV/JM/MONO(2001)15. 

Padgette, S.R., N.B. Taylor, D.L. Nida, M.R. Bailey, J. MacDonald, L.R. Holden, and 

R.L. Fuchs. 1996. The composition of glyphosate-tolerant soybean seeds is equivalent to 

that of conventional soybeans. Journal of Nutrition 126:702-716. 

Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. 

Comparison of the nutritional profile of glyphosate-tolerant corn event NK603 with that 

of conventional corn (Zea mays L.). Journal of Agricultural and Food Chemistry 

50:7235-7243. 

Wolff, R.L. 1993. Further studies on artificial geometrical isomers of α-linolenic acid in 

edible linolenic acid-containing oils. Journal fo the American Oil Chemists’ Society 

3:219-224. 

 
Monsanto Company 09-SY-201U Page 335 of 471

Appendix F. Materials and Methods for Seed Dormancy and Germination 

Analyses of MON 87705 

F.1 Materials 

MON 87705, a conventional soybean control (A3525), and commercial soybean 

reference variety starting seed were produced in Jefferson County, IA; Boone County, 

IN; and Macon County, MO in 2007. 

F.2 Characterization of the Materials 

For the MON 87705, conventional soybean control, and commercial soybean reference 

variety starting seed, the presence or absence of MON 87705 was verified by eventspecific polymerase chain reaction (PCR) analyses. The results of these analyses 

confirmed the presence of MON 87705 in the MON 87705 starting seed and the absence 

of MON 87705 in the control and reference variety seed. 

F.3 Performing Facility and Experimental Methods 

Dormancy and germination evaluations were conducted at BioDiagnostics, Inc. in River 

Falls, WI. The principal investigator was qualified to conduct seed dormancy and 

germination testing consistent with the standards established by the Association of 

Official Seed Analysts, a seed trade association (AOSA, 2000; AOSA, 2006; AOSA, 

2007). 

Six germination chambers were used in the evaluation and each chamber was maintained 

dark under one of the following six temperature regimes: constant temperature of 

approximately 10, 20 or 30 o

C or alternating temperatures of approximately 10/20, 10/30, 

or 20/30 o

C. The alternating temperature regimes were maintained at the lower 

temperature for 16 hours and the higher temperature for eight hours. The temperature 

inside each germination chamber was monitored and recorded every 15 minutes 

throughout the duration of the study. 

Germination towels for MON 87705, control, and reference materials were prepared per 

facility SOPs. Each germination towel represented one replication. The types of data 

collected depended on the temperature regime. Each rolled germination towel in the 

AOSA-recommended temperature regime (i.e., 20/30 o

C) was assessed periodically 

during the study for normal germinated, abnormal germinated, hard (viable and 

nonviable), dead, and firm swollen (viable and nonviable) seed as defined by AOSA 

guidelines (AOSA, 2006; AOSA, 2007). Each rolled germination towel in the additional 

temperature regimes (i.e., 10, 20, 30, 10/20 and 10/30 o

C) was assessed periodically 

during the study for germinated, hard (viable and nonviable), dead, and firm swollen 

(viable and nonviable) seed. 

 
Monsanto Company 09-SY-201U Page 336 of 471

F.4 Statistical Analysis 

Statistical analyses were performed by the Monsanto Statistics Technology Center. 

Analysis of variance was conducted according to three randomized complete block 

designs with four replications, with the exception that the germination towels were 

arbitrarily arranged in each bucket and not necessarily randomized. SAS was used to 

compare MON 87705 to the conventional soybean control within each seed production 

site for the following germination characteristics: percent germinated (categorized as 

percent normal germinated and percent abnormal germinated for the AOSA temperature 

regime), percent viable hard seed, percent dead, and percent viable firm-swollen seed. 

The level of statistical significance was predetermined to be α = 0.05. The MON 87705 

substance was not statistically compared to the reference substances, nor were 

comparisons made across temperature regimes. The minimum and maximum mean 

values (reference range) were determined from the reference substances at each site. 

F.5 Individual Site Seed Dormancy and Germination Analysis 

There were no statistically significant differences detected between MON 87705 and the 

control for any of the measured characteristics (i.e., percent germinated, viable hard, or 

viable firm swollen seed) in any temperature regime for seed produced at the IN site. 

Seven statistically significant differences were detected between MON 87705 and the 

control (Table F-2) for seed produced at the IA and MO sites. MON 87705 had lower 

percent normal germination than the control at 20/30 oC for seed produced at the IA site 

(89.3 vs. 94.0%). At 30 °C, MON 87705 had lower percent germination than the control 

for seed produced at the IA site (82.8 vs. 95.5%) and higher percent germination than the 

control for seed produced at the MO site (100.0 vs. 96.3%). MON 87705 had lower 

percent viable hard seed than the control at 20° C for seed produced at the MO site (0.0 

vs. 0.8%). Percent dead seed was higher for MON 87705 than the control at 30 oC (17.3 

vs. 4.5%) and 20/30 oC (6.0 vs. 2.5%) for seed produced at the IA site and lower for 

MON 87705 than the control at 30 oC for seed produced at the MO site (0.0 vs. 3.8%). 
Monsanto Company 09-SY-201U Page 337 of 471

Table F-1. Starting Seed of MON 87705, Control and Commercial Reference Soybean Varieties Used in Assessment 

Production Site Substance Name Substance Type Phenotype1 Sample ID IA MON 87705 Test Modified oil profile RPN0727IA A3525 Control Conventional RPN0727IA Hoegemeyer 333 Reference Conventional RPN0727

IA Stewart 3454 Reference Conventional RPN0727IA Stine 3600-0 Reference Conventional RPN0727IA Lewis 372 Reference Conventional RPN0727IN MON 87705 Test Modified oil profile RPN0727

IN A3525 Control Conventional RPN0727

IN Midland 363 Reference Conventional RPN0727IN NK S33A8 Reference Glyphosate tolerant2 RPN0727IN Asgrow AG3505 Reference Glyphosate tolerant2 RPN0727IN FS 3591 Reference Conventional RPN0727MO MON 87705 Test Modified oil profile RPN0727

MO A3525 Control Conventional RPN0727

MO Hoegemeyer 333 Reference Conventional RPN0727MO Stewart 3454 Reference Conventional RPN0727MO Stine 3600-0 Reference Conventional RPN0727MO Lewis 372 Reference Conventional RPN0727

1 MON 87705 possesses the improved fatty acid profile trait; the control and reference soybean varieties do not possess the trait. 2 Glyphosate tolerant = Commercially available Roundup Ready soybean 40-3-2 variety. 
Monsanto Company 09-SY-201U Page 338 of 471

Table F-2. Comparison of MON 87705 to the Control for Dormancy and Germination Characteristics 

Temp. Regime Category 

IA1

Mean % (S.E.)2

IN1 

Mean % (S.E.)2

Mea

MON 87705 Control 

Reference 

Range3 MON 87705 Control 

Reference 

Range3 MON 87705 

10 °C Germinated 96.8 (1.5) 98.0 (1.2) 99.3 – 100.0 97.5 (1.7) 98.8 (0.5) 98.5 – 99.3 98.8 (0.6)  Viable Hard 0.0 (0.0) 0.3 (0.3) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.3 (0.3)  Dead 3.0 (1.6) 1.8 (1.0) 0.0 – 0.8 2.3 (1.7) 0.8 (0.3) 0.8 – 1.3 1.0 (0.7) 

Viable Firm Swollen 0.3 (0.3) 0.0 (0.0) 0.0 – 0.0 0.3 (0.3) 0.5 (0.3) 0.0 – 0.5 0.0 (0.0) 

20 °C Germinated 97.5 (1.3) 98.5 (0.6) 98.8 – 99.5 99.8 (0.3) 99.8 (0.3) 99.5 – 99.8 100.0 (0.0)  Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.3 (0.3) 0.0 – 0.0 0.0 (0.0) *  Dead 2.5 (1.3) 1.5 (0.6) 0.5 – 1.3 0.3 (0.3) 0.0 (0.0) 0.3 – 0.5 0.0 (0.0) 

Viable Firm Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 

30 °C Germinated 82.8 (3.1)* 95.5 (1.3) 97.5 – 98.5 95.5 (1.2) 94.8 (0.9) 96.0 – 99.5 100.0 (0.0) * 

 Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.0 (0.0)  Dead 17.3 (3.1) * 4.5 (1.3) 1.5 – 2.5 4.5 (1.2) 5.3 (0.9) 0.3 – 4.0 0.0 (0.0) * 

Viable Firm Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 
Monsanto Company 09-SY-201U Page 339 of 471

Table F-2 (cont.). Comparison of MON 87705 to the Control for Dormancy and Germination Characteris

Temp. Regime Category 

IA1

Mean % (S.E.)2

IN1

Mean % (S.E.)2

Mea

MON 87705 Control 

Reference 

Range3 MON 87705 Control 

Reference 

Range3 MON 87705 

10/20°C Germinated 99.3 (0.3) 99.5 (0.5) 98.8 – 99.5 99.5 (0.3) 99.8 (0.3) 99.3 – 100.0 99.3 (0.5)  Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.3 (0.3)  Dead 0.8 (0.3) 0.5 (0.5) 0.5 – 1.3 0.5 (0.3) 0.3 (0.3) 0.0 – 0.5 0.5 (0.3) 

Viable Firm Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 

10/30°C Germinated 95.3 (1.3) 97.5 (1.0) 99.3 – 99.5 99.3 (0.5) 99.3 (0.3) 98.8 – 99.5 99.5 (0.3) 

 Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.0 (0.0)  Dead 4.8 (1.3) 2.5 (1.0) 0.5 – 0.8 0.8 (0.5) 0.8 (0.3) 0.5 – 1.3 0.5 (0.3) 

Viable Firm Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 

20/30°C 

Normal Germinated 89.3 (1.3) * 94.0 (1.1) 95.8 – 97.3 96.0 (0.8) 94.5 (1.0) 57.0 – 98.5 92.0 (0.9) 

(AOSA) 

Abnormal Germinated 4.8 (1.3) 3.5 (1.4) 2.0 – 3.8 3.5 (1.0) 5.5 (1.0) 1.3 – 42.5 6.5 (0.6) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 0.0 (0.0) 0.0 (0.0) 0.0 – 0.0 0.0 (0.0)  Dead 6.0 (0.4) * 2.5 (0.5) 0.0 – 1.0 0.5 (0.3) 0.0 (0.0) 0.3 – 1.0 1.5 (0.5) 

Viable Firm Swollen 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) † 0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 

* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05). † No statistical comparison could be made due to lack of variability in the data. 1 Site codes are as follows: IA = Jefferson County, IA; IN = Boone County, IN; MO = Macon County, MO. 2 Means based on four replicates (n = 4) of approximately 100 seeds. In some instances, the total percentage of both MON 87705 and t

exactly 100% due to numerical rounding of the means. S.E. = Standard Error. 

3 Minimum and maximum mean values determined from commercially available reference soybean varieties
Monsanto Company 09-SY-201U Page 340 of 471

Appendix F References 

AOSA. 2000. Tetrazolium Testing Handbook -Contribution No. 29 to the Handbook on 

Seed Testing. Association of Official Seed Analysts, Lincoln, Nebraska. 

AOSA. 2006. Seedling Evaluation Handbook, Contribution No. 35 to the Handbook on 

Seed Testing. Association of Official Seed Analysts, Lincoln, Nebraska. 

AOSA. 2007. Rules for Testing Seeds. Association of Official Seed Analysts, Lincoln, 

Nebraska. 
Monsanto Company 09-SY-201U Page 341 of 471

Appendix G. Material, Methods and Individual Site Results from Phenotypic, 

Agronomic and Ecological Interactions Analyses of MON 87705 

G.1 Materials 

The materials for phenotypic assessments include: MON 87705, a conventional soybean 

control (A3525), and 17 commercially available soybean varieties as references. The 

references contain both conventional soybean and Roundup Ready soybean 40-3-2 

varieties. The list of soybean varieties planted in each site is presented in Table G-1. 

The identities of MON 87705 and control (A3525) seed were confirmed by PCR analysis 

prior to use. 

G.2 Field Sites and Plot Design 

Data were collected from field trials conducted in 2007 at 17 sites within U.S. soybean 

production regions (Section VIII, Table VIII-3). The 17 sites provided a range of 

environmental and agronomic conditions representative of major U. S. soybean-growing 

regions. The field cooperators at each site were familiar with the growth, production, and 

evaluation of soybean characteristics. 

The experiment was established at each of the 17 sites in a randomized complete block 

design with three replications. Each plot at the IA1, IL2, IN1, and MO2 sites consisted of 

eight 30 ft long rows with inter-row spacing of approximately 30 inches. Rows # 2 and 3 

were designated for the collection of phenotypic, abiotic stress response, disease damage, 

and arthropod damage data. Rows # 5–7 were designated for the collection of arthropod 

samples. Rows # 1, 4, and 8 were used as buffer rows. Each plot was surrounded by an 

approximately 10 ft, four-row border of a commercially available soybean variety to 

create a continuous soybean stand across the plot area to ensure collection of more robust 

arthropod abundance data within the test area. 

Each plot at the AR, IA2, IL1, IL3, IN2, KS, MI, MO3, MO4, NE, OH, PA, and WI sites 

consisted of four 20 ft long rows with inter-row spacing of approximately 30 inches. 

Rows # 2 and 3 were designated for the collection of phenotypic, abiotic stress response, 

disease damage, and arthropod damage data. Rows # 1 and 4 were used as buffer rows. 

The entire plot area was surrounded by an approximately 10 ft, four-row border of a 

commercially available soybean variety. 
Monsanto Company 09-SY-201U Page 342 of 471

Table G-1. Starting Seed for Phenotypic Assessments 

Substance Substance type

Relative Maturity Group Phenotype1 Monsanto Lot # Sites2

MON 87705 Test 3.5 Improved Fatty Acid Profile GLP-0702-18254-S All sitesA3525 Control 3.5 Conventional GLP-0702-18252-S All sitesA3244 Reference 3.2 Conventional GLP-0703-18458-S All sitesCroplan HT3596STS Reference 3.5 Conventional GLP-0703-18386-S AR, IL2, KPioneer 93M50 Reference 3.5 Roundup Ready 3 GLP-0604-17240-S AR, IL2, KChannel Bio 3461 Reference 3.4 Conventional GLP-0605-17332-S AR, IL2, KPioneer 93M52 Reference 3.5 Conventional GLP-0703-18407-S AR, IL2, KHoegemeyer 333 Reference 3.2 Conventional GLP-0703-18383-S IA1, IL3, Stewart 3454 Reference 3.4 Conventional GLP-0703-18426-S IA1, IL3, Stine 3600-0 Reference 3.6 Conventional GLP-0605-17336-S IA1, IL3, Lewis 372 Reference 3.7 Conventional GLP-0604-17261-S IA1, IL3, Midland 363 Reference 3.3 Conventional GLP-0703-18384-S IA2, IN1, NK S33A8 Reference 3.3 Roundup Ready 3 GLP-0604-17243-S IA2, IN1, Asgrow AG3505 Reference 3.5 Roundup Ready 3 GLP-0604-17250-S IA2, IN1, FS 3591 Reference 3.5 Conventional GLP-0703-18387-S IA2, IN1, Stine 3300-0 Reference 3.3 Conventional GLP-0605-17335-S IL1, IN2, DeKalb DKB34-51 Reference 3.4 Roundup Ready 3 GLP-0604-17254-S IL1, IN2, Garst 3585N Reference 3.5 Conventional GLP-0703-18388-S IL1, IN2, Crows C37003N Reference 3.7 Conventional GLP-0703-18390-S IL1, IN2, 

1 MON 87705 expresses the improved fatty acid profile trait; whereas the conventional soybean control and reference varieties do not e

acid profile trait. 

2 MON 87705, the control, and reference material A3244 were planted at all field sites; the remaining reference varieties were site-spec

follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, IL; IL2 = Stark CouCounty, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO2 = Lincoln CLouis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA; WI = W

3 Commercially available glyphosate-tolerant (Roundup Ready 40-3-2) soybean variety.
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Table G-2. Field and Planting Information 

Site1 

Planting 

date

(mm/dd/yr)

Seeding 

rate 

(seeds/ft)2

Planting 

depth 

(in)

Plot 

size 

(ft)3

Rows/

plot Soil series, organic matter, pH

Cr

20

AR 05/26/2007 8 0.75 10 x 20 4 Crowley silt loam, 2.5%, 6.2 RIA1 05/23/2007 8 1.25 20 x 30 8 Tainter/Mahaska silty clay loam, 4.02%, 6.75 C

IA2 05/26/2007 8 1.5 10 x 20 4 Tama-Muscatine silty clay loam, 2.8%, 6.3 Milk IL1 06/04/2007 8 1.0 10 x 20 4 Silt loam, 2.65%, 6.59 M

IL2 05/31/2007 8 1.5 20 x 30 8 Plano silt loam, 3.5%, 6.4 C

IL3 05/23/2007 8 1.0 10 x 20 4 Sable silty clay loam, 6.0%, 5.6-7.3 C

IN1 05/30/2007 8 1.5 20 x 30 8 Crosby silty clay loam, 2.4%, 6.8 CIN2 06/08/2007 8 1.25 10 x 20 4 Reesville silt loam, 1.4%, 5.8 C

KS 05/22/2007 9 1.0 10 x 20 4 Farnum loam, 2.6%, 7.6 W

MI 05/23/2007 8 1.5 10 x 20 4 Nester loam, 2.1%, 6.5 C

MO2 05/30/2007 8 1.5 20 x 30 8 Crider silt loam, 2.3%, 6.7 CMO3 05/25/2007 8 1.5 10 x 20 4 Kennebec silt loam, 2.6%, 6.3 C

MO4 06/07/2007 8 1.0 10 x 20 4 Gorin silt loam, 4.2%, 5.8 Fe

NE 05/21/2007 8 1.0 10 x 20 4 Hastings silt loam, 3.0%, 6.6 Soy

OH 05/22/2007 8 1.0 10 x 20 4 Crosby silt loam, 1.8%, 6.6 CPA 06/09/2007 8 1.5 10 x 20 4 Philo/Atkins silt loam, 2.4%, 5.8 Tom

WI 05/29/2007 8 1.0 10 x 20 4 Radford silt loam, not available, not available W

1 Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, I

IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO

MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, P

County, WI. 

2 The KS site planted the production at nine seeds per foot as specified in the study protocol. 3 Width × length. 
Monsanto Company 09-SY-201U Page 344 of 471

G.3 Planting and Field Operations 

Planting information is listed in Table G-2. Agronomic practices used to prepare and 

maintain each study site were characteristic of those used in each respective geographic 

region. Herbicides containing glyphosate were not used to avoid injury to the 

conventional soybean control or conventional soybean reference varieties and to ensure 

all plants were managed uniformly. 

G.4 Phenotypic Observations 

The description of the characteristics measured and the designated developmental stages 

where observations occurred are listed in Section VIII, Table VIII-1. 

G.5 Ecological Observations 

Ecological interactions (i.e., interactions between the crop plants and their receiving 

environment) were used to characterize MON 87705 by evaluating plant response to 

abiotic stressors, disease damage, arthropod damage, and pest and beneficial arthropod 

abundance in the plots using the following methods: 

G.6 Abiotic Stress Response, Disease Damage, and Arthropod Damage 

MON 87705 and the conventional soybean control were evaluated at each of 17 sites 

(AR, IA1, IA2, IL1, IL2, IL3, IN1, IN2, KS, MI, MO2, MO3, MO4, NE, OH, PA, and 

WI) for differences in plant response to abiotic stressors, disease damage, and arthropod 

damage. Three abiotic stressors, three diseases, and three arthropod pests were evaluated 

four times during the growing season at the following intervals: 

Observation 1: V2 – V4 growth stage 

Observation 2: R1 – R2 growth stage 

Observation 3: R3 – R5 growth stage 

Observation 4: R6 – R8 growth stage 

The principal investigator at each site chose abiotic stressors, diseases, and arthropod 

pests that were either actively causing plant injury in the study area or were likely to 

occur in soybean during a given observation period. Therefore, abiotic stressors, 

diseases, and arthropod pests assessed often varied between observations at a site and 

between sites. 

Abiotic stressors and disease damage were assessed in Rows # 2 and 3 of each plot using 

a continuous 0 – 9 rating scale of increasing symptomology. Data were collected 

numerically and then placed into one of the following categories for reporting purposes: 

Rating Severity of plant damage 

0 none (no symptoms observed) 

1 – 3 slight (symptoms not damaging to plant development) 

4 – 6 moderate (intermediate between slight and severe) 

7 – 9 severe (symptoms damaging to plant development) 
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Arthropod damage was assessed in Rows # 2 and 3 of each plot on the upper four nodes 

of 10 non-systematically selected plants using arthropod-specific 0 – 5 rating scales of 

increasing symptomology listed below. Data were collected numerically and then placed 

into one of the categories in the following rating scales for reporting purposes: 

Defoliating arthropods (e.g., corn earworm, bean leaf beetle, Japanese beetle, soybean 

looper)

Rating Defoliation (%) Severity of plant damage 

0 none none (no symptoms observed) 

1 1 – 20 % slight (symptoms not damaging to plant development) 

2 21 – 40% moderate (intermediate between slight and severe) 3 41 – 60% 

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80% 

Pod feeding arthropods (e.g., corn earworm, bean leaf beetle, stink bug, Lygus bug on 

reproductive plant parts)

Rating Damaged pods (%) Severity of plant damage 

0 none none (no symptoms observed) 

1 1 – 20 % slight (symptoms not damaging to plant 

development) 

2 21 – 40% moderate (intermediate between slight and severe) 3 41 – 60% 

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80% 

Leafhoppers (e.g., potato leafhopper)

Rating Foliar damage (%) Severity of plant damage 

0 none none (no symptoms observed) 

1 1 – 50% of foliage with leaf yellowing; no 

leaf puckering or leaf margin necrosis 

slight (symptoms not damaging 

to plant development) 

2 1 – 50% of foliage with leaf yellowing, 

leaf puckering and/or leaf margin necrosis moderate (intermediate between 

slight and severe) 3 > 50% of foliage with leaf yellowing; no 

leaf puckering or leaf margin necrosis 

4 > 50% of foliage with leaf yellowing, leaf 

puckering, and/or leaf margin necrosis severe (symptoms damaging to 

plant development) 5 > 50% of foliage with necrotic leaves 

(leaves dead due to leafhopper damage) 

 
Monsanto Company 09-SY-201U Page 346 of 471

Aphids (e.g., soybean aphid)

Rating Aphids present Severity of plant damage 

0 none none (no symptoms observed) 

1 1 – 100 aphids per plant; 

no leaf puckering 

slight (symptoms not damaging to plant 

development) 

2 101 – 250 aphids per plant; 

no leaf puckering moderate (intermediate between slight and 

severe) 3 ≥ 250 aphids per plant with 

leaf puckering 

4 

≥ 250 aphids per plant with 

leaf puckering and leaf 

yellowing and/or necrosis severe (symptoms damaging to plant 

development) 

5 ≥ 250 aphids per plant with 

plant stunting 

For each abiotic stress response, disease damage, and arthropod damage observation at a 

site, the range of injury severity ratings observed across all three replications for each of 

MON 87705, the conventional soybean control and reference soybean varieties at the site 

was determined, and the numeric ranges were then converted to categorical ranges (e.g., 

none, slight, moderate, severe) for reporting purposes. 

G.7 Arthropod Abundance 

Pest and beneficial arthropods were collected at the IA1, IL2, IN1, and MO2 sites three 

times during the growing season at the following intervals: 

Collection 1: R1 – R2 growth stage 

Collection 2: R3 – R5 growth stage 

Collection 3: R6 – R8 growth stage 

Arthropods were collected using a beat sheet sampling method (Kogan and Pitre, 1980). 

The beat sheet was an approximately 36 × 42 inch white, vinyl sheet spread between the 

plants of two adjacent rows. Plants were shaken vigorously along the length of each side 

of the beat sheet to dislodge arthropods from the plants. A total of four subsamples were 

collected in this way from each plot. Specifically, two subsamples were collected from 

Rows # 5 and 6 of each plot (subsamples 1 and 3) and two subsamples were collected 

from Rows # 6 and 7 of each plot (subsamples 2 and 4). The subsamples collected from 

the same row were at least 10 ft apart and at least 3 ft from the edge of each plot. The 

four subsamples were combined into one pre-labelled container and placed on dry ice. 

The samples were then sent overnight to the Monsanto Regulatory Environmental 

Science Center for arthropod identification and enumeration. 

A maximum of the six most abundant pest and six most abundant beneficial arthropods 

were determined for each collection interval from each individual site. These specific 

arthropods were then enumerated across all samples from a given collection interval at 

each individual site. The arthropods assessed often varied between collection intervals 

from a site and between sites due to differences in temporal activity and geographical 

distribution of the taxa. 
Monsanto Company 09-SY-201U Page 347 of 471

G.8 Ecological Interactions Evaluation Criteria 

For the assessments of abiotic stress response, disease damage, and arthropod damage, 

MON 87705 and the conventional soybean control were considered different in 

susceptibility or tolerance to an abiotic stressor, disease, or arthropod pest on a particular 

observation date if the range of injury severity to MON 87705 did not overlap with the 

range of injury severity to the control across all three replications. These data are 

categorical and, therefore, were not subjected to statistical analysis. For each observation 

at a site, the range of injury severity across the commercially available reference soybean 

varieties provided data that are representative of commercial soybean varieties. Pest and 

beneficial arthropod abundance data were quantitatively evaluated and subjected to 

statistical analysis, as appropriate. 

G.9 Data Assessment 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all components of data collection, summarization, and analysis. Personnel 

assessed that measurements were taken properly, data were consistent with expectations 

based on experience with the crop, and the experiment was carefully monitored. Prior to 

analysis, the overall dataset was evaluated for evidence of biologically relevant changes 

and for possible evidence of an unexpected plant response. Any unexpected observations 

or issues that would impact the evaluation objectives were noted. Data were then 

subjected to statistical analysis as indicated below. 

G.10 Statistical Analysis 

Analysis of variance was conducted according to a randomized complete block design 

using SAS (Version 9.2). The level of significance was α = 0.05. MON 87705 was 

compared to the control substance within each site (individual-site analysis) and pooled 

across all sites (combined-site analysis) for early stand count, seedling vigor, days to 50% 

flowering, plant height, lodging, shattering, final stand count, seed moisture, 100 seed 

weight, seed test weight, and yield. Growth stage, flower color, plant pubescence, abiotic 

stress response, disease damage, and arthropod damage data were not statistically 

analyzed. Arthropod pest abundance and beneficial arthropod abundance data were 

statistically analyzed only within individual collection intervals and sites due to the 

variation in temporal activity and geographical distribution of the taxa. 

No statistical comparisons were made between MON 87705 and reference soybean 

varieties. The reference range for each measured phenotypic characteristic was 

determined from the minimum and maximum mean values collected from the 17 

reference soybean varieties planted among the sites. The reference range for the plant 

response to abiotic stressor, disease, arthropod damage, and abundance of each arthropod 

evaluated from a given collection and site was determined from the minimum and 

maximum mean values collected from the reference varieties at the site. Thus, reference 

ranges for abiotic stressor, disease susceptibility, arthropod damage, and arthropod 

abundance were specific to each collection/observation interval and site. 

G.11 Individual Field Site Plant Growth and Development Results and Discussion 
Monsanto Company 09-SY-201U Page 348 of 471

In the individual-site analysis, lack of variability in the data precluded statistical 

comparisons between MON 87705 and the control for seedling vigor at the IL1, IL3, 

MO4, and PA sites; days to 50% flowering at the IA2, IN1, NE, and WI sites; plant 

lodging ratings at the IL1, IN1, MO2, MO3, and NE sites; pod shattering at the IL1, IL2, 

IL3, IN2, MI, MO2, MO3, NE, OH, PA, and WI sites; and test weight at the WI site. In 

each of these cases, however, the mean for MON 87705 and the mean for the control 

were the same value or nearly the same value (Table G-3). 

In the individual-site analysis, a total of 30 statistically significant differences were 

detected out of 161 comparisons between MON 87705 and the conventional soybean 

control (Table G-3). These differences were distributed among nine of the 13 phenotypic 

characteristics. Early stand count was lower for MON 87705 than the control at the AR 

site (101.7 vs. 155.3 plants/plot), the IL1 site (253.3 vs. 271.0 plants/plot), the IL2 site 

(240.6 vs. 284.6 plants/plot), the OH site (112.0 vs. 159.0 plants/plot), and the PA site 

(262.3 vs. 288.0 plants/plot). MON 87705 flowered later than the control at the IL2 site 

(208.0 vs. 205.3 days after 1 Jan. 2007), the IN2 site (204.7 vs. 203.0 days after 1 Jan. 

2007), the MO2 site (193.7 vs. 191.0 days after 1 Jan. 2007), the MO4 site (204.3 vs. 

201.3 days after 1 Jan. 2007), and the PA site (209.0 vs. 207.3 days after 1 Jan. 2007). 

Plants of MON 87705 were taller than the control at the IN2 site (35.4 vs. 33.6 inches) 

but shorter than the control at the IA1 (40.3 vs. 42.9 inches), the MO3 (29.6 vs. 32.0 

inches), and the PA (28.6 vs. 32.0 inches) sites. MON 87705 had more lodging than the 

control at the IN2 site (4.0 vs. 1.7 rating). Final stand count was lower for MON 87705 

than the control at the AR (98.0 vs. 153.7 plants/plot), the IA1 (172.8 vs. 213.0 

plants/plot), the IL1 (254.3 vs. 276.0 plants/plot), the IL2 (206.6 vs. 248.1 plants/plot), 

the IN1 (226.3 vs. 255.7 plants/plot), and the OH (116.7 vs. 156.7 plants/plot) sites. Seed 

moisture was lower for MON 87705 than the control at the KS (11.8 vs. 13.4%) and the 

MO3 (12.0 vs. 14.4%) sites. The weight of 100 seeds was lower for MON 87705 than 

the control at the IA2 (13.8 vs. 14.4 g), MI (19.6 vs. 21.3 g), and MO4 (13.3 vs. 14.5 g) 

sites. Test weight was greater for MON 87705 than the control at the IN1 site (50.1 vs. 

42.1 lb/bu), but less than the control at the MO4 site (50.7 vs. 53.8 lb/bu). Yield was 

lower for MON 87705 than the control at the IL2 (43.5 vs. 51.2 bu/ac) and MO3 (21.7 vs. 

31.8 bu/ac) sites. Considering that the statistical differences for plant height, lodging, 

seed moisture, test weight, and yield that were detected in the individual-site analyses 

were not detected in the combined site analysis, this suggests these differences were not 

indicative of a consistent plant response associated with the trait and are unlikely to be 

biologically meaningful in terms of increased weed potential of MON 87705 compared to 

the conventional soybean control. While some statistical differences were detected in the 

combined-site analysis, the assessed phenotypic values of MON 87705 were within the 

range of values expected for commercial soybean. 
Monsanto Company 09-SY-201U Page 349 of 471

Table G-3. Phenotypic Comparison of MON 87705 to the Conventional Soybean Control within Each Site 

 Phenotypic Characteristic (units)  Early stand count (#/plot)

Seedling vigor 

(1-9 scale) Days to 50% flowering Flower color2 

Site1

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) MON 87705 Control 

AR 101.7* (13.9) 155.3 (17.5) 6.0 (0.6) 5.3 (0.3) 180.0 (1.2) 178.0 (1.2) Purple Purple IA1 261.7 (8.9) 282.6 (5.4) 3.0 (0.6) 2.7 (0.7) 192.7 (0.3) 191.7 (0.3) Purple Purple 

IA2 266.7 (5.6) 282.3 (2.3) 3.3 (0.3) 3.0 (0.0) 197.0‡ (0.0) 197.0 (0.0) Purple Purple 

IL1 253.3* (2.7) 271.0 (5.1) 1.0‡ (0.0) 1.0 (0.0) 201.0 (0.0) 201.3 (0.3) Purple Purple 

IL2 240.6* (8.5) 284.6 (3.5) 1.0 (0.0) 1.0 (0.0) 208.0* (1.0) 205.3 (0.3) Purple Purple 

IL3 283.3 (11.0) 302.0 (9.9) 2.0‡ (0.0) 2.0 (0.0) 190.0 (1.0) 188.3 (0.7) Purple Purple 

IN1 222.8 (15.1) 248.6 (5.8) 2.3 (0.3) 3.0 (1.0) 207.0‡ (0.0) 207.0 (0.0) Purple Purple 

IN2 257.3 (6.9) 259.7 (7.2) 3.7 (0.3) 3.3 (0.7) 204.7* (0.3) 203.0 (0.0) Purple Purple 

KS 158.3 (15.9) 180.7 (8.7) 4.0 (0.0) 4.0 (0.0) 191.0 (0.6) 191.0 (0.0) Purple Purple 

MI 320.3 (1.5) 303.7 (14.3) 3.3 (0.3) 4.0 (0.6) 198.7 (0.7) 198.0 (0.0) Purple Purple 

MO2 288.6 (8.0) 299.5 (6.7) 5.3 (0.3) 5.0 (0.0) 193.7* (0.3) 191.0 (0.6) Purple Purple 

MO3 237.3 (17.5) 280.0 (5.8) 4.7 (0.7) 3.3 (0.3) 191.7 (1.3) 190.0 (1.5) Purple Purple 

MO4 201.3 (12.8) 218.3 (6.6) 2.0‡ (0.0) 2.0 (0.0) 204.3* (0.7) 201.3 (0.3) Purple Purple 

NE 308.0 (6.1) 289.3 (13.1) 3.0 (0.0) 3.0 (0.0) 192.0‡ (0.0) 192.0 (0.0) Purple Purple 

OH 112.0* (11.0) 159.0 (10.2) 3.7 (0.9) 3.3 (0.7) 199.3 (0.7) 197.3 (0.9) Purple Purple 

PA 262.3* (6.6) 288.0 (4.2) 2.0‡ (0.0) 2.0 (0.0) 209.0* (0.0) 207.3 (0.3) Purple Purple 

WI 221.0 (20.0) 249.7 (2.9) 1.3 (0.3) 1.7 (0.7) 207.0‡ (0.0) 207.0 (0.0) Purple Purple 

* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05). 

‡ Indicates a lack of variability in the data which precluded statistical analysis. N/A = Data not available or excluded from the data analysis. 

1 Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, IL; I

IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO2 = 

MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, PA; 

WI. 

2 Flower color and plant pubescence data were categorical and were not statistically analyzed. 

S.E. = standard error. Means based on n = 3. 
Monsanto Company 09-SY-201U Page 350 of 471

Table G-3 (cont.). Phenotypic Comparison of MON 87705 to the Conventional Soybean Control within Ea

 Phenotypic Characteristic (units) 

Plant height 

(in)

Lodging 

(0-9 scale)

Pod Shattering 

(0-9 scale) Final stand count (#/plot) 

Site1

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) 

AR 25.0 (0.6) 24.1 (0.5) 2.3 (0.3) 1.0 (0.6) 0.7 (0.3) 0.3 (0.3) 98.0* (12.1) 153.7 (16.5) IA1 40.3* (0.1) 42.9 (0.6) 5.0 (0.6) 5.0 (0.6) 0.0 (0.0) 0.3 (0.3) 172.8* (12.5) 213.0 (8.2) 

IA2 42.7 (1.1) 44.3 (1.7) 4.3 (0.3) 4.3 (0.3) 0.7 (0.3) 0.7 (0.3) 238.3 (11.3) 257.7 (8.4) 

IL1 24.7 (0.3) 25.1 (0.4) 0.0‡ (0.0) 0.0 (0.0) 0.0‡ (0.0) 0.0 (0.0) 254.3* (1.8) 276.0 (2.0) 

IL2 41.3 (0.9) 43.1 (0.5) 3.7 (0.7) 3.7 (0.3) 0.0‡ (0.0) 0.0 (0.0) 206.6* (8.9) 248.1 (9.7) 

IL3 42.8 (2.3) 43.5 (0.6) 2.3 (0.3) 2.3 (0.3) 0.0‡ (0.0) 0.0 (0.0) 264.7 (18.0) 293.3 (12.7) 

IN1 33.0 (2.0) 33.3 (0.6) 0.0‡ (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 226.3* (19.3) 255.7 (11.2) 

IN2 35.4* (1.0) 33.6 (0.6) 4.0* (0.6) 1.7 (0.3) 0.0‡ (0.0) 0.0 (0.0) 231.7 (19.6) 236.0 (6.4) 

KS 31.2 (0.6) 31.1 (0.1) 0.3 (0.3) 1.0 (0.0) 0.0 (0.0) 0.0 (0.0) 139.3 (16.6) 157.3 (7.3) 

MI 30.5 (1.3) 31.3 (2.1) 0.0 (0.0) 0.0 (0.0) 0.0‡ (0.0) 0.0 (0.0) 285.0 (3.8) 290.3 (2.9) 

MO2 32.4 (0.4) 32.4 (0.9) 0.0‡ (0.0) 0.0 (0.0) 0.0‡ (0.0) 0.0 (0.0) 288.6 (7.3) 302.4 (6.6) 

MO3 29.6* (0.5) 32.0 (0.4) 0.0‡ (0.0) 0.0 (0.0) 0.0‡ (0.0) 0.0 (0.0) 241.0 (17.8) 285.0 (3.6) 

MO4 29.4 (0.8) 30.2 (1.2) 0.3 (0.3) 0.7 (0.3) 0.3 (0.3) 0.3 (0.3) 189.7 (12.1) 202.7 (7.2) 

NE 39.3 (0.4) 39.4 (1.4) 0.0‡ (0.0) 0.0 (0.0) 0.0‡ (0.0) 0.0 (0.0) 300.0 (2.3) 286.7 (5.8) 

OH 30.0 (0.6) 33.3 (1.8) 0.0 (0.0) 0.7 (0.3) 0.0‡ (0.0) 0.0 (0.0) 116.7* (7.7) 156.7 (3.4) 

PA 28.6* (0.0) 32.0 (0.6) 0.3 (0.3) 0.3 (0.3) 0.0‡ (0.0) 0.0 (0.0) 253.7 (7.8) 268.7 (3.5) 

WI 45.3 (0.1) 45.0 (1.4) 7.0 (0.0) 6.3 (0.7) 1.0‡ (0.0) 1.0 (0.0) 170.3 (11.6) 185.7 (10.5) 

* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05). 

‡ Indicates a lack of variability in the data which precluded statistical analysis. 

1 Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, I

IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO

MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, P

County, WI. - Data not available or excluded from the data analysis. S.E. = standard error. Means based on n = 3.
Monsanto Company 09-SY-201U Page 351 of 471

Table G-3 (cont.). Phenotypic Comparison of MON 87705 to the Conventional Soybean Control within Ea

 Phenotypic Characteristic (units)  100 seed weight (g) Test weight (lb/bu) Yield (bu/ac) Site1

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) 

MON 87705 

(S.E.) 

Control 

(S.E.) 

AR 15.1 (0.1) 16.0 (0.2) 53.4 (0.1) 53.3 (0.7) 40.8 (8.9) 36.1 (7.5) IA1 23.8 (0.0) 24.3 (0.2) 53.1 (1.7) 50.9 (1.2) 69.0 (3.8) 72.0 (2.0) IA2 13.8* (0.1) 14.4 (0.2) 55.2 (0.2) 55.0 (0.0) 75.6 (8.5) 68.1 (1.0) IL1 13.7 (0.9) 15.2 (0.3) 57.0 (1.0) 56.0 (0.6) 24.4 (4.4) 24.5 (2.2) IL2 13.5 (0.1) 13.7 (0.6) 53.3 (0.9) 52.3 (0.3) 43.5* (0.5) 51.2 (2.3) 

IL3 14.1 (0.3) 14.0 (0.4) 53.7 (0.8) 55.6 (0.9) 77.1 (3.5) 78.8 (5.4) IN1 15.3 (0.3) 15.9 (0.2) 50.1* (1.6) 42.1 (0.6) 56.2 (1.7) 66.0 (5.0) IN2 16.5 (0.1) 16.7 (0.5) 46.4 (0.6) 46.3 (0.1) 71.9 (0.9) 68.1 (3.9) KS 13.8 (0.1) 14.1 (0.2) 56.3 (0.7) 56.0 (0.7) 65.9 (6.1) 63.6 (2.8) MI 19.6* (0.2) 21.3 (0.6) 56.7 (0.3) 57.6 (0.2) 54.6 (1.3) 52.1 (1.3) MO2 14.8 (0.1) 14.9 (0.2) 59.7 (0.3) 60.0 (0.5) 31.4 (1.5) 28.9 (6.9) MO3 14.9 (0.5) 13.9 (0.6) 59.7 (0.5) 58.7 (0.2) 21.7* (0.8) 31.8 (1.1) MO4 13.3* (0.3) 14.5 (0.4) 50.7* (1.2) 53.8 (0.4) 63.2 (4.0) 58.2 (1.9) NE 15.0 (0.1) 14.9 (0.1) 59.0 (0.1) 59.0 (0.0) 66.1 (2.8) 67.8 (3.7) OH 16.0 (0.4) 16.9 (0.1) 47.4 (0.6) 48.7 (1.0) 52.0 (4.3) 50.2 (1.6) PA 16.3 (0.9) 16.7 (0.3) 55.8 (0.6) 55.8 (0.8) 54.6 (2.9) 61.0 (1.5) WI N/A N/A 56.0‡ (0.0) 56.0 (0.0) 55.6 (1.9) 52.8 (1.0) 

* Indicates a statistically significant difference between MON 87705 and the conventional soybean control (p < 0.05). ‡ Indicates a lack of variability in the data which precluded statistical analysis. N/A = Data not available or excluded from the data analysis. 1 Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, I

IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO

MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York County, NE; OH = Fayette County, OH; PA = Berks County, P

County, WI. S.E. = standard error. Means based on n = 3.
Monsanto Company 09-SY-201U Page 352 of 471

Table G-4. Growth Stage Monitoring of MON 87705, the Conventional Soybean Control, and the ReferVarieties 

Date and Range of Growth Stages Observed2

Site1 Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. AR 06/20/2007 07/09/2007 07/27/2007 08/13/2007 09/04/2007 09/25/2007 — —  MON 87705 V2-V3 V8-V9 R4-R5 R5-R6 R7 R8 — —  Control V3 V8-V9 R4-R5 R6 R7 R8 — —  References V2-V3 V7-V9 R4-R5 R5-R6 R7 R8 — — IA1 06/18/2007 07/09/2007 07/30/2007 08/20/2007 09/11/2007 10/02/2007 — —  MON 87705 V3 V8 R3 R5 R6-R7 R8 — —  Control V3 V8 R3 R5 R6-R7 R8 — —  References V3 V8 R3 R5 R6-R7 R8 — — IA2 06/20/2007 07/10/2007 07/31/2007 08/17/2007 09/05/2007 09/23/2007 10/11/2007 —  MON 87705 V2 V6 R3-R4 R5 R6 R7 R8 —  Control V2 V7 R3 R5 R6 R7 R8 —  References V2 V6-V7 R3-R4 R5 R6 R7 R8 — IL1 06/20/2007 07/19/2007 08/06/2007 08/27/2007 09/10/2007 09/27/2007 — —  MON 87705 V2 R1 R3 R6 R6 R8 — —  Control V2-V3 R1 R3 R6 R6-R7 R8 — —  References V2-V3 R1 R3 R6 R6 R7-R8 — — IL2 06/27/2007 07/11/2007 07/24/2007 08/08/2007 08/27/2007 09/17/2007 10/08/2007 —  MON 87705 V2 V3-V4 R1-R2 R2-R3 R5 R7 R7-R8 — 

 Control V2 V3-V4 R1-R2 R3-R4 R5 R7 R7-R8 —  References V2 V3-V4 R1-R2 R2-R4 R5 R7 R7-R8 — 

1 Site codes are as follows: AR = Jackson County, AR; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IL1 = Clinton County, I2 Obs. = Observation number; dates in month/day/year format. 

- Indicates information not available. 
Monsanto Company 09-SY-201U Page 353 of 471

Table G-4 (continued). Growth Stage Monitoring of MON 87705, the Conventional Soybean Control, and Soybean Varieties

Date and Range of Growth Stages Observed2

Site1 Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 IL3 06/05/2007 06/20/2007 07/11/2007 07/27/2007 08/10/2007 08/30/2007 09/18/2007 10/04/20 MON 87705 VE V3 R1-R2 R3 R5 R6 R7 R8  Control VE V3 R2 R3 R5 R6 R7 R8  References VE V3 R1-R2 R3 R5 R6 R7 R8 IN1 06/26/2007 07/16/2007 07/25/2007 08/17/2007 09/12/2007 10/17/2007 — —  MON 87705 V2-V3 R1 R1-R2 R3-R4 R6 R8 — —  Control V2-V3 R1 R1-R2 R3-R4 R6 R8 — —  References V2-V3 R1 R1-R2 R3-R4 R6-R7 R8 — — IN2 07/02/2007 07/27/2007 08/01/2007 08/16/2007 09/12/2007 09/28/2007 10/19/2007 —  MON 87705 V2 R1 R2-R4 R5 R6 R8 R8 —  Control V2 R1 R2-R4 R5 R6 R8 R8 —  References V2 R1 R2-R3 R5 R6 R8 R8 — KS 06/12/2007 06/29/2007 07/13/2007 08/01/2007 08/13/2007 08/24/2007 09/05/2007 10/05/20 MON 87705 V3 V8 R2 R5 R5 R6 R7 R8  Control V3 V8 R2 R5 R5 R6 R7 R8  References V3 V8 R2 R5 R5 R6 R7 R8 MI 06/20/2007 07/05/2007 07/18/2007 08/01/2007 08/14/2007 08/29/2007 09/12/2007 09/26/20 MON 87705 V2 V5 R1-R2 R3 R5 R5 R6 R7  Control V2-V3 V5 R2 R3 R5 R5 R6 R7  References V2-V3 V5-V6 R1-R2 R2-R3 R5 R5 R6 R7 1 Site codes are as follows: IL3 = Warren County, IL; IN1 = Boone County, IN; IN2 = Parke County, IN; KS = Pawnee County, KS; M 2 Obs. = Observation number; dates in month/day/year format. 

 - Indicates information not available. 
Monsanto Company 09-SY-201U Page 354 of 471

Table G-4 (continued). Growth Stage Monitoring of MON 87705, the Conventional Soybean Control, and Soybean Varieties 

Date and Range of Growth Stages Observed2 

Site1 Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. MO2 06/26/2007 07/13/2007 07/25/2007 08/05/207 08/26/2007 09/17/2007 — —  MON 87705 V2-V3 V6-V8 R3-R4 R5-R6 R6 R8 — —  Control V2-V3 V7-V8 R3-R4 R5 R6 R8 — —  References V2-V3 V7-V9 R3-R4 R5-R6 R6 R7-R8 — — MO3 06/06/2007 06/21/2007 07/09/2007 07/24/2007 08/05/2007 08/26/2007 09/17/2007 —  MON 87705 VC V2 V7-V8 R4 R5 R6 R8 —  Control VC V2 V7-V8 R4 R5 R6 R8 —  References VC V2 V7-V8 R4 R5-R6 R6-R7 R8 — MO4 06/29/2007 07/13/2007 08/01/2007 08/14/2007 09/11/2007 10/08/2007 — —  MON 87705 V2-V3 V6-V7 R3 R5 R6 R8 — —  Control V3 V6-V7 R3 R5 R6 R8 — —  References V2-V3 V6-V7 R2-R3 R5 R6 R8 — — NE 06/13/2007 07/02/2007 07/23/2007 08/13/2007 09/05/2007 09/25/2007 — —  MON 87705 V2 V6 R3 R5 R6 R7 — —  Control V2 V6 R3 R5 R6 R7 — —  References V2 V6 R3 R5 R6 R7 — — OH 06/15/2007 07/02/2007 07/20/2007 08/14/2007 09/04/2007 09/25/2007 — —  MON 87705 V1-V2 V3-V4 R1-R2 R3-R4 R6 R8 — —  Control V1-V2 V3-V4 R1-R2 R3-R4 R6 R8 — —  References V1-V2 V3-V4 R1-R2 R3-R4 R6 R8 — — 1 Site codes are as follows: MO2 = Lincoln County, MO, MO3 = St. Louis County, MO; MO4 = Macon County, MO; NE = York Coun

County, OH. 

2 Obs. = Observation number; dates in month/day/year format 

 - Indicates information not available. 
Monsanto Company 09-SY-201U Page 355 of 471

Table G-4 (continued). Growth Stage Monitoring of MON 87705, the Conventional Soybean Control, and Soybean Varieties 

Date and Range of Growth Stages Observed2 

Site1 Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 ObPA 06/29/2007 07/13/2007 07/27/2007 08/13/2007 08/31/2007 09/19/2007 10/10/2007 —  MON 87705 V1 V4 V7 R4 R5-R6 R6 R8 —  Control V1 V4 V7-V8 R4 R5-R6 R6 R8 —  References V1 V4 V7-V8 R3-R4 R5-R6 R6 R8 — WI 06/20/2007 07/11/2007 08/16/2007 08/31/2007 09/27/2007 10/24/2007 — —  MON 87705 V2 V6 R3-R4 R5 R6 R8 — —  Control V2 V6 R3-R4 R5 R6 R8 — —  References V2 V6-V7 R3-R4 R5 R6 R8 — — 1 Site codes are as follows: PA = Berks County, PA; WI = Walworth County, WI.2 Obs. = Observation number; dates in month/day/year format. 

- Indicates information not available. 
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Table G-5. Abiotic Stressor Evaluation Using Observational Severity Scale for 

MON 87705 and the Conventional Soybean Control 

Abiotic stressor

Number of observations 

across the sites

Number of observations

where no differences were 

detected between MON 87705 

and the control

Total 167 167

Chloride toxicity 4 4

Cold 2 2

Drought 37 37

Flood 3 3

Frost 4 4

Hail 30 30

Heat stress 27 27

Moisture stress 4 4

Nutrient deficiency 12 12

Soil compaction 8 8

Wet soil 4 4

Wind 32 32

Note: The experimental design was a randomized complete block with three replications. Observational 

data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2, 

Observation 3 = R3-R5, and Observation 4 = R6-R8. No differences were observed between MON 87705 

and the control during any observation. Data were not subjected to statistical analysis. 

 
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Table G-6. Disease Damage Evaluations Using an Observational Severity Scale for 

MON 87705 and the Conventional Soybean Control 

Disease

Number of observations 

across the sites

Number of observations

where no differences 

were detected between 

MON 87705 and the control

Total 206 203

Alternaria leaf spot 14 14

Anthracnose 2 2

Asian rust 3 3

Bacterial blight 22 19*

Brown stem rot 5 5

Brown stem rust 1 1

Charcoal rot 2 2

Downy mildew 14 14

Cercospora 6 6

Frogeye leaf spot 31 31

Phytophthora1 10 10

Powdery mildew 11 11

Pythium 7 7

Rhizoctonia2 8 8

Sclerotinia 2 2

Seedling blight 1 1

Septoria (brown spot) 25 25

Soybean cyst nematode 1 1

Sudden death 12 12

Soybean mosaic virus 9 9

White mold 9 9

Soybean rust 11 11

*Indicates a difference observed between test and the control for bacterial blight at MO2 site (slight vs. 

none; Observation 1, Observation 2, and Observation 3). Data were not subjected to statistical analysis. 

Note: The experimental design was a randomized complete block with three replications. Observational 

data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2, 

Observation 3 = R3-R5, and Observation 4 = R6-R8. 

1

 includes Phytophthora root rot. 2

 includes Rhizoctonia root rot. 

 
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Table G-7. Arthropod Damage Evaluated Using an Observational Severity Scale for 

MON 87705 and the Conventional Soybean Control 

Arthropod

Number of 

observations across 

the sites

Number of observations

where no differences were 

detected between MON 87705 

and the control

Total 206 204

Aphids1 28 27*

Armyworms 7 7

Bean leaf beetles 46 46

Flea beetles 1 1

Grasshoppers 26 26

Green cloverworms 20 20

Japanese beetles 28 28

Leafhoppers2 14 13*

Leafrollers 3 3

Mexican bean beetles 3 3

Soybean loopers 5 5

Spider mites 4 4

Stink bugs 11 11

Thistle caterpillars 2 2

Thrips 1 1

Whiteflies 2 2

Wireworms 1 1

Yellow wooly bear 4 4

*Indicates a difference observed between MON 87705 and the control for aphids (none vs. slight; 

Observation 2) and leafhoppers (none vs. slight; Observation 2) at the WI site. Data were not subjected to 

statistical analysis. 

Note: The experimental design was a randomized complete block with three replications. Observational 

data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2, 

Observation 3 = R3-R5, and Observation 4 = R6-R8. Data were not subjected to statistical analysis. 1 Includes soybean aphids. 

2

 Includes potato leafhoppers. 
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Table G-8. Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87705, the Conven

Control, and the Reference Soybean Varieties 

Abundance of Pest Arthropods2

Collection 1 Collection 2 

Arthropod Site1 MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Aphid IL2 31.7 (31.7) 18.3 (14.5) 5.0 – 25.3 — — — — 

MO2 — — — 145.0 (123.2) 35.3 (32.4) 16.7 – 199.0 — 

Bean leaf beetle 

IA1 0.0 (0.0) 0.3 (0.3) 0.0 – 1.0 1.3 (0.3) 0.7 (0.3) 0.0 – 1.3 0.0* (0.0) 

IL2 12.0 (3.5) 12.0 (2.6) 8.7 – 12.0 11.0 (1.0) 10.3 (2.9) 7.3 – 16.0 17.0 (0.6) 

IN1 3.7 (1.5) 2.0 (1.0) 1.7 – 3.0 1.0 (0.6) 0.0 (0.0) 0.3 – 1.0 62.0 (18.0) 

MO2 3.7(1.2) 2.3(0.7) 2.3 – 5.0 0.7(0.3) 0.7(0.7) 0.0 – 1.3 4.7 (2.2) 

Corn flea beetle 

IN1 — — — — — — 0.3 (0.3) 

Green cloverworm 

IA1 1.0 (0.0) 1.7 (0.3) 0.7 – 1.3 1.7 (1.2) 0.3 (0.3) 0.0 – 1.0 0.0 (0.0) 

IL2 0.0† (0.0) 0.0 (0.0) 0.0 – 0.0 2.0 (0.6) 1.3 (0.9) 0.0 – 2.0 0.0 (0.0) 

IN1 0.7 (0.3) 0.0 (0.0) 0.0 – 0.3 5.7 (2.0) 8.3 (2.6) 1.7 – 7.3 0.0 (0.0) 

MO2 7.7 (1.8) 4.3 (1.7) 6.7 – 8.7 3.3 (2.4) 1.7 (1.7) 1.0 – 5.3 1.3 (0.9) 

Japanese beetle 

IN1 0.3 (0.3) 1.3 (0.7) 0.0 – 2.0 2.3 (1.9) 2.0 (0.6) 0.7 – 4.7 — 

Potato leafhopper 

IA1 1.3 (0.3) 1.0 (1.0) 0.0 – 2.7 — — — — 

IL2 16.0 (3.5) 30.0 (3.5) 25.7 – 117.3 — — — — 

IN1 2.3 (1.5) 5.7 (2.8) 0.0 – 5.7 2.3 (2.3) 2.3 (0.3) 3.0 – 8.3 — 

* Indicates a statistically significant difference between MON 87705 and the control (p < 0.05). Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections perfordevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8. 1 Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO2 MON 87705 and control values represent mean number of arthropods collected across three replications. S.E. = standard error. Means † No statistical comparisons were made due to lack of variability in the data. 

- Indicates the arthropod was not evaluated.
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Table G-8 (continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87705

Soybean Control, and the Reference Soybean Varieties 

Abundance of Pest Arthropods2

Collection 1 Collection 2 C

Arthropod Site1 MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Stink bug IA1 0.3(0.3) 0.0 (0.0) 0.0 – 1.0 0.0 (0.0) 0.7 (0.7) 0.0 – 1.0 0.3 (0.3) 

IL2 0.3 (0.3) 0.0 (0.0) 0.0 – 0.7 2.0 (1.0) 0.7 (0.7) 1.3 – 3.7 2.7 (0.9) 

IN1 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 1.0 (0.0) 0.3 (0.3) 0.0 – 0.3 4.3 (1.9) 

MO2 0.3 (0.3) 0.3 (0.3) 0.0 – 1.0 0.3 (0.3) 1.0 (0.6) 0.0 – 1.7 1.0 (1.0) 

Thrips IA1 23.0 (3.1) 11.7 (6.6) 5.0 – 29.3 — — — — 

MO2 42.0 (21.0) 66.0 (54.9) 15.3 –34.0 — — — — 

Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections perfordevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8. 1 Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO2 MON 87705 and control values represent mean number of arthropods collected across three replications. S.E. = standard error. Means 

- Indicates the arthropod was not evaluated. 
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Table G-9. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87705, the CSoybean Control, and the Reference Soybean Varieties 

Abundance of Beneficial Arthropods2

Collection 1 Collection 2 

Arthropod Site1 MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Big-eyed bug MO2 1.0 (0.6) 1.0 (0.6) 0.3 – 3.0 5.3 (2.3) 7.7 (3.2) 8.0 – 9.0 7.3 (6.4) Carabid beetle 

IL2 — — — — — — 0.3 (0.3) 

IN1 — — — — — — 0.7 (0.7) 

Lacewing IA1 — — — 0.3 (0.3) 0.3 (0.3) 0.0 – 0.7 — 

MO2 — — — 1.0 (0.6) 1.0 (1.0) 0.7 – 1.7 0.0 (0.0) 

Ladybird beetle 

IA1 — — — 0.7 (0.3) 0.3 (0.3) 0.0 – 1.0 — 

IL2 — — — 0.0 (0.0) 0.7 (0.7) 0.3 – 4.3 0.7 (0.7) 

MO2 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 1.7 (1.7) 0.7 (0.7) 0.3 – 3.0 — 

Microparasitic hymenoptera 

IL2 — — — 1.3 (0.7) 0.7 (0.3) 0.7 – 2.3 — 

Nabis IA1 0.3 (0.3) 2.3 (1.9) 0.7 – 2.0 2.0 (1.2) 2.0 (0.6) 1.0 – 2.7 0.3 (0.3) 

IL2 0.0 (0.0) 0.7 (0.3) 0.0 – 2.3 0.7 (0.3) 1.3 (0.7) 0.3 – 2.3 0.7 (0.7) 

IN1 0.3 (0.3) 0.3 (0.3) 0.0 – 1.3 1.0 (0.0) 1.3 (0.7) 0.3 – 2.0 5.0 (2.0) 

MO2 0.3 (0.3) 1.0 (0.6) 0.3 – 2.3 1.3 (0.9) 1.7 (0.3) 0.0 – 1.7 0.3 (0.3) 

Orius IA1 0.3 (0.3) 0.0 (0.0) 0.0 – 0.3 0.3 (0.3) 0.3 (0.3) 0.0 – 0.7 0.0 (0.0) 

IL2 25.7 (15.7) 23.3 (4.8) 21.3 – 35.0 32.0 (4.0) 32.0 (7.5) 25.3 – 65.0 4.0 (1.0) 

IN1 3.0 (0.6) 5.7 (1.7) 1.0 – 3.3 14.3 (5.2) 17.3 (0.9) 8.7 – 23.0 7.3 (3.3) 

MO2 1.0 (1.0) 2.0 (2.0) 0.0 – 1.3 3.3 (1.8) 1.3 (0.7) 1.0 – 4.0 0.0 (0.0) 

Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections performdevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8. 1 Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO2 MON 87705 and control values represent mean number of arthropods collected across three replications. S.E. = standard error. Means 

- Indicates arthropod not evaluated.
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Table G-9 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON Conventional Soybean Control, and the Reference Soybean Varieties

Abundance of Beneficial Arthropods2

Collection 1 Collection 2 Co

Arthropod Sites1 MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) Control Mean (S.E.) Reference Range MON 87705 Mean (S.E.) C

Me

Spider IA1 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 0.3 (0.3) 1.0 (1.0) 0.0 – 0.3 0.0 (0.0) 0

IL2 1.7 (0.9) 1.3 (0.7) 0.7 – 3.3 1.0 (1.0) 1.7 (0.3) 1.0 – 2.3 0.0 (0.0) 1

IN1 0.3 (0.3) 1.0 (0.6) 0.0 – 2.3 0.3 (0.3) 0.3 (0.3) 0.3 – 1.0 1.7 (0.9) 2

MO2 0.3 (0.3) 0.7 (0.3) 0.0 – 2.0 1.3 (0.3) 2.0 (0.6) 0.0 – 2.3 2.0 (1.2) 3

Note: The experimental design was a randomized complete block with three replications. Data were from arthropod collections performdevelopmental stages: Collection 1 = R1-R2, Collection 2 = R3-R5, and Collection 3 = R6-R8. 1 Site codes are as follows: IA1 = Jefferson County, IA; IL2 = Stark County, IL; IN1 = Boone County, IN; MO2 = Lincoln County, MO2 MON 87705 and control values represent mean number of arthropods collected across three replications. S.E. = standard error. Means 

- Indicates arthropod not evaluated.
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Appendix H. Materials and Methods for Pollen Morphology and Viability 

Evaluation 

H.1 Plant Production 

MON 87705, a conventional soybean control (A3525), and five commercially available 

reference soybean varieties were grown in St. Louis County, MO, in a randomized 

complete block design with three replications. Each plot consisted of four rows 

approximately 20 ft in length. 

H.2 Flower Collection 

When soybean plants were at flowering stage, whole flowers were collected from five 

non-systematically selected plants from the first and fourth row of each plot. All flowers 

from all plots were collected on the same day. Four flowers were collected from each of 

the five plants per plot: one flower from the bottom, two flowers from the middle, and 

one flower from the top of each plant. Up to five additional flowers were collected from 

each plot to ensure a sufficient quantity of pollen for evaluation. All flowers selected 

from a plot were transferred into a single, clean container and labelled with the plot 

number from which the sample originated, the entry number, and the entry name. The 

containers were kept on wet ice or refrigerated for less than eight hours until the pollen 

was prepared and stained. 

H.3 Pollen Sample Preparation 

Pollen samples were prepared in a laboratory. Clean microscope slides were labelled 

with the plot number. A circle of approximately 1 cm diameter was drawn in the center 

of the slide with a pap hydrophobic barrier pen. Tweezers and a dissecting needle were 

used to open each of the collected flowers from a plot and brush the pollen into the circle 

on the slide. The tweezers were cleaned between extractions. Approximately 20 μl of 

Alexander’s stain (Alexander, 1980) was added to the center of the circle containing the 

pollen. The pollen was stained at ambient temperature for at least ten minutes prior to 

examination. Pollen samples from all plots within a replicate were stained and evaluated 

on the same day. 

H.4 Data Collection 

Pollen characteristics were assessed by viewing samples under an Olympus Provis AX70 

light/fluorescence microscope equipped with an Olympus DP70 digital color camera. 

The microscope and camera were connected to a computer running Microsoft Windows 

2000 Professional (© 1981-1999, Microsoft Corp.) and installed with associated camera 

software (DP Controller v1.2.1.108 and DP Manager v1.2.1.107 [Olympus Optical Co., 

Ltd.] and imaging software Image-Pro Plus v4.5.1.27 [Media Cybernetics, Inc.]). 

Pollen Viability: When exposed to the stain solution, viable pollen grains stained red to 

purple due to the presence of living cytoplasmic content. Nonviable pollen grains stained 

blue to green and may have appeared round to collapse in shape, depending on the degree 

of hydration. For each pollen sample, the number of viable and nonviable pollen grains 

was counted from a minimum of 75 pollen grains from a random field of view under the 
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microscope. Dense clusters of pollen or pollen grains adhering to flower parts were not 

counted because they may not have absorbed the stain solution uniformly. 

Pollen Diameter: Micrographs (400X resolution) of ten representative pollen grains from 

each plot were taken and imported into the imaging software. The software was used to 

measure pollen grain diameter along two perpendicular axes for each selected pollen 

grain. Mean pollen diameter for each plot was calculated from the 20 total 

measurements. 

General Pollen Morphology: General pollen morphology was observed from 

micrographs of MON 87705, the conventional soybean control, and commercial 

reference soybean varieties that were also used for pollen diameter measurements. 

H.5 Statistical Analysis 

An analysis of variance was conducted according to a randomized complete block design 

using SAS (SAS, 2002-2003). The level of statistical significance was predetermined to 

be 5% (p<0.05). MON 87705 was compared to the conventional soybean control for 

percent viable pollen and pollen diameter. No statistical comparisons were made 

between MON 87705 and the reference soybean varieties. Instead, a reference range for 

each measured characteristic was determined from the minimum and maximum mean 

values from among the reference soybean varieties. General pollen morphology was 

qualitative; therefore, no statistical analysis was conducted on these observations. 

Appendix H References 

Alexander, M.P. 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain 

Technology 55:13-18. 

SAS. 2002-2003. SAS Software Release 9.1 (TS1M0), SAS Institute, Inc., Cary, North 

Carolina. 
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Appendix I. Materials and Methods for Symbiont Evaluation 

I.1 Materials 

The starting seed of MON 87705 was produced in Puerto Rico in 2006-2007 under 

Protocol LS272 by Monsanto Trait Development. The control substance starting seed 

was produced in Illinois in 2007 under Monsanto Production Plan 07-01-83-18. 

Reference substance starting seed was obtained from commercial sources or was 

produced in Puerto Rico in 2007 under Seed Increase 07-SI-71-01. Nodules, root tissue, 

and shoot tissue collected from MON 87705, the conventional soybean control, and 

reference soybean varieties were evaluated in the study. 

Table I-1 Starting seed of MON 87705, Control, and Commercial Soybean 

Reference Varieties Used in the Symbiont Evaluation 

Materials Material Type Material Source1 Phenotype

MON 87705 Test Monsanto TD Improved fatty acid 

profile 

A3525 Control 07-01-83-18 Conventional 

A2553 Reference Commercial Conventional 

Stine 3300-0 Reference 07-SI-71-01 Conventional 

Stewart SB3454 Reference Commercial Conventional 

Garst 3585N Reference Commercial Conventional 

Hartz H5218 Reference 07-SI-71-01 Conventional 

A5560 Reference 07-SI-71-01 Conventional 1

 Starting seed were obtained from commercial sources, Monsanto Trait Development (TD), Monsanto 

Field Production 07-01-83-18, or Monsanto Seed Increase 07-SI-71-01. 

The presence or absence of MON 87705 in the test and control starting seed was verified 

by event-specific polymerase chain reaction (PCR) analyses. Results of PCR analyses 

were as expected. 

I.2 Greenhouse Phase and Experimental Design 

MON 87705, the conventional soybean control, and reference soybean varieties starting 

seed were planted in 6-inch pots containing nitrogen-deficient potting medium (Sunshine 

Mix #2 Basic/LB2, Sun Gro Horticulture, Inc., Garland, TX) comprised of primarily peat, 

vermiculite, and perlite. Plants from MON 87705, the conventional soybean control, and 

reference soybean varieties starting seed were grown in a greenhouse where actual 

temperatures ranged from approximately 21 o

C to approximately 42 oC. Eight replicate 

pots were planted with three seeds per pot for each of MON 87705, the conventional 

soybean control, and reference soybean varieties. At planting, each seed was inoculated 
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with approximately 1 x 107 cells of Bradyrhizobium japonicum (VAULT NP, Becker 

Underwood, Ames, IA) in phosphate-buffered saline. Pots were arranged after thinning 

in eight replicated blocks for the 6-week sampling period using a randomized complete 

block design. 

The reference soybean varieties starting seed were planted on September 9 and 10, 2008, 

and MON 87705 and the conventional soybean control starting seed were planted on 

September 11, 2008. In all cases, replicate pots had a minimum of one plant emerge 

within one week. A solution of nitrogen-free nutrient solution (~250 ml) was added 

weekly after plant emergence. 

I.3 Plant Harvesting/Data Collection 

Six weeks after emergence, plants were excised at the surface of the potting medium and 

shoot and root plus nodule material were removed from the pots. The shoot material was 

cut into smaller pieces and placed in labelled bags. The plant roots with nodules were 

separated from the potting medium by washing with water. Excess moisture was 

removed using absorbent paper towels and the roots plus nodules were placed in labeled 

bags. The same day that plants were harvested, nodules were removed by hand from the 

roots of each plant, enumerated, and the fresh weight (fwt) was determined. Nodules 

from each plant were then dried for at least 72 hours at approximately 65 °C, and dry 

weights were determined. 

The remaining root and shoot mass (fresh weight) were determined for each plant. Root 

and shoot material from each plant was then dried for at least 72 hours at approximately 

65 °C for dry weight determination. The shoot tissue was ground after drying with a 

Harbil 5G high-speed paint shaker prior to total nitrogen analysis. Shoot total nitrogen 

was determined by combustion using a nitrogen analyzer (Rapid N Cube, Elementar 

Americas, Inc.). 

I.4 Statistical Analysis 

The data consisted of six measurement endpoints taken at the six week sampling period: 

nodule number, nodule dwt (g), shoot dwt (g), root dwt (g), and shoot total nitrogen (% 

and g/plant). Data obtained from MON 87705, the control (A3525), and the reference 

soybean varieties were analyzed. 

An analysis of variance was conducted using a randomized complete block design with 

eight replications for each test, control, and reference substance. Data were analyzed 

using the Statistical Analysis System (SAS Version 9.2, SAS Institute, Inc. 2002-2008) 

with the level of statistical significance predetermined to be 5% (p<0.05). 
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Appendix J. Materials and Methods for Analysis of FAD2-1A and FATB1-A RNA 

levels in MON 87705 immature seeds 

J.1 Test Substance 

The test substance was MON 87705. As listed below in the table, four replicates of immature 

seed from a production plan, generated from seed lot GLP-0702-18254-S were used in this 

study. 

Sample ID Seed Lot # Tissue Type Replicate # 

REG07189-0015 GLP-0702-18254-S Immature seed 1 

REG07189-0016 GLP-0702-18254-S Immature seed 2 

REG07189-0017 GLP-0702-18254-S Immature seed 3 

REG07189-0018 GLP-0702-18254-S Immature seed 4 

J.2 Control Substance 

The control substance was conventional soybean variety A3525, the same genetic 

background as the test substance. As listed below in the table, four replicates of immature 

seed from a production plan, generated from seed lot GLP-0702-18252-S were used in this 

study. 

Sample ID Seed Lot # Tissue Type Replicate # 

REG07189-0001 GLP-0702-18252-S Immature seed 1 

REG07189-0002 GLP-0702-18252-S Immature seed 2 

REG07189-0003 GLP-0702-18252-S Immature seed 3 

REG07189-0004 GLP-0702-18252-S Immature seed 4 

J.3 Reference Substance 

Probe templates were used as positive hybridization controls in northern blot analyses. Prior 

to probe generation, DNA segments from the FAD2-1A (Monsanto Sequence Database) and 

FATB1-A (Monsanto Sequence Database) genes were generated from conventional soybean 

genomic DNA using a forward primer specific to the coding region and a reverse primer 

specific to the 3'UTR region of each gene. The resulting PCR segments of the FAD2-1A and 

FATB1-A genes were cloned into pGEM-T Easy vectors (pGEM-T Easy Vector System, 

Invitrogen, Carlsbad, CA) according to the manufacture's manual with minor modifications 

documented in the raw data. The modifications were approved by the study director and 

documented in the raw data. The sequence of the plasmid DNA was confirmed by 

sequencing and comparison to the expected sequences using pair wise alignments. The 

plasmid DNAs then were used as templates for PCR reactions to amplify the FAD2-1A and 

FATB1-A probe templates. The actin probe template was generated by PCR from 

conventional soybean genomic DNA using a forward primer specific to the coding region 

and a reverse primer specific to the 3'UTR region of the soybean actin gene (Soy57, GI: 

1498333). All PCR reactions and purification of DNA segments from agarose gels were 

performed according to in house SOPs. RiboRulerTM RNA ladders (high range) from 
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Fermentas Co (Glen Burnie, MD) were used for size estimations on northern blots and 

formaldehyde/agarose gels. The unique identities of the probes, as well as molecular weight 

markers are documented in the raw data. 

J.4 Characterization of Test, Control and Reference Substances 

The identity of the test and control substances was determined by event-specific PCR prior to 

planting the seed in the green house. The study director reviewed the chain of custody 

(COC) documentation and certificate of analysis (COA) to confirm the identity of the test 

and control substances prior to use of the materials in the study and a copy of the COAs was 

archived with the study data. Immature seed was harvested from the plants and identity 

confirmation was performed on each sample by event-specific PCR prior to use in the study. 

The study director reviewed the data prior to use of the materials in the study and the raw 

data was archived with the study. Mature seed was harvested from the plants and tested by an 

event-specific PCR. These results were reviewed by the study director prior to the start of the 

study and a copy of the Verification of Identity (VOI) was archived with the study. 

Prior to the start of the study, the FAD2-1A, FATB1-A, and actin probe templates were 

sequenced by the Monsanto Genomics Sequencing Center and the sequences were aligned 

with the expected sequences to confirm the identities of the probes. The raw data are 

archived with the study. 

Extracted RNA and RNA markers were stored in a -80° C freezer. DNA probes were stored in 

a -20° C freezer. The test, control, and reference substances were deemed stable during 

storage because there were no signs of degradation on formaldehyde/agarose gels and 

probing with endogenous genes yielded interpretable signals on northern blots. 

J.5 Total RNA extraction 

Total RNA from test and control substances was extracted according to an in-house SOP. 

All extracted RNA was stored in -80° C freezers. 

J.6 PolyA+ RNA Isolation 

PolyA+ RNA10 from the test and control substances was extracted from total RNA using the 

Poly(A) Purist MAG Kit (Ambion Co., Austin, TX) according to the manufacturer's protocol 

with minor modifications approved by the study director prior to performing the analysis. The 

procedure was documented in the raw data. The resulting polyA+ RNA was stored in a -80 °C 

freezer. 

J.7 Formaldehyde/Agarose Gel Electrophoresis 

PolyA+ RNA samples from test and control substances for FAD2-1A northern blot analysis 

were extracted from approximately 20-50 μg of total RNA, and resolved in 0.8% 

formaldehyde/agarose gels (0.8% with regard to agarose). PolyA+ RNA samples from test 

and control substances for FATB1-A northern blot analyses were extracted from approximately 

100 μg total RNA, and resolved on 1.0% formaldehyde/agarose gels (1.0% with regard to 

agarose). 

 

10Poly A enriched RNA 
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J.8 Northern Blot Analyses 

Northern blot analyses were performed by transferring polyA+ RNA from formaldehyde/ 

agarose gels onto nylon membranes and probing with radiolabeled DNA. FAD2-1A and 

FATB1-A hybridization signals were stripped from the blots and the stripped blots were 

hybridized to a 32P-labeled actin probe in order to show the relative amount of RNA loaded in 

each lane. Probes were prepared by random prime labeling. Each probe was added at 

approximately 1-2 × 106

 cpm per ml of the hybridization solution. 

DNA probe templates were run on the formaldehyde/agarose gels to serve as positive controls 

for hybridization. On northern blots the probe templates migrated slightly differently than 

predicted by the RNA ladder. The difference in migration is probably due to the inaccuracy of 

comparing a DNA probe to an RNA ladder. The following table lists the DNA probes, the 

positive hybridization controls, and the hybridization and wash temperatures for each northern 

blot analysis. 

Analysis 

Positive hybridization 

controls Probe 

Hybridization and 

Wash Temperature 

Expression of 

FAD2-1A

FAD2-1A probe template FAD2-1A 65 oC 

Expression of 

FATB1-A

FATB1-A probe 

template 

FATB1-A 60 oC 

Expression of 

actin

actin probe template actin 55 oC 
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Appendix K. Manufacturing Process for Soybean 

MON 87705 may be processed using conventional industry standard processing methods, 

which include extrusion methods (Erickson et al., 1980). Following cracking and aspiration 

of the soybean to separate the hulls, the hulls are screened to recover the fines generated 

during cracking, and the cracked soybean meats are conditioned and flaked to rupture oil 

cells and prep are a thin flake with a large surface area for solvent extraction. The soybean 

flakes then undergo solvent extraction with iso-hexane/hexane to yield crude soybean oil and 

soy meal. The crude oil is processed through a series of steps known as refining. In the first 

step, phospholipids are removed through a process known as degumming. This involves 

mixing with water to form gums. followed by centrifugation. The fatty acids in the 

degummed oil are neutralized through the addition of a caustic solution (sodium hydroxide) 

to form soaps soluble in water in a process known as alkali-refining. This process is 

preceded by a pretreatment step with phosphoric acid. The resulting soap solution is 

removed by centrifugation. Water washing and centrifugation further removes soaps to 

levels compatible with bleaching. Alternatively, oil can be further treated with trisyl to 

remove any residual soap content and then filtered. In the next step, the oil is “bleached” by 

mixing with a citric acid solution, followed by treatment with adsorbent clay to remove the 

peroxides, phosphatides, color bodies and traces of soap. Under vacuum to inhibit oxidation, 

the pigments are adsorbed and removed by filtration. In the final refining step, odoriferous 

components, flavor components and additional free fatty acids are removed by steam 

distillation to produce refined, bleached and deodorized (RBD) oil. This process of 

deodorization is carried out at high temperatures (250°C) and under vacuum. Anti-oxidants 

are utilized to inhibit oxidation of the oil. A pictorial representation of the manufacturing 

process described above is shown below in Figure K-1. 

Figure K-1. Schematic of Manufacturing Process for Soybean Oil. 

Mill

Ground 

Seed

Extractor

Seed

Meal

Miscella

Hexane Stripping Degumming

Caustic 

Refining

Bleaching

Deodorization Volatiles

RBD Oil

Optional

Pressing
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Appendix K References 

Erickson, D.R., E.H. Pryde, O.L. Brekke, T.L. Mounts and R.A. Falb, eds. 1980. Handbook 

of Soy Oil Processing and Utilization. American Soybean Association and American Oil 

Chemist’s Society, St. Louis, Missouri and Champaign, Illinois. Pp. 49-130. 
Monsanto Company 09-SY-201U Page 372 of 471

Appendix L. Glyphosate Resistance 

L.1 Introduction 

Monsanto considers product stewardship to be a fundamental component of customer service 

and business practices. Stewardship of the glyphosate molecule to preserve its usefulness for 

growers is an important aspect of Monsanto’s stewardship commitments. While herbicide 

resistance may eventually occur when any herbicide is widely used, resistance can be 

postponed, contained and managed through research, education and good management 

practices. These are key elements of Monsanto’s approach to glyphosate stewardship. 

The development of plant populations resistant to glyphosate can adversely impact the utility 

and life cycle of glyphosate products if it is not managed properly. As leaders in the 

development and stewardship of glyphosate products for over 30 years, Monsanto makes a 

significant investment in research to understand and develop the proper use and stewardship 

of glyphosate. Monsanto also invests in grower/retailer education and training programs to 

provide information on best practices to manage glyphosate resistance. This research 

includes an evaluation of factors that can contribute to the development of weed resistance 

and develop ways to mitigate the risk. The risk of weeds developing resistance and the 

potential impact of resistance on grower weed management practices vary greatly across 

different herbicide modes of action and they depend on a combination of factors. Therefore, 

there is no single set of best management practices to mitigate resistance for all herbicides, 

nor is there a single set of best management practices to mitigate resistance to a single 

herbicide for all weeds in all cropping systems. However, this document provides an 

overview of Monsanto’s approach to the development of best management practices to 

mitigate glyphosate resistance. 

L.2 The Herbicide Glyphosate

Glyphosate (N-phosphonomethyl-glycine) (CAS Registry #: 1071-83-6), the active 

ingredient in the Roundup family of nonselective, foliar-applied, postemergent agricultural 

herbicides, is among the world’s most widely used herbicidal active ingredients. Glyphosate 

is highly effective against the majority of economically significant annual and perennial 

grasses and broadleaf weeds. Currently glyphosate is labeled for control of more than 300 

weed species. Glyphosate kills plant cells by inhibition of 5-enolpyruvylshikimate-3-

phosphate synthase (EPSPS), an enzyme involved in the shikimic acid pathway for aromatic 

amino acid biosynthesis in plants and microorganisms (Franz et al., 1997). Glyphosate is the 

only known herbicide with this mode of action (Franz et al., 1997). The relevant aromatic 

amino acid pathway is not present in mammalian metabolic systems (Cole, 1985). A 

comprehensive human safety evaluation and risk assessment concluded that glyphosate has 

low toxicity to mammals, is not a carcinogen, does not adversely affect reproduction and 

development, and does not bioaccumulate in mammals (Williams et al., 2000). An 

ecotoxicological risk assessment concluded that the use of glyphosate does not pose an 

unreasonable risk of adverse effects to nontarget species, such as birds and fish, when used 

according to label directions (Giesy et al., 2000). Glyphosate has favorable environmental 

characteristics, including a low potential to move through the soil to reach ground water and 

is degraded over time by soil microbes. Because it binds tightly to soil, glyphosate’s 
Monsanto Company 09-SY-201U Page 373 of 471

bioavailability is reduced immediately after application, which is why glyphosate has no 

residual soil activity. 

L.3 Herbicide Use and Herbicide-resistant Weeds 

The vast majority of U.S. soybean growers depend, at least in part, on herbicides to control 

weeds. According to a report by USDA-ERS, herbicides were used on more than 97 percent 

of the total U.S. soybean acreage in 1997 (Fernandez-Cornejo and McBride, 2002).

Control of weeds in a crop is essential because weeds compete with the crop for the same 

limited resources in the field, including sunlight, water and nutrients (Ross and Lembi, 1985; 

Wilcut et al., 2003). Lack of effective weed control in soybean fields can result in yield 

losses of 50-90%. Because failure to control weeds within the crop can result in decreased 

yields and reduced crop quality, an intensive program for weed control is essential to ensure 

profitability (Dalley et al., 2001). When failure to control weeds in a crop increases weed 

populations, additional herbicide applications or cultural practices are then required to reduce 

the weed population. 

With any herbicide use, however, comes the potential for the selection of weeds resistant to 

that herbicide. Within a weed species individuals may possess an inherent ability to 

withstand the effects of a particular herbicide. Repeated use of that herbicide will expose the 

weed population to a "selection pressure," which may lead to an increase in the number of 

surviving resistant individuals in the population (HRAC, 2009). In other words, plants 

susceptible to the applied herbicide will die, while those few having some type of natural 

resistance may survive and reproduce. 

Weed resistance is generally defined as the naturally occurring heritable ability of some weed 

biotypes within a given weed population to survive a herbicide treatment that should, under 

normal use conditions, effectively control that weed population. Thus, a resistant weed must 

demonstrate two criteria as defined by the Weed Science Society of America website at 

www.wssa.net: (1) the ability to survive application rates of a herbicide product that once 

were effective in controlling it, and (2) resistance is heritable. Procedures to confirm 

resistance generally require both field and greenhouse analysis. Obtaining a correlation 

between field and greenhouse studies has been particularly important for the accurate 

detection of glyphosate resistance, for which the levels of resistance observed have been as 

low as 2X the susceptible biotypes (Lee and Ngim, 2000; Lorraine-Colwill et al., 1999). 

Herbicide-resistant weeds are neither a new phenomena nor is resistance unique to 

glyphosate. In 1952, the first U.S. herbicide-resistant weed, a spreading dayflower species 

resistant to the synthetic auxin 2,4-D, was identified in Hawaii (Heap, 2009). There are 

currently 72 herbicide-resistant weed species in the U.S. For example there are 38 weed 

species resistant to ALS herbicides, 15 species resistant to ACCase inhibitors, 23 weed 

species resistant to triazine herbicides, and nine (9) weeds species resistant to glyphosate 

herbicides (Heap, 2009). Growers have been managing these herbicide-resistant weeds for 

decades with the use of alternative herbicides and/or cultural methods such as tillage or crop 

rotation. 
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The occurrence of a herbicide-resistant weed biotype generally does not end the useful 

lifespan or preclude the effective use of the herbicide in question as part of an overall weed 

management system. This is particularly true for glyphosate, which remains a very useful 

tool despite its lack of effectiveness on specific resistant weed biotypes due to its ability to 

control a weed at different growth stages and the wide spectrum of weeds it effectively 

controls. Even for other herbicides that do not have the spectrum of control or the wide 

window of application, growers have continued to have a need to use these products even 

with the presence of resistant populations. For example, in 1995, imazethapyr and 

imazaquin, both in the imidazolinone herbicide family, were used on almost 60 percent of the 

soybean acres in the U.S., even though, at that time, nine weed species that were resistant to 

those herbicides were present in soybean producing areas (Heap, 2009). 

It is important to distinguish herbicide resistance, explained above, from herbicide tolerance. 

In contrast to species for which resistant biotypes have occurred, herbicide tolerant weed 

species has the inherent ability to survive applications of a particular herbicide at 

recommended rates. In other words, the species does not develop tolerance through selection 

but is innately tolerant to that herbicide. It may also be the case that a certain weed species, 

while neither resistant nor tolerant, is difficult to control with a particular herbicide, requiring 

more careful herbicide use and weed management. Section E herein discusses resistant, 

tolerant, and hard-to-control species in the context of glyphosate. 

L.4 Characteristics of Herbicides and Herbicide Use Influencing Resistance 

While the incidence of weed resistance is often associated with repeated applications of a 

herbicide product, the actual onset of resistance within a population depends very much on 

the specific herbicide chemistry in question, as well as the inherent presence of gene(s) that 

confer the ability of a plant to be resistant to a particular chemical within a specific weed 

species and even a specific population of that species (Sammons et al., 2007). Some 

herbicide products are much more prone to develop herbicide resistance than others (Heap, 

2009). Glyphosate has been used extensively for over three decades with relatively few 

cases of resistance development, particularly when compared to many other herbicides, 

considering the substantial acreage glyphosate-treated worldwide, and the total number of 

weeds that glyphosate can control. The graph in Figure 1 illustrates the instances of weed 

resistance to various herbicide families. The different slopes observed are largely due to the 

factors described above, which relate to chemistry and function, in addition to levels of 

exposure in the field. The summary below describes herbicide-specific factors determined to 

be important in the process of selecting for individuals that are inherently resistant to a 

herbicide. 
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Figure L-1. Weed Resistance to Various Herbicide Chemical Families 

L.5 Mechanisms of Resistance 

The application of a herbicide to a weed does not, itself, cause a mutation in later generations 

of the plant. Rather, over time, those few biotypes that are not susceptible to the herbicide 

may become dominant within a population with the repeated use of that herbicide. To date, 

the three known avenues by which a weed species is resistant to an herbicide have been 

identified as target site alteration (target site), enhanced metabolism of the herbicides 

(metabolism), and reduced absorption and/or translocation of the herbicide such that the 

herbicide does not get to the site of action within the plant cell (exclusion) (Sammons et al., 

2007). 

Herbicide resistance via target site is the most common resistance mechanism among the 

various herbicide classes. It has been found that a target site mechanism is the most common 

mechanism for ALS inhibitors, ACCase inhibitors, and triazines, but is less common for 

glyphosate. A target site alteration is one where there is/are amino acid substitution(s) in the 

protein that is the target of a herbicide such that the alteration prevents the binding of the 

herbicide to the protein and thus the activity of the protein is not altered and the plant grows 

normally. For ALS inhibitors, the level of resistance conferred by a target site mechanism 

has been found to be as high as 3,400 X (Ferguson et al., 2001). (Note: X is the labeled or 

recommended rate for a herbicide on a particular weed species.) For glyphosate, species 

found to exhibit a target site mechanism often show low levels of resistance (2-3X) due to the 

fact that glyphosate is a true transition state inhibitor (Schonbrunn et al., 2001; Sammons et 

al., 2007). In addition, multiple alterations of the same enzyme have been found for ALS and 

ACCase inhibitors (Tranal and Wright, 2002). This may explain the apparent high frequency 

of resistance and the short time in which resistance developed to herbicides in these two 
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classes of chemistries. Only one altered site in the targeted plant EPSPS enzyme has been 

found for glyphosate (Baerson et al., 2002). 

Herbicide resistance as a result of exclusion mechanisms is the glyphosate resistant 

mechanism among the majority of the weed species studied to date. This resistance 

mechanism has also been found to be associated with 2,4 D and paraquat. Within this 

category, there are two types of translocation alterations that have been observed for 

glyphosate; (a) restricted movement of glyphosate from leaf cells into the meristematic cells 

of the plant and (b) restricted movement of glyphosate within a cell into the chloroplast due 

to accumulation within the vacuole. The level of glyphosate resistance conferred with this 

mechanism is higher (6-8X) than for species exhibiting target site mutations (2-3X). 

The third type of herbicide resistance mechanism, metabolism, has not been found to be a 

resistance mechanism associated with glyphosate in any of the weed species studies thus far. 

However, legumes have been shown to degrade glyphosate and therefore this type of 

resistance mechanism may be active in some species (Reddy et al., 2008). 

In some species the experimental evidence suggests that multiple mechanisms of glyphosate 

resistance may occurr within the same plant and multiple mechanisms are needed to protect 

the plant from the phytotoxic effects of glyphosate (Yu et al., 2007). This implies that 

multiple genes (polygenic resistance) are necessary and thus the selection of plants with 

multiple genes needed to confer resistance would be expected to occur at a low frequency. 

In summary, the overall low occurrence of glyphosate resistance may be in part explained by: 

(1) the nature of the target site inhibition by glyphosate relative to other herbicides, (2) the 

lack of metabolism as a mechanism of selectivity for weed resistance, and (3) evidence of 

multiple mechanisms being necessary for resistance and thus resistance is polygenic and 

difficult to assemble and maintain. 

L.6 Use of Recommended Glyphosate Rate 

The interaction between herbicide application rate and resistance for postemergence 

herbicides, such as glyphosate, is dependent upon the nature of the plant gene(s) conferring 

resistance to the chemical. In general, herbicide rate has more effect on selecting for 

resistant individuals in a population if the resistant gene is semi-dominant or recessive as 

compared to the resistant gene being dominant. Likewise, herbicide rates would have more 

of an effect on the onset of resistance if commercially significant resistance required the 

additive effect of multiple genes (i.e. quantitative or polygenic resistance). Low rates would 

tend to allow certain biotypes to survive and mate with other biotypes of the same or an 

alternate resistant gene. The offspring of this mating may then be able to survive a full rate. 

Less-than-recommended or suboptimal rates have been implicated or speculated as the causal 

factor in herbicide resistance for several different herbicides, including chlortoluron-resistant 

blackgrass, diclofop-resistant ryegrass and dicamba-resistant kochia (Beckie, 2006). Busi 

(2009) demonstrated that, in three generations of a ryegrass biotype sprayed at sublethal rates 

of diclofop-methyl or glyphosate, a high level of resistance evolved to diclofop-methyl and a 

moderate level to glyphosate. The conclusion of this work was that growers should avoid 

lowering the application rate of herbicides, especially where major cross-pollinating weed 

species, such as lolium, are present. 
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L.7 Weeds Resistant to Glyphosate 

As with any other herbicide, the use of glyphosate may lead to the development of 

glyphosate-resistant weed species, and a list of glyphosate resistant weeds is provided below 

in Table L-1. However, the potential for the development of a glyphosate-resistant weed 

needs to be considered in the following context: (1) if a glyphosate-based weed control 

system were not available, other herbicide(s) with equal or greater potential for resistance 

would be used to control weeds and (2) other herbicides and cultural practices can be used to 

manage the glyphosate resistant species (Neve, 2008; Gustafson, 2008). 

To date, biotypes of fifteen weed species resistant to glyphosate have been identified and 

confirmed worldwide. Nine species resistant to glyphosate have been confirmed in the U.S., 

two of which were identified outside of Roundup Ready cropping systems. The speed of 

spread and geographical distribution of the resistant species has varied. Some species with 

resistant biotypes, such as common ragweed (Ambrosia artemisfolia) have been found in a 

limited number of sites across the mid-west, whereas marestail (Conyza canadensis) has been 

found in many states in the northeast, mid-west and the south. The reproductive biology of 

the particular weed species involved appears to be a factor contributing to the spread of 

resistant biotypes. In the above examples, marestail produces a large number of winddispersed seeds, which contributes to rapid spread, while ragweed seeds do not have features 

that allow for such easy distribution by the wind (Weaver, 2001; Pennsylvania State 

University Cooperative Extension Service Weed Identification Bulletin #8, 1986). 

Table L-1. U.S. Glyphosate Resistant Weeds through June 2009 

Weeds identified outside of Roundup Ready 

Systems

Rigid ryegrass (Lolium rigidum)

Hairy fleabane (Conyza bonariensis)

Weeds identified in Roundup Ready Systems Horseweed (Conyza canadensis)

Common ragweed (Ambrosia artemisiifolia)

Giant ragweed (Ambrosia trifida)

Palmer amaranth (Amaranthus palmeri)

Common waterhemp (Amaranthus rudis)

Italian ryegrass (Lolium multiflorum)

Johnson grass (Sorghum halepense)
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Some weed species, such as Equisetum arvensis (field horseweed), are tolerant, as opposed to 

resistant, to herbicides. In addition, some species are more difficult to control with 

glyphosate than others (e.g. lambsquarters (Chenopodium album) and morninglory (Ipomea 

sp.) and require more care to make sure the correct amount of glyphosate is applied at the 

right growth stage. For these difficult-to-control weeds, environmental conditions can affect 

herbicide performance more than for weeds that are easier to control and therefore it is more 

critical that the correct rate be applied at the right growth stage when making applications to 

weeds in the difficult-to-control category. Weed control situations involving tolerant or 

difficult-to-control species are often confused with resistance. 

L.8 Use of Glyphosate for In-crop Weed Management 

Monsanto has developed plants through biotechnology to be tolerant to glyphosate. The 

development, approval and cultivation of these Roundup Ready crops have facilitated 

additional uses of glyphosate in crops where such uses were not previously possible given the 

non-selective nature of glyphosate. This development has provided growers with an 

additional weed management option and benefits relative to existing weed management 

options. The glyphosate-tolerant trait in Roundup Ready crops has no effect per se on the 

control of weeds. From a weed resistance standpoint, the use of glyphosate with glyphosatetolerant soybean is no different than the use of a selective herbicide in a conventional 

soybean crop. 

The most often cited benefits of glyphosate as an in-crop weed management option are 

simplicity, flexibility of application timing, weed spectrum, crop safety, and environmental 

safety (Dill, 2005). The ability to use glyphosate in-crop has allowed farmers to change their 

farming practices in some cases. For example, Roundup Ready cotton and Roundup Ready 

soybean have often been cited as a major reason for an increase in no-till practices (Dill et 

al., 2008). 

Since Monsanto commercialized the first Roundup Ready soybean variety in 1996, growers 

have enthusiastically adopted the technology. Currently, biotechnology-derived herbicide 

tolerant soybean is planted on over 92% of U.S. soybean acreage (USDA-NASS, 2008). The 

Roundup Ready soybean system, (i.e., planting Roundup Ready soybean and applying 

glyphosate in-crop), has become the standard weed control program in U.S. soybean 

production. In addition, weed control in a Roundup Ready soybean system likely will 

involve not only glyphosate-based herbicides but also other herbicides and weed 

management practices to effectively manage weeds, thus increasing crop yield and reducing 

development of resistant weeds. State Universities/Cooperative Extension Services (CES) 

publish information on best weed management practices in Roundup Ready crops to address 

both of these objectives (see Table L-2). In addition Monsanto and other companies selling 

glyphosate products provide information on these same best management practices as 

detailed later in this Appendix. 

L.9 Weed Resistance Management Strategies for Glyphosate 

As part of Monsanto’s stewardship of Roundup agricultural herbicides and Roundup Ready 

crop systems, the company has conducted investigations and worked extensively with 
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academics and other herbicide manufactures to understand the best practices to manage 

resistance. These investigations have demonstrated that one of the major factors contributing 

to the development of resistant weeds is poor weed control management practices. These 

include application of herbicides at rates below those indicated on the EPA-approved label 

for the weed species and sole reliance on a particular herbicide for weed control without the 

use of other herbicides or cultural control methods (i.e. pre-plant and in-crop tillage) (Beckie, 

2006; Peterson et al., 2007). 

The weed resistance management recommendations that will be made for the use of 

glyphosate in conjunction with varieties developed from MON 87705 will be consistent with 

the Herbicide Resistance Action Committee’s guidelines for prevention and management of 

herbicide resistance (HRAC, 2009). These guidelines recommend an integrated approach to 

weed resistance management including crop management (i.e. row spacings, etc), cultural 

techniques and herbicides. 

EPA is the U.S. federal regulatory agency that administers the federal law governing 

pesticide sale and use (FIFRA). EPA encourages pesticide manufacturers to provide growers 

with information regarding a herbicide’s mode of action to aid growers in planning herbicide 

use practices and to foster the adoption of effective weed-resistance management practices as 

specified by EPA in PR Notice 2001-5. In that document EPA states that “this approach to 

resistance management is sound and would be highly beneficial to pesticide manufacturers 

and pesticide users” (EPA PR Notice 2001-5 at 

http://www.epa.gov/opppmsd1/PR_Notices/pr2001-5.pdf). EPA approves all pesticide label 

use instructions based on the agency’s evaluation of supporting data supplied by the pesticide 

registrant or manufacturer. After EPA approves a pesticide label, it is a violation of federal 

law to use the pesticide for a use or in a manner not in accordance with the label directions.

Monsanto incorporates EPA’s guidelines for pesticide resistance management labeling on its 

glyphosate-based agricultural herbicide labels, and will do so on the label for products to be 

applied over the top of varieties developed from MON 87705 (An example of current 

Roundup WeatherMAX product label is available at www.cdms.net/ldat/ld5UJ029.pdf). 

EPA-approved labels for Roundup branded herbicide weed-resistant management 

recommendations are designed to minimize the potential for the development of glyphosateresistant weeds. By approving a label for a glyphosate-based agricultural herbicide, EPA has 

concluded that the product will not cause unreasonable adverse effects to the environment or 

human health when used in accordance with the label’s directions. 

The weed resistance management guidelines on the labels of Roundup agricultural herbicides 

include recommendations that are well-documented in the scientific literature as being 

appropriate and effective for weed control and to mitigate weed resistance. Significant 

research has been conducted to identify the appropriate application rate of glyphosate 

required to control a particular weed at various growth stages under various agronomic and 

environmental conditions. These rates are based on over 35 years of ongoing research at 

Monsanto to evaluate the efficacy of Roundup agricultural herbicides. Studies have included 

efficacy of weed control for a broad spectrum of weeds and under a wide range of conditions. 

Research has also involved the study of tolerance of the Roundup Ready crops to over-thetop applications of Roundup agricultural herbicides. Delaying herbicide application allows 

weeds to grow larger, requiring higher herbicide rates for control. At lower glyphosate 

application rates, larger weeds are controlled less consistently (Gubbiga et al., 2002). A key 
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element of effective weed control and weed resistance management, therefore, is using the 

correct rate of glyphosate at the right time for the weed species and the size of the weed (i.e., 

using a lethal dose which avoids the need for subsequent applications). This important 

strategy is well-supported by field research studies at several universities (Wilson et al., 

2003; Jeschke et al., 2006; Stoltenberg, 2002; Wilson et al., 2006). Additionally, it is 

accepted in the weed science community that the use of multiple herbicide modes of action 

via tank mixtures, use of herbicides with different modes of action in a rotational crop, or 

using multiple herbicides in sequence within a crop will reduce the risk of developing weed 

resistance (Gessel and Stegal, 1990; Beckie, 2006). Tank-mixing involves mixing two or 

more herbicides in the spray tank immediately prior to application. Simultaneously using 

two herbicides with different modes of action significantly reduces the probability of weeds 

developing resistance to either or both chemistries. To provide growers with the tools 

needed to minimize resistant weed development, Monsanto will continue to investigate and 

recommend appropriate residual and postemergence herbicide products that have a different 

mode of action from glyphosate. As an example, the herbicide metolachlor (tradename 

PARRLAYTM) is a residual herbicide that will help reduce flushes of annual grasses and 

pigweed which could slow the selection and potential spread of glyphosate-resistant weeds in 

Roundup Ready cotton and Roundup Ready Flex cotton systems. 

Crop rotation and management of the fallow period and cover crops, has been found to be an 

important consideration in managing resistance. In general, crop rotation fosters use of 

alternate herbicide modes of action and, potentially, use of additional cultural practices to 

manage weeds over time (Beckie et al., 2004). Several authors have referred to this general 

concept as applying “diversity” across cropping/fallow seasons to manage weed resistance 

(Beckie, 2006; Powles, 2008). In general, conservation tillage practices (minimum-till and 

no-till) are viewed as creating environments where herbicide resistance is likely to develop. 

This is probably due to heavy selection pressure put on weeds by herbicides in these 

environments and the absence of tillage as a cultural practice to supplement herbicides. 

However, this is not always the case. Legere et al. (2000) found that an increase in ACCase 

inhibitors (aryloxyphenosy propionate and cyclohexanedinone chemical families) use in 

conservation tillage system did not result in an increased incidence of wild oat populations 

resistant to ACCase inhibitors. In addition, preplant and/or in-crop cultivation will actually 

promote the spread of some species such as Johnsongrass. Therefore, no-till would be a 

better cropping system to contain Johnsongrass populations that could contain a resistant 

gene (McWhorter, 1972). 

The stewardship program for managing weed resistance in MON 87705 soybean is 

essentially the same program that is already in place for Roundup Ready soybean event 40-3-

2 and for Roundup Ready 2 Yield soybean. The weed-resistance management guidelines 

used for varieties developed from MON 87705 will be consistent with those that growers of 

Monsanto’s products are contractually obligated to abide by through the Monsanto 

Technology Stewardship Agreement (MTSA). Each soybean grower who will purchase a 

variety developed from MON 87705, or any other variety containing the Roundup Ready 

trait, must enter into a limited use license with Monsanto and must sign and comply with the 

MTSA. The MTSA incorporates by reference and requires the grower to follow Monsanto’s 

Technology Use Guide (TUG), which sets forth the requirements and best practices for the 

cultivation of crops with the Roundup Ready trait including recommendations on weedresistance management practices. Among each grower’s legal commitments is the federal 
Monsanto Company 09-SY-201U Page 381 of 471

requirement to use all Roundup herbicides in accordance with the directions for use on their 

labels, which incorporate weed resistance management practices. These are inclusive of the 

weed resistance management guidelines that are found on the EPA approved Roundup 

agricultural herbicide labels. 

The weed-resistance management practices articulated in the TUG are broadly 

communicated to growers and retailers in order to minimize the potential for the 

development of glyphosate-resistant weeds. These practices are communicated through a 

variety of means, including direct mailings to each grower purchasing a Roundup Ready crop 

product, a public website (www.weedresistancemanagement.com), and reports in farm media 

publications. The overall stewardship program is reinforced through collaborations with U.S. 

academics who provide their recommendations for appropriate stewardship of the use of 

Roundup agricultural herbicides with Roundup Ready crops, as well as by crop commodity 

groups who have launched several weed-resistance educational modules available on their 

websites. Finally, Monsanto urges farmers to report any incidence of repeated 

nonperformance of Roundup agricultural herbicides on a particular weed, and Monsanto 

investigates cases of unsatisfactory weed control to determine the cause as defined in Section 

L-10 of this Appendix. 

In cases where resistance is confirmed, Monsanto and University/CES provide 

recommendations for alternative control methods for farmers (see Table L-2). These 

recommendations are made available through Monsanto supplemental labels, Monsanto 

TUG, Monsanto and University publications and internet sites to growers, consultants, 

retailers and distributors. In all cases of glyphosate-resistant weeds in the U.S. and globally, 

there are alternative herbicides and cultural methods available to farmers to effectively 

control these species. Some examples of these recommendations from University/CES 

personnel are found in Table L-2. It is important to note that there are many alternative 

options in each situation. 
Monsanto Company 09-SY-201U Page 382 of 471

Table L-2. Management Reccomendations for Control of Glyphosate Resistant Weeds Glyphosate Resistant Weed 

State Crop Recommendations for alternative herbicides to 

manage glyphosate resistant weeds1

Reference (Bulletin No.) 

Palmer amaranth 

AR Soybean Burndown: flumioxazin and 

Pre: flumioxazin or metolachlor, and/or Post: fomesafin 

U of AR (FSA2152www.uaex.edu 

 AR Cotton PPI: triflualin or pendimethalin and/or 

Pre: diuron or fluometuron and/or E. Post: metolachlor and/or Post directed: diuron or prometryn or Layby: flumioxazin 

U of AR (FSA2152www.uaex.edu 

Waterhemp MO Corn Pre: metolachlor or acetachlor or isoxaflutole or 

mesotrione or atrazine, and/or Post: atrazine or dicamba or 2,4D 

U of MO (IPM1030www.extension.mis

 MO Soybean Pre: metribuzine or sulfentrazone or metolachlor or 

flumioxazin and/or Post: lactofen or fomesafen or aciflorfen 

U of MO (IPM1030www.extension.mis

Common ragweed 

OH / IN 

Corn Pre: atrazine or dicamba or acetochlor and/or 

Post: dicamba or tembotrione or mesotrione, or troprmezone 

2009 OH/IN Weed (789) www.btny.purdue.e

 OH / 

IN 

Soybean Burndown: 2,4D and 

Pre: metribuzine or flumioxazin or cloransulam and/or Post: cloransulam or fomesafen or lactofen 

2009 OH/IN Weed (789) www.btny.purdue.e

Giant ragweed 

OH / IN 

Corn Burndown: 2,4D +atrazine and 

Pre: Lumax or atrazine+isoxaflutole and/or 

Post: atrazine or dicamba or tembotrione or mesotrione, or troprmezone 

2009 OH/IN Weed (789) www.btny.purdue.e
Monsanto Company 09-SY-201U Page 383 of 471

Table L-2 (cont.). Management Reccomendations for Control of Glyphosate Resistant Weeds Glyphosate Resistant Weed

State Crop Recommendations for alternative herbicides to 

manage glyphosate resistant weeds1

Reference (Bulletin No.) 

Giant ragweed 

OH / IN 

Soybean Burndown: 2,4D and 

Pre: Canopy or Envive or imazaquin or Authority or flumioxazin or cloransulam and/or Post: cloransulam or fomesafen or lactofen+bentazon 

2009 OH/IN Weed (789) www.btny.purdue.

Marestail TN Corn Burndown: 2,4D or dicamba or 

Pre: atrazine and/or Post: dicamba 

2009 TN Weed Co(PB1580) www.weeds.utk.ed

 Soybean Burndown: 2,4D or dicamba or flumioxizin or 

Pre: metribuzin or fluioxazin and/or Post:cloransulam 

2009 TN Weed Co(PB1580) www.weeds.utk.ed

 Cotton Burndown: dicamba or flumioxazin or trifloxysulfuron 

or Pre: fluometuron or diron or prometryn and/or Post:trifloxysulfuron and/or Post-directed: flometuron+MSMA or diuron+MSMA or prometryn+trifloxysulfuron 

2009 TN Weed Co(PB1580) www.weeds.utk.ed

1(Burndown=before planting; Pre= preemergence; Post= postemergence; Post-directed= applied postemergence 

the crop; PPI=Pre Plant Incorporated) 
Monsanto Company 09-SY-201U Page 384 of 471

L.10 Monsanto Weed Performance Evaluation and Weed Resistance Management Plan 

To support and enhance Monsanto’s weed management principles, stewardship program, 

and grower recommendations, Monsanto implements a Weed Performance Evaluation 

Program (WPEP) based on grower performance inquiries and field trial observations. In 

addition, a Monsanto Resistance Management Plan (MRMP) will be implemented as 

warranted. Each of these is discussed below. 

The goal of the WPEP program is to continue to adapt, modify, and improve Monsanto’s 

weed control recommendations to growers, with a focus on: (a) identifying performance 

issues with particular weeds and growing conditions; (b) providing product support to 

growers who are not satisfied with their level of weed control; and (c) identifying and 

investigating potential cases of glyphosate resistance early so that mitigation strategies can be 

implemented. 

The WPEP is initiated when a grower reports to Monsanto, a retailer and/or University/CES 

personnel instances of unsatisfactory weed control following a glyphosate application. 

Follow-up with the grower is initiated by any of the three parties listed above to understand 

the situation, understand the reason for the inquiry and resolve it to the satisfaction of the 

customer. By virtue of Monsanto’s presence in the marketplace, in cases where the initial 

contact with the grower is a retailer or University/CES person, Monsanto will be contacted 

and will engage in the technical investigation. It is important to Monsanto, as part of its 

proactive stewardship commitment, that these product performance inquiries are acted upon 

and investigated quickly. 

The vast majority of these reported herbicide performance issues are determined to be due to 

application error or environmental conditions and are resolved through a phone conversation 

with the grower. However, a system is in place to investigate isolated or recurring 

performance problems for a weed species in a specific field. As warranted, a Monsanto, 

retailer or University/CES technical representative will arrange an on-site visit. If resistance 

is suspected to be a cause of the performance problem and has not already been confirmed 

for the specific weed and/or geography, additional steps will be taken to further explore the 

possibility of a new resistant weed species or population. These are the first steps of the 

Monsanto Resistance Management Plan (MRMP). When the reported problem involves a 

weed where resistance is common in the geography, the technical representative may simply 

confirm that the grower is managing a resistant population in an appropriate manner. 

The MRMP consists of two primary elements: (a) initiation of testing steps to verify cases of 

suspected resistance, and (b) development and communication of Monsanto and/or 

University/CES guidelines to incorporate resistance mitigation into weed management 

programs where resistance is confirmed. 

As part of the steps to verify cases of suspected resistance and if the on-site technical 

investigation cannot rule out potential resistance, seed may be collected from the subject site 

for follow-up greenhouse trials at Monsanto or at another University or third-party testing 

facilities that are set up to do this type of evaluation. If greenhouse trials do not repeat the 

observation and the weed is clearly controlled at label rates, then a thorough follow-up visit 

is conducted with the grower to re-evaluate the application recommendations and conditions 

of the operation that may be impacting weed control. If the greenhouse efficacy trials do 
Monsanto Company 09-SY-201U Page 385 of 471

indicate insufficient control at label rates, then additional studies may be conducted to verify 

if the weed is resistant or, if the Monsanto, University/CES or third-party researcher has 

sufficient evidence, resistance may be considered to be confirmed without further testing. 

Resistance is considered to be confirmed if the two resistance criteria outlined in the Weed 

Science Society of America (www.wssa.net) (described in section L.3) are deemed to be 

fulfilled either through field and/or greenhouse data. 

When resistance is confirmed, the scientific and grower communities are notified and a weed 

resistance mitigation plan is implemented by Monsanto in cooperation with the 

University/CES. The mitigation plan is designed to manage the resistant biotype through 

effective and economical weed management recommendations implemented by the grower. 

The scope and level of intensity of the mitigation plan may vary depending on a combination 

of the following factors: (a) biology and field characteristics of the weed (seed shed, seed 

dormancy, etc.), (b) importance of the weed in the agricultural system, (c) resistance status of 

the weed to other herbicides with alternate modes of action, and (d) availability of alternative 

control options. These factors are analyzed by Monsanto and University/CES personnel in 

combination with economic and practical management considerations to develop a tailored 

mitigation strategy. The plan considers what is technically appropriate for the particular 

weed and incorporates practical management strategies that can be implemented by the 

grower. 

After a mitigation plan is developed, Monsanto communicates the plan to the grower 

community through the use of supplemental labeling (labeling which includes newly 

approved uses, use directions, or other instructions which have been added since the last 

EPA-approved Master label), informational fact sheets, retailer training programs, agriculture 

media and/or other means, as appropriate. 

The final step of the MRMP may include extensive genetic, biochemical or physiological 

analyses of confirmed cases of glyphosate resistant weeds in order to elucidate the 

mechanism of resistance. The research findings are communicated to the scientific 

community through meetings and publications, and information pertinent to field 

applications is incorporated into weed management recommendations. 

In addition to the grower inquiry initiated process, Monsanto, alone and in cooperation with 

University/CES, conducts field studies to understand the potential for weed resistance and 

weed shifts as the result of various weed management programs implemented in a Roundup 

Ready crop. These studies allow researchers to better track specific factors that can influence 

the development of resistance to specific weeds. 

L.11 Summary 

Development of weed resistance is a complex process that can be difficult to accurately 

predict. No single agronomic practice will mitigate resistance for all herbicides or all weeds. 

As a result, weed resistance needs to be managed on a case-by-case basis and tailored for the 

particular herbicide and weed in order to meet grower needs. Using good weed management 

principles, built upon achieving high levels of control through proper application rate, choice 

of cultural practices, and appropriate companion weed control tools will allow Roundup 

agricultural herbicides to continue to be used effectively. In cases where weed populations 

have developed resistance to glyphosate, effective management options are available and 
Monsanto Company 09-SY-201U Page 386 of 471

experience has shown that growers continue to find value in using glyphosate in their weed 

control programs. 

The key principles for effective stewardship of glyphosate use, including Roundup Ready 

crops, include: (1) basing weed management and weed resistance management practices on 

local needs and using the tools necessary to optimize crop yield, (2) using proper rate and 

timing of application, (3) not relying solely on one herbicide weed control option across a 

cropping system, (4) responding rapidly to instances of unsatisfactory weed control, and (5) 

providing up to date weed management and weed resistance management training. 

 
Monsanto Company 09-SY-201U Page 387 of 471

Appendix L References 

Baerson, S.R., D.J. Rodriguez, M. Tran, Y.M. Feng, N.A. Biest, and G.M. Dill. 2002. 

Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-

enolpyruvylshikimate-3 phosphate synthase. Plant Physiology 129:1265-1275. 

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed 

Technology 20:793-814. 

Beckie, H.J., L.M. Hall, S. Meers, J.J. Laso, and F.C. Stevenson. 2004. Management 

practices influencing herbicide resistance in wild oat. Weed Technology 18:853-859. 

Busi, R., S. M. Velayudhan, and S.B. Powles. 2009. Use of below label herbicide rates can 

lead to evolution of herbicide resistant weeds. Abstract No. 296. Proceedings of the Weed 

Science Society of America 2009. 

Cole, D.J. 1985. Mode of action of glyphosate-a literature analysis. The herbicide 

glyphosate:48-74. 

Dalley, C.D., K.A. Renner, and J.J. Kells. 2001. Weed competition in Roundup Ready 

soybean and corn. Michigan State University, Dept of Crop and Soil Science. 

Dill, G.M., C.A. Jacob, and S.R. Padgette. 2008. Glyphosate resistant crops: Adoption, use 

and future considerations. Pest Management Science 64:326-331. 

Dill, G.M. 2005. Glyphosate-resistant crops: History, status and future. Pest Management 

Science 61:219-224. 

Ferguson, Gabrielle M., Allan S. Hamill, and François J. Tardif. 2001. ALS inhibitor 

resistance in populations of Powell amaranth and redroot pigweed. Weed Science 49:448-453. 

Fernandez-Cornejo, J. and W.D. McBride. 2002. Adoption of bioengineered crops. 

Agricultural Economic Report AER810, U.S. Department of Agriculture, Washington, D.C. 

Franz, J., M.K. Mao, and J.A. Sikorski. 1997. Glyphosate: a Unique Global Herbicide. 

American Chemical Society, Washington, D.C. 

Giesy, J.P., S. Dobson, and K.R. Solomon. 2000. Ecotoxicological risk assessment for 

Roundup herbicide. Rev. Environ. Contam. Toxicol. 167:35-120. 

Gustafson, D.I. 2008. Sustainable use of glyphosate in North American cropping systems. 

Pest Management Science 64:409-416. 

Heap, I. 2009. International survey of herbicide resistant weeds. 

http://www.weedscience.org/in.asp [Accessed July 1, 2009]. 

HRAC. 2009. Classification of herbicides according to mode of action. Herbicide Resistance 

Action Committee. http://www.hracglobal.com/ [Accessed July 1, 2009]. 
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Jeschke, M.R. and D.E. Stoltenberg. 2006. Weed community composition over eight years of 

continuous glyphosate use in a corn-soybean rotation. Pages 33-34 in Weed Science Society 

of America Proceedings. 

Lee, L.J. and J. Ngim. 2000. A first report of glyphosate-resistant goosegrass (Eleusine 

indica L. Gaertn) in Malaysia. Pest Management Science 56:336-339. 

Legere, A., H.J. Beckie, F.C. Stevenson, and A.G. Thomas. 2000. Survey of management 

practices affecting the occurrence of wild oat (Avena fatua) resistant to acetyl-coA 

carboxylase inhibitors. Weed Technology 14:366-376. 

Lorraine-Colwill, D.F., T.R. Hawkes, P.H. Williams, S.A.J. Warner, P.B. Sutton, S.B. 

Powles, and C. Preston. 1999. Resistance to glyphosate in Lolium rigidum. Pesticide Science 

55:489-491. 

McWhorter, C.G. 1972. Factors affecting johnsongrass rhizome production and germination. 

Weed Science 20:41-45. 

Neve, P. 2008. Simulation modeling to understand the evolution and management of 

glyphosate resistance in weeds. Pest Management Science 64:392-401. 

Peterson, D., B. Olson, K. Al-Khatib, R. Currie, J.A. Dille, J. Falk, P. Geier, D. Regehr, P. 

Stahlman, C. Thompson. 2007. Glyphosate stewardship. Kansas State University 

Agricultural Expewriment Station and Cooperative Extension Service Bulletin MF2767. 

Powles, S.B. 2008. Evolved glyphosate-resistant weeds around the world: Lessons to be 

learnt. Pest Management Science 64:360-365. 

Reddy, K.N., A.M. Rimando, S.O. Duke, and V.K. Nandula. 2008. Aminomethylphosphonic 

acid accumulation in plant species treated with glyphosate. Journal of Agricultural and Food 

Chemistry 56:2125-2130. 

Ross, M.A. and C.A. Lembi. 1985. Applied Weed Science. Burgess Publishing Co., 

Minneapolis. 

Sammons, R.D., D.C. Heering, N. DiNicola, H. Glick, and G.A. Elmore. 2007. Sustainability 

and stewardship of glyphosate and glyphosate-resistant crops. Weed Technology 21(2):347-

354. 

Schonbrunn, E., S. Eschenburg, W.A. Shuttleworth, J.V. Schloss, N. Amrhein, J.N.S. Evans, 

and W. Kabsch. 2001. Interaction of the herbicide glyphosate with its target enzyme EPSP 

synthase in atomic detail. Proceedings of the National Academy of Sciences U.S.A. 98:1376-

1380. 

Stoltenberg, D.E. 2002. Weed management and agronomic risks associated with glyphosateresistant corn and soybean cropping systems. Proceedings Fertilizer Aglime and Pest 

Management Conference Cooperative Extension Service, University of Wisconsin, Madison 

41:200-208. 
Monsanto Company 09-SY-201U Page 389 of 471

Tranel, P.J. and T.R. Wright. 2002. Resistance of weeds to ALS-inhibiting herbicides: What 

have we learned? Weed Science 50:700-712. 

USDA-NASS. 2007. Acreage 2007 (June report). U.S. Department of Agriculture National 

Agricultural Statistics Service, Washington, D.C. 

Weaver, S.E. 2001. The biology of Canadian weeds. 115. Conyza Canadensis. Canadian 

Journal of Plant Science 81:867-875. 

Wilcut, J.W., R.M. Hayes, R.L. Nichols, S.B. Clawis, J. Summerlin, D.K. Miller, A. Kendig, 

J.M. Chandler, D.C. Bridges, B. Bracke, C.E. Snipes, and S.M. Brown. 2003. Weed 

management in transgenic cotton. North Carolina State University Technical Bulletin 319. 

Williams, G.M., R. Kroes, and I. Munro. 2000. Safety evaluation and risk assessment of the 

herbicide Roundup and its active ingredient, glyphosate for humans. Regulatory Toxicology 

and Pharmacology 31:117-165. 

Wilson, R.G., S. Miller, P. Weestra, and P. Stahlman. 2006. Sustainability of glyphosate 

resistant cropping systems. Page 60 in Weed Science Society of America Proceedings. 

Wilson, R. and P. Stahlman. 2003. Maintain the rate. Pages 12-13 in Nebraska Farmer, June 

2003. 

Yu, Q., A. Cairns, and S. Powles. 2007. Glyphosate, paraquat and ACCase multiple herbicide 

resistance evolved in a Lolium rigidum biotype. Planta 225:499-513. 

 
Monsanto Company 09-SY-201U Page 390 of 471

Appendix M. Nutritional Impact Evaluation of MON 87705 Soybean Oil 

M.1 Intended Food Uses of MON 87705 Soybean Oil 

It is anticipated that MON 87705 soybean oil would substitute for the liquid soybean oil in 

the market place. The use of liquid soybean oil is mainly in salad dressings, mayonnaise and 

spreads, home use liquid soybean oil, and margarines (stick, tub, light variety). These four 

food categories represent approximately 94% (5,498 million pounds) of the replaceable 

liquid soybean oil market in the U.S. (HyQuest, 2009). 

In order to assess the nutritional impact from the introduction of MON 87705 into the 

marketplace, a dietary replacement model was used. In this model, MON 87705 soybean oil 

was substituted for the liquid soybean oil fraction in a subset of foods in the U.S. diet, 

including salad dressings, mayonnaise and spreads, home use liquid soybean oil, and 

margarines. This assessment assumed that all of the targeted oil components of the foods 

proposed for replacement at all eating occasions in the U.S. are replaced with MON 87705 

soybean oil. The results presented in this assessment represent a conservative estimation of 

the effect of MON 87705 soybean oil on fatty acid (FA) composition of the diet. The 

assessment is exclusive of frying applications, which generally utilize hydrogenated or low 

linolenic oils. Fatty acid intake estimates in this dietary replacement model were prepared 

for Monsanto by Exponent (Washington D.C.) 

A baseline FA composition for the soybean oil component of the four target food categories 

(mentioned above) was created using a combination of published FA compositions of liquid 

and partially hydrogenated soybean oils (ISEO, 2006; Exler et al., 1993), and market 

research analysis on currently utilized oil blends for the target foods. 

M.2 Estimated Dietary Intake of MON 87705 Soybean Oil based on Proposed Food 

Uses 

Four major sources of data were used to conduct the analysis: (1) Food intake data and 

nutrient composition from National Health and Nutrition Examination Survey (NHANES, 

CDC, 2007) 2003-2004 and 2005-2006, (2) Target foods and associated oils and Exponent’s 

proprietary recipes, (3) Baseline fatty acid profile for the oils associated with target foods, 

and (4) Fatty acid profile for MON 87705 soybean oil. Exponent’s proprietary software 

package, FARETM version 8.43 was used to implement the analysis. The fatty acid 

composition of MON 87705 used in the dietary replacement model is summarized in Table 

M-1. 
Monsanto Company 09-SY-201U Page 391 of 471

Table M-1. Fatty Acid Profile for MON 87705 Used in the Intake Estimations 

Fatty Acids Composition 

(% wt of Total FA) 

16:0 2.4 

18:0 3.3 

18:1 76.5 

18:2 10.1 

18:3 6.7 

Substitutions of MON 87705 soybean oil were done at the 100% substitution level, i.e.,

assuming 100% of the liquid soybean oil contained in these foods was replaced with the 

MON 87705 soybean oil, and every eating occasion of a target food was replaced by a 

comparable food containing MON 87705 soybean oil. 

M.3 Estimated Daily Intake of Total Fat and Selected Fatty Acids before and after 

Replacement of Conventional Soybean Oil with MON 87705 Soybean Oil in Targeted 

Foods 

The mean and 90th percentile intakes of total fat and five selected FA from conventional 

soybean (prior to substitution with MON 87705 soybean oil) were estimated for the target 

foods, and are reported in Tables M-2 and M-3. For the U.S.population, the soybean oil 

component of the target foods contributes an average of 4.27 g/day per capita of total fat; this 

amounts to approximately 5.4% of total fat from the whole diet. At the 90th percentile, per 

user, the total fat contribution from the soybean oil component of the target foods is 16.28 

g/day, or approximately 12.6% of total fat from the whole diet. To normalize consumption 

of nutrients for the range of caloric intake across a population, percent of energy/caloric 

intake (% en) is used (IOM, 2002). Therefore, on a percent energy (% en) basis, the mean 

per capita contribution from the liquid soybean oil component of the target foods is 1.8% and 

the 90th percentile per user is 6.83%. 

In MON 87705 soybean oil, intake of 18:1 oleic acid is increased the most among the five 

fatty acids evaluated. For the U.S. population, the mean per capita intake of 18:1 oleic acid 

from the MON 87705 soybean oil component from proposed foods increased from 1.04 

g/day to 3.13 g/day (1.35 % en). The 90th percentile per user intake of 18:1 oleic acid 

increased from 3.86 g/day to 12.22 g/day (Tables M-2 and M-3). 

 
Monsanto Company 09-SY-201U Page 392 of 471

Table M-2. Mean Per Capita Fatty Acid Intake from Target Foods Pre and Post 

MON 87705 Replacement, U.S.population 

Fatty Acids g/day % En

Pre 

replacement

Post 

replacement

% 

change

Pre 

replacement

Post 

replacement

% 

change

Palmitic 16:0 0.45 0.13 -71.11 0.19 0.06 -68.42

Stearic 18:0 0.18 0.19 5.56 0.08 0.08 0.00

Oleic 18:1 1.04 3.13 200.96 0.45 1.35 200.00

Linoleic 18:2 1.93 0.39 -79.79 0.83 0.17 -79.52

Linolenic 18:3 0.25 0.26 4.00 0.11 0.11 0.00

Total Fat Intake 4.27 4.27 0.00 1.84 1.84 0.00

Table M-3. 90th Percentile Per User Fatty Acid Intake from Target foods, Pre and Post 

MON 87705 Replacement, U.S.Population 

Fatty Acids g/day % En

Pre 

replacement

Post 

replacement

% 

change

Pre 

replacement

Post 

replacement

% 

change

Palmitic 16:0 1.71 0.48 -71.93 0.72 0.21 -70.83

Stearic 18:0 0.66 0.7 6.06 0.27 0.31 14.81

Oleic 18:1 3.86 12.22 216.58 1.64 5.12 212.20

Linoleic 18:2 7.9 1.58 -80.00 3.26 0.66 -79.75

Linolenic 18:3 1.03 1.05 1.84 0.42 0.44 4.76

Total Fat Intake 16.28 16.28 0.000 6.83 6.83 0.00
Monsanto Company 09-SY-201U Page 393 of 471

M.4 Impact of Replacement on Total Diet 

The impact of oil replacement under the proposed food uses with MON 87705 soybean oil on 

the total daily intake (g/day and % en) of total fat and the five major fatty acids (palmitic, 

stearic, oleic, linoleic, linolenic) in the diet of the U.S. population are summarized in Tables 

M-4 and M-5. As expected, the replacement with MON 87705 soybean oil does not change 

the total daily fat intake, which remains at 79.58 g/day on the mean per capita basis and at 

129.4 g/day in the 90th percentile per user group. 

Table M-4. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, U.S.Population 

(g/day) 

Fatty Acids Mean Per Capita 90th Percentile Per User

Pre 

MON 87705 

(g/day) 

Post 

MON 87705 

(g/day) 

% 

change 

Pre 

MON 87705 

(g/day) 

Post 

MON 87705 

(g/day) 

% 

change 

 

Palmitic 16:0 14.38 13.83 -3.80 23.62 21.48 -9.00

Stearic 18:0 6.96 6.98 0.30 11.75 11.84 0.80

Oleic 18:1 27.34 30.29 10.80 45.20 57.52 27.20

Linoleic 18:2 14.75 14.12 -4.30 25.66 23.08 -10.10

Linolenic 18:3 1.42 1.42 0.00 2.49 2.50 0.20

Total Fat 

Intake

79.58 79.58 0.00 129.40 129.40 0.00

Table M-5. Fatty Acid Intake from Total Diet, Pre and Post MON 87705, U.S.Population 

(% en) 

Fatty Acids Mean Per Capita 90th Percentile Per User

Pre 

MON 87705 

(% en) 

Post 

MON 87705 

(% en) 

% 

change 

Pre 

MON 87705 

(% en) 

Post 

MON 87705 

(% en) 

% 

change 

 

Palmitic 16:0 6.07 5.84 -3.70 8.00 7.10 -11.30

Stearic 18:0 2.91 2.91 0.00 3.94 4.03 2.40

Oleic 18:1 11.49 12.75 10.90 15.16 20.28 33.80

Linoleic 18:2 6.23 5.96 -4.40 9.22 8.05 -12.70

Linolenic 18:3 0.61 0.61 0.00 0.95 0.96 0.80

Total Fat 

Intake

33.61 33.61 0.00 42.88 42.88 0.00

 
Monsanto Company 09-SY-201U Page 394 of 471

Table M-6. Mean Per Capita and 90th Percentile Per User Fatty Acid Intake from Total 

Diet, Post MON 87705, Age and Gender Groups (g/day) 

Population Total 

Fat 

Palmitic 

16:0 

Stearic 

18:0 

Oleic 

18:1 

Linoleic 

18:2 

Linolenic 

18:3 

U.S. Mean 79.58 13.83 6.98 30.29 14.12 1.42 

90th percentile 129.40 21.48 11.84 57.52 23.08 2.50 

Males 1-8 Mean 64.76 12.09 5.95 23.07 10.48 1.00 

90th percentile 100.53 17.73 9.40 38.23 16.54 1.63 

Males 9-19 Mean 93.45 16.95 8.54 34.47 15.87 1.46 

90th percentile 142.14 24.38 13.45 61.62 24.93 2.48 

Males 20-49 Mean 102.91 18.08 9.10 39.27 18.02 1.77 

90th percentile 155.60 26.78 14.44 68.31 27.48 2.97 

Males 50+ Mean 87.93 14.82 7.56 34.33 15.92 1.64 

90th percentile 139.74 22.57 12.27 62.39 26.17 2.83 

Females 1-8 Mean 60.85 11.36 5.62 21.84 9.80 0.97 

90th percentile 91.46 16.47 8.71 36.82 15.75 1.55 

Females 9-19 Mean 72.86 12.96 6.57 27.22 12.92 1.24 

90th percentile 111.56 18.71 10.24 48.62 20.25 2.08 

Females 20-49 Mean 73.65 12.57 6.39 28.27 13.40 1.39 

90th percentile 114.17 18.48 10.26 52.51 20.70 2.35 

Females 50+ Mean 63.30 10.43 5.38 24.76 11.80 1.31 

90th percentile 100.54 15.75 8.75 45.77 19.21 2.29 

Following replacement with MON 87705 soybean oil, there is an overall increase in the total 

daily intake of 18:1 oleic acid from 27.34 g/day (11.49 % en) to 30.29 g/day (12.75 % en) on a 

mean per capita basis and an increase from 45.2 g/day (15.16 % en) to 57.52 g/day (20.28 

% en) at the 90th percentile per user (Tables M-4 and M-5). These increases post-replacement 

are expected, since the 18:1 oleic acid fraction in conventional soybean oil is much lower 

compared to the 76.5% oleic acid observed in MON 87705. The pattern of total daily intake of 

fatty acids in sub groups stratified by age and gender are similar to that of the overall U.S. 

population, with 18:1 oleic acid having the largest increase following replacement of 

conventional soybean oil with MON 87705 soybean oil (Table M-6). The Dietary Guidelines 

for Americans Technical Report (USHHS, 2005) stated that MUFAs are not required in the 

diet; however, they provide a vehicle to achieving total fat intake recommendations within the 

context of saturated fat and PUFA recommendations. Further, substitution of oleic acid for 

saturated fat in the diet can reduce low density lipoprotein cholesterol (LDL-C). 

Total daily intakes of 16:0 palmitic acid decreased as a consequence of dietary replacement 

with MON 87705 soybean oil (Tables M-4 and M-5). On a mean per capita basis, 16:0 

palmitic acid intake decreased from 14.38 g/day (6.07 % en) to 13.83 g/day (5.84 % en), while 

the decrease was predictably greater in the per user 90th percentile group, where daily intake 

decreased from 23.62 g/day (8.00 % en) to 21.48 g/day (7.10 % en) (Table M-6). Total dietary 

intakes of 18:0 stearic acid were similar in pre- and post- MON 87705 replacement analysis, in 

both mean per capita (6.96 g/day and 6.98 g/day, respectively) and 90th percentile per user 

groups (11.75 g/day and 11.84 g/day), respectively. A decrease in dietary saturated fat intake, 
Monsanto Company 09-SY-201U Page 395 of 471

as shown here for 16:0 palmitic acid, is consistent with current dietary recommendations 

(Lichtenstein et al., 2006; USDA-ERS, 2005; WHO/FAO, 2003). 

Linoleic acid (18:2) intake levels decreased by a similar rate, from 14.75 g/day (6.23 % en) to 

14.12 g/day (5.96 % en) on a mean per capita basis; and from 25.66 g/day (9.22 % en) to 23.08 

g/day (8.05 % en) in the 90th percentile per user group. An adequate intake (AI) for 18:2 

linoleic acid of 17 g/day for men (19-50 years) and 12 g/day for women (19-50 years) has been 

established by the Institute of Medicine (IOM, 2002). The values observed post-replacement 

for the respective subgroups are consistent with these recommendations. For example, post 

replacement values are 18.02 g/day (males 20-49) and 13.40 g/day (females 20-49) (Table 

VII-8). Further, the population and adult subgroup 18:2 linoleic intakes post replacement are 

still in line with U.S. Dietary Guidelines (USHHS, 2005) and the Acceptable Macronutrient 

Distribution Range of 5-10% energy (IOM, 2002), which is designed to reduce risk for chronic 

disease. For example, the population mean intake of linoleic acid was 6.23% energy before 

replacement and 5.96% energy post-replacement (Table M-5). 

There was no change in 18:3 linolenic acid after replacement with MON 87705 oil (Table M-4, 

M-5). An adequate intake (AI) for 18:3 linolenic acid of 1.6 g/day for men (19-50 years) and 

1.1 g/day for women (19-50 years) has been established by the Institute of Medicine (IOM, 

2002). The values observed both pre and post replacement for the respective subgroups are 

consistent with these recommendations. For example, post replacement values are 1.77 g/day 

(males 20-49) and 1.39 g/day (females 20-49) (Table M-6). 

In summary, replacement of conventional soybean oil with MON 87705 soybean oil under the 

proposed food uses results in changes in the fatty acid composition in the U.S. diet that lead to 

higher oleic acid intake, and lower consumption of saturated fats (i.e., 16:0 palmitic) and 

linoleic acid, with no impact on total fat intake. Given that this assessment assumes that all of 

the targeted oil components of the foods proposed for replacement at all eating occasions in the 

U.S. are replaced with MON 87705 soybean oil, the results presented here represent a 

theoretical maximal effect of MON 87705 on fatty acid composition of the diet. The 

nutritional impact of exposure to MON 87705 soybean oil in targeted foods under the intended 

conditions of use is estimated to result in changes in fatty acid consumption that are in line 

with current dietary guidelines for fatty acid intake (Lichtenstein et al., 2006; USDA-ERS, 

2005; WHO/FAO, 2003). 

M.5 Composition and Nutrition Assessment Conclusion 

In conclusion, the nutritional impact from the use of MON 87705 soybean oil in targeted foods 

under the intended conditions of use, is estimated to result in changes in fatty acid consumption 

that are in line with current dietary guidelines for fatty acid intake. Therefore, MON 87705 is 

regarded to be as safe and nutritious as conventional soybean for food use. 

 
Monsanto Company 09-SY-201U Page 396 of 471

Appendix M References 

CDC. 2007. National health and nutrition survey examination survey data. US department of 

health and human services (DHHS), Centers for Disease Control and Prevention (CDC), 

National Center for Health Statistics (NCHS). Data sets available from: 

http://www.cdc.gov/nchs/about/major/nhanes/datalink.htm 

Exler, J., L. Lemar, and J. Smith. Fat and Fatty Acid Content of Selected Foods Containing 

Trans-Fatty Acids. U.S. Department of Agriculture, ARS. Washington, D.C. 

http://www.nal.usda.gov/fnic/foodcomp/Data/Other/trans_fa.pdf [Accessed July 9, 2009]. 

Lichtenstein, A.H., L.J. Appel, M. Brand, M. Carnethon, S. Daniels, H.A. Franch, B. Franklin, 

P. Kris-Etherton, W.S. Harris, B. Howard, N. Karanja, M. Lefevre, L. Rudel, F. Sacks, L. Van 

Horn, M. Winston, and J. Wylie-Rosett. 2006. Diet and lifestyle recommendations revision 

2006. Circulation 114:82-96. 

HyQuest Partners, Soybean Oil Utilization Report, 2009 

IOM. 2002. Dietary fats: Total fats and fatty acids. Pages 8-57 and 422-541 in Dietary 

Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and 

Amino Acids. Panel on Macronutrients, . Institute of Medicine. National Academies Press, 

Washington D.C. 

ISEO. 2006. Foods and fats. Pages 1-37 in Food Fats and Oils. Institute of Shortening and 

Edible Oils, Washington, D.C. 

USDA-ERS. 2005. Soybeans and oil crops: Background. U.S. Department of Agriculture, 

Washington, D.C. 

U.S. Department of Health and Human Services and U.S. Department of Agriculture 

(USDHHS-USDA). Dietary Guidelines for Americans, 2005. 6th Edition, Washington, DC: 

U.S. Government Printing Office, January 2005. 

USHHS. 2005. Dietary Guidelines for Americans. HHS Publication No: HHS-ODPHP-2005-

01-DGA-A. U.S. Department of Health and Human Services, Washington, D.C. 

www.health.gov/dietaryguidelines [Accessed June 7, 2009]. 

WHO/FAO. 2003. Diet, nutrition and the prevention of chronic diseases. 

http://whqlibdoc.who.int/trs/WHO_TRS_916.pdf WHO technical report: 916. 
Monsanto Company 09-SY-201U Page 397 of 471

Appendix N. Glyphosate-Tolerant Crops: Potential Impacts on Land Use and 

Agricultural Practices 

N.1 Introduction 

Since 1994, USDA has granted nonregulated status to 13 glyphosate-tolerant biotechnologyderived products (Table N-1). Petitions for three additional glyphosate-tolerant products have 

been submitted to the agency, with deregulation decisions pending for alfalfa, corn and 

creeping bentgrass. The crops for which petitions have been approved or are pending include: 

corn, cotton, creeping bentgrass, rapeseed (canola), soybean, and sugar beet. This document 

discusses the cumulative impacts on land use and agricultural practices from deregulation of 

glyphosate-tolerant crop products, considering the existing glyphosate-tolerant crops and those 

potentially available in the reasonably foreseeable future based on pending petitions. 

N.2 Impact on Land Use 

USDA has granted nonregulated status to events conveying the glyphosate-tolerance trait in 

five different crops (Table N-1). These crops have been rapidly adopted by U.S. farmers and 

since their introduction have been grown cumulatively on more than 778 million acres. In 

2007 alone, glyphosate-tolerant crops were planted on approximately 120 million acres in the 

U.S.11 The sections below assess the impact of the adoption of glyphosate-tolerant crops on 

cropping patterns. 

Table N-1. Herbicide-Tolerant Crops Undergoing Review or Previously Deregulated by 

USDA-APHIS. 

Petition Crop Species Trait(s) Status 

93-258-01p Soybean Glyphosate-tolerant Deregulated 

95-045-01p Cotton Glyphosate-tolerant Deregulated 

96-317-01p Corn Glyphosate-tolerant and 

Corn Borer-resistant Deregulated 

97-099-01p Corn Glyphosate-tolerant Deregulated 

98-173-01p Sugar Beet Glyphosate-tolerant Deregulated 

98-216-01p Canola Glyphosate-tolerant Deregulated 

00-011-01p Corn Glyphosate-tolerant Deregulated 

01-324-01p Canola Glyphosate-tolerant Deregulated 

03-104-01p Creeping Bentgrass Glyphosate-tolerant Pending 

03-323-01p Sugar Beet Glyphosate-tolerant Deregulated 

04-086-01p Cotton Glyphosate-tolerant Deregulated 

04-110-01 Alfalfa Glyphosate-tolerant Pending 

06-178-01p Soybean Glyphosate-tolerant Deregulated 

06-271-01p Soybean Glyphosate- and Acetolactate 

Synthase Inhibitor-tolerant Deregulated 

06-332-01p Cotton Glyphosate-tolerant Deregulated 

07-152-01p Corn Glyphosate- and Imidazolinone-tolerant Pending 

 

11 Calculated from datasets found at www.ers.usda.gov/data/biotechcrops. 
Monsanto Company 09-SY-201U Page 398 of 471

N.3 Total Cropland Acres 

The overall U.S. area planted to principal crops, which include corn, sorghum, oats, barley, 

winter wheat, rye, durum, wheat, rice, soybean, peanuts, sunflower, cotton, dry edible beans, 

potatoes, canola, proso millet, and sugar beets, has remained relatively constant over the past 

25 years. From 1983 to 1995, the average yearly acreage of principal crops was 328 million. 

This average is statistically unchanged at 326 million acres since the introduction of 

glyphosate-tolerant crops in 199612. If fact, the total U.S. cropland acres have been relatively 

constant since the 1940s (Vesterby and Krupa, 1997). Therefore, the introduction of 

glyphosate-tolerant crops has not resulted in a significant change to the total cropland acres in 

the U.S. 

Following deregulation and commercial introduction, the acres of glyphosate-tolerant 

varieties/hybrids planted for each crop has steadily increased (see Figures N-1 through N-3 

below). However, rapid adoption of glyphosate-tolerant crops has not correlated with an 

increase in the total acres for that particular crop. Specific crop acres do vary from year-toyear, but these fluctuations occur routinely and are due to a myriad of factors based largely 

around grower economic returns for each crop, crop rotation practices, and government 

programs. The impact of glyphosate-tolerant crop deregulation or pending deregulation and 

adoption on crop acres is discussed below. 

N.3.1 Canola 

Glyphosate-tolerant canola was commercialized in the U.S. in 1999. In 2007, U.S. growers 

planted 1.2 million acres of canola (USDA-NASS, 2007) and of those acres, 0.61 million acres, 

or approximately 50%, contained the glyphosate-tolerance trait (Monsanto, 2007). 

The total canola acreage since 1990 and the acreage of glyphosate-tolerant canola since 

commercialization in 1999 are provided in Figure N-1. The total U.S. acreage of canola 

increased 200% from 367,000 acres in 1996 to 1.1 million acres in 1998, coinciding with 

passage of the 1996 Federal Agriculture Improvement Act. 13 This acreage peaked at 1.55 

million in 2000 and has remained near 1.l million acres since 2005. 

The acreage of glyphosate-tolerant canola increased rapidly following commercial introduction 

in 1999 and has remained between 50 and 70% of planted canola acres since 2001. The 

dramatic fluctuation in total canola acreage before and after glyphosate-tolerant canola was 

commercialized indicates that factors unrelated to the availability of the glyphosate-tolerant 

trait play a larger role in acres planted than the availability of the glyphosate-tolerant trait. 

 

12 Calculated from datasets at http://usda.mannlib.cornell.edu/usda/current/htrcp/ by comparing the total acres 

from 1983 to 1995 and from 1996 to 2007. 

13 The 1996 Federal Agriculture Improvement Act, 7 U.S.C. § 7201 et seq., gave growers almost complete 

flexibility in selecting the crops they could plant. 
Monsanto Company 09-SY-201U Page 399 of 471

Figure N-1. Total Acres of Canola Relative to Acres of Glyphosate-tolerant Varieties 

planted. Data obtained from http://usda.mannlib.cornell.edu/usda/current/htrcp/ for total 

canola acres and www.monsanto.com/pdf/investors/2007/FY2007BiotechAcres.pdf for 

glyphosate-tolerant canola acres. 

N.3.2 Corn 

Glyphosate-tolerant corn was commercialized in the U.S. in 1996. From 1996 to 2007, 

glyphosate-tolerant corn hybrids were grown on 151 million cumulative acres. In 2007, U.S. 

growers planted 93.6 million acres of field corn (USDA-NASS, 2007); 48 million acres, or 

52%, contained a glyphosate-tolerance trait (USDA- ERS, 2007). 

Since its introduction, the percentage of glyphosate-tolerant corn has increased steadily to 52% 

in 2007. The total corn acreage, however, remained relatively steady from 1996 to 2006, with 

a yearly average of 78 million acres (see Figure N-2), indicating that the glyphosate-tolerant 

trait has had little impact on overall corn acreage. In 2007, the total corn acreage was up 15% 

from 2006 to 92.9 million acres. This increase was attributed to increased demand from 

ethanol producers and strong exports sales14. The increase in total corn acres, including an 

increase in glyphosate-tolerant corn acres, resulted in fewer acres of soybean planted in the 

Corn Belt and Great Plains and fewer acres of cotton and rice planted in the Delta and 

Southeast regions. 

 

 

14 http://www.usda.gov/nass/PUBS/TODAYRPT/acrg0607.txt 

Total and Glyphosate-Tolerant Canola Acres in the US

0

200

400

600

800

1000

1200

1400

1600

1800

1990

1992

1994

1996

1998

2000

2002

2004

2006

Year

1000 Acres

Canola

RR Canola
Monsanto Company 09-SY-201U Page 400 of 471

Figure N-2. Total Acres of Corn and Soybean Relative to Acres of Herbicide-tolerant 

(HT) Varieties Planted. The varieties are predominately glyphosate-tolerant varieties, but 

also include phosphinothricin-tolerant hybrids for corn. Data obtained from 

http://usda.mannlib.cornell.edu/usda/current/htrcp/ for total crop acres and USDA-ERS (2007) 

for herbicide-tolerant crops acres. 

N.3.3 Soybean 

Glyphosate-tolerant soybean was commercialized in the U.S. in 1996. From 1996 to 2007, 

glyphosate-tolerant soybean was grown on more than 509 million cumulative acres. In 2007, 

growers planted 64 million acres of soybean in the U.S. (USDA-NASS, 2007); 58 million 

acres, or 91%, contained the glyphosate-tolerance trait (USDA-ERS, 2007). 

Since its introduction, the percentage of glyphosate-tolerant soybean has increased steadily to 

91% in 2007 (Figure 2). The total acres of soybean increased 23% (62.4 to 76.9 million acres) 

from 1996 to 2006. This increase was due to higher global demand, but was facilitated by 

increased planting flexibility from: (1) the 2002 Farm Bill; (2) steadily rising yield 

improvements from narrow-rowed seeding practices; (3) a greater number of 50-50 cornsoybean rotations; and (4) lower production costs, partly due to widespread adoption of 

herbicide-tolerant varieties (Ash et al., 2006). In 2007, acres of soybean were down 17% to 

64.1 million compared to 2006 due to growers planting more corn, as discussed above. 

 

Total and Herbicide-Tolerant Corn and Soybean

Acres in the U.S.

0

20,000

40,000

60,000

80,000

100,000

1990

19921994

199619982000

20022004

2006

Year

1000 Acres

Corn

Soybean

HT Corn

HT Soybean

Corn

Soybean HT Soybean

HT Corn

Total and Herbicide-Tolerant Corn and Soybean

Acres in the U.S.

0

20,000

40,000

60,000

80,000

100,000

1990

19921994

199619982000

20022004

2006

Year

1000 Acres

Corn

Soybean

HT Corn

HT Soybean

Corn

Soybean HT Soybean

HT Corn
Monsanto Company 09-SY-201U Page 401 of 471

Figure N-3. Total Acres of Cotton Relative to Acres of Herbicide-tolerant (HT) Varieties 

Planted. The varieties are predominately glyphosate-tolerant varieties, but also include 

phosphinothricin-tolerant varieties. Data obtained from 

http://usda.mannlib.cornell.edu/usda/current/htrcp/ for total crop acres and USDA-ERS (2007) 

for herbicide-tolerant crops acres. 

N.3.4 Cotton 

Glyphosate-tolerant cotton was commercialized in the U.S. in 1997. From 1997 to 2007, 

glyphosate-tolerant cotton was grown on more than 76 million cumulative acres. In 2007, 

growers planted 11 million acres of cotton in the U.S. (USDA-NASS, 2007) of which 7.7 

million acres, or 70%, contained a glyphosate-tolerance trait (USDA-ERS, 2007). 

Since its introduction, the percentage of glyphosate-tolerant cotton increased to approximately 

60% in 2001 and remained near that level until reaching 70% in 2007 (Figure N-3). The total 

cotton acreage, however, remained relatively constant from 1997 to 2006 at 14.1 million +/- 

1.3 million acres (Figure N-3). In 2007, the total cotton acres were down 28% due to a shift to 

corn and soybean across the Southeast U.S.15

N.3.5 Sugar beet 

Since 1990, the total U.S. sugar beet acreage has remained relatively constant at 1.4 million 

acres (USDA-NASS, 2007). In 2007, approximately 1.3 million acres of sugar beet were 

planted in the 11 largest sugar beet production states in the U.S. Glyphosate-tolerant sugar 

beet has only been grown on limited acres since its deregulation in 2005, but is expected to be 

grown on substantially more acres in 2008. Regardless, the acres forecasted by USDA for 

sugar beet in 2008 are 1.08 million acres16, down slightly from 1.3 million acres in 2007, again 

due to the market factors described above. 

 

15 http://www.usda.gov/nass/PUBS/TODAYRPT/acrg0607.txt 16 http://usda.mannlib.cornell.edu/usda/current/Acre/ 

Total Cotton and Herbicide-Tolerant Cotton Acres in the U.S. 

0

5000

10000

15000

20000

1990

1992

1994

1996

1998

2000

2002

2004

2006

Year

1000 Acres

Cotton

HT Cotton
Monsanto Company 09-SY-201U Page 402 of 471

N.3.6 Bentgrass 

Creeping bentgrass is widely grown on about 77,000 acres on golf course putting greens, tees, 

and fairways in the cooler climates throughout the U.S., and exclusively for greens in the south 

where specialized root zone management systems allow survival of creeping bentgrass under 

summer heat conditions. After deregulation, glyphosate-tolerant creeping bentgrass is 

expected to replace conventional creeping bentgrass on some golf courses. The glyphosatetolerance trait does not alter the overall biology and ecology of creeping bentgrass17 therefore, 

glyphosate-tolerant creeping bentgrass is not expected to impact land use any differently than 

conventional creeping bentgrass. 

N.3.7 Alfalfa 

Alfalfa grown for forage is a major crop in the U.S. More than 20 million acres have been 

planted annually since 1950. Acreage peaked in the mid-1960s at approximately 30 million 

acres and declined gradually to approximately 21 million acres in 2007 (USDA-NASS 2007), 

in part due to increased use of corn silage as a forage. 

Data from the previously deregulated glyphosate-tolerant crops shows that market forces and 

governments programs (1996 and 2002 Farm Bills), not the presence of glyphosate-tolerant 

crop varieties, are primarily responsible for any significant changes in crop acreage. Based on 

the adoption rates of other glyphosate-tolerant crops, it is expected that the deregulation and 

commercial availability of glyphosate-tolerant alfalfa would replace a significant portion of 

conventional alfalfa varieties. The availability of these glyphosate-tolerant varieties provides 

for more efficient weed control and thus allows growers more flexibility when choosing what 

crop varieties to plant on their farms. This may result in a shift back from corn silage to alfalfa 

hay for animal feed; thus increasing the alfalfa acreage to the level seen in the 1980s and 

1990s. However, based on the experience with the other glyphosate-tolerant crops, 

deregulation of glyphosate-tolerant alfalfa is expected to have little impact on the overall 

acreage historically seen for alfalfa or other crops. 

N.3.8 Conventional and Organic Production 

Since the introduction of glyphosate-tolerant crops, the total acreage of principal crops and 

acreage of each specific crop have remained relatively constant, as discussed above. 

Therefore, the increase in acreage of glyphosate-tolerant crops comes at the expense of crop 

varieties used for conventional production, although conventional varieties are still widely 

available to those who choose to plant them (see Section XI.D.3). USDA data show that the 

acreage of organic corn and soybean has actually increased since the introduction of 

glyphosate-tolerant crops in 1996 (Figure N-4). The acreage of organic cotton has decreased 

from a high of 32,850 acres in 1995 to 9,537 acres in 2005, which is comparable to the acreage 

planted in 1993 and 1994 before the introduction of glyphosate-tolerant cotton. The decrease 

in acreage is attributed to increased market competition from imported organic cotton (USDAERS, 2006). Therefore, the use of glyphosate-tolerant varieties has replaced conventional 

 

17 http://www.aphis.usda.gov/brs/aphisdocs/03_10401p.pdf 
Monsanto Company 09-SY-201U Page 403 of 471

varieties, resulting in a change in agricultural practices related to weed control and soil tillage, 

as discussed below. Based on adoption rates of other glyphosate-tolerant crops, it is expected 

that deregulation of glyphosate-tolerant alfalfa will result in a replacement of a significant 

percentage of conventional alfalfa with glyphosate-tolerant alfalfa. Less than one percent of 

alfalfa hay acres were harvested in 2005 as organic (USDA-NASS, 2006). Depending on 

market demand, and again based on the experiences of the corn and soybean markets, a similar 

increase in organic alfalfa acres is possible. 

 

Figure N-4. Percentage of Certified Organic Acres for Corn, Cotton, and Soybean. Data 

obtained from www.ers.usda.gov/Data/organic and www.nass.usda.gov for total acres. 

N.4 Impact on Herbicide Use and Costs 

N.4.1 Canola 

For a description of weed control practices in conventional canola, please see the 

recommendations from the North Dakota State University Extension Service18. 

For glyphosate-tolerant canola, it is recommended that glyphosate be applied at a maximum 

rate of 0.38 lb acid equivalent (ae)/acre (A) with no more than two applications to glyphosatetolerant canola from emergence to bolting (Zollinger, 2008). On average, a typical weed 

management program in conventional canola (that could provide control comparable to the 

program in herbicide-tolerant canola) cost about $39/A in 2005. In contrast, weedmanagement costs in glyphosate-tolerant canola, inclusive of technology fee/seed premium, 

were about $24/A, for a total cost reduction of 62%. The net impact on gross margins has been 

an increase of between $47/ha and $55/ha for glyphosate-tolerant canola (Brooks and Barfoot, 

2006). 

In 2005 in North Dakota, which contained 92% of the total U.S. canola acreage, glyphosatetolerant canola was planted on 676,000 acres. Use of the glyphosate-tolerant varieties resulted 

 

18http://www.ndsu.edu/weeds/weed_control_guides/2009_weed_control_guide/ 

Percentage of Certified Organic Acres

0

0.05

0.1

0.15

0.2

0.25

1995 1997 2000 2001 2002 2003 2004 2005

Year

Percent of Crop Acres

 Corn

 Soybean

 Cotton
Monsanto Company 09-SY-201U Page 404 of 471

in reduction in total weed management costs by $10 million and a reduction in herbicide use by 

426,000 total pounds (Sankula, 2006). 

Therefore, glyphosate-tolerant canola provides significant cost savings to the growers and 

reduces the amount of herbicide used. 

N.4.2 Corn 

A description of weed control practices in conventional corn can be found in USDA petition 

04-125-01p, p. 12819. 

A mixture of the herbicides registered for use in corn provides fair to excellent control of most 

weeds in a conventional weed control system. However, growers must know the specific weed 

problems present on their farms or fields to select the most effective herbicide program. 

Typical weed management in conventional corn includes a preemergence application of 

metalachlor+atrazine followed by a postemergence application of mesotrione plus a premix of 

nicosulfuron and rimsulfuron (Sankula, 2006). 

Two major options are used for weed management in glyphosate-tolerant corn (Sankula, 2006). 

The first and most widely used option is the use of half-rate of metolachlor + atrazine as a 

preemergence herbicide, followed by glyphosate as a postemergence application. The second 

approach involves a total postemergence-based program with either one or two applications of 

glyphosate or tank-mix applications of glyphosate with atrazine. 

Sankula (2006) compared weed management programs and costs associated with glyphosatetolerant and conventional corn. Weed management costs in 2005 were 25% lower in 

glyphosate-tolerant corn compared to conventional corn. In 2005 alone, glyphosate-tolerant 

corn reduced the herbicide use in corn by 18.3 million pounds of active ingredient and reduced 

weed management costs by $238.2 million (Sankula, 2006). 

Brookes and Barfoot (2006) compared a typical herbicide treatment regime for glyphosatetolerant corn and conventional corn. They calculated the herbicide use rate and the 

Environmental Impact Quotient (EIQ), a measure of impact to the environment. The EIQ 

integrates the various environmental impacts of a single pesticide into a single field value per 

hectare. The EIQ value is then multiplied by the amount of pesticide used per hectare to get 

the overall EIQ rating. A lower EIQ rating indicates a lower impact on the environment. 

The conventional herbicide program used a mix of herbicides, such as acetochlor, atrazine, 

primsulfuron, and dicamba. The average herbicide use rate by growers of conventional corn 

was 3.74 kg active ingredient (ai)/hectare (ha), with a field EIQ rating of 76.6/ha. The 

glyphosate-tolerant corn program used glyphosate plus lower rates of acetochlor and atrazine 

than those used on conventional crops. The values for the glyphosate-tolerant corn program 

included an average herbicide use rate of 2.59 kg ai/ha, with a field EIQ rating of 48.4/ha. 

Based on these analyses, the glyphosate-tolerant corn program has a significantly better 

environmental profile than the conventional herbicide program. 

 

19http://www.aphis.usda.gov/brs/aphisdocs/04_12501p.pdf

 
Monsanto Company 09-SY-201U Page 405 of 471

N.4.3 Cotton 

For a description of weed control practices in conventional cotton, please see USDA petition 

04-086-01p20. 

For glyphosate-tolerant cotton, typical weed management programs consist of a preemergence 

application followed by postemergence applications of glyphosate or a glyphosate only system 

for pre- and postemergence applications. The overall impact of herbicide-tolerant cotton on 

U.S. agriculture in 2005 alone was a reduction in crop production costs of $39 million and 

pesticide use of 18 million pounds of active ingredient (Sankula, 2006). 

According to Brooks and Barfoot (2006), the use of herbicide-tolerant cotton21 (of which 98% 

was tolerant to glyphosate) led to reduced production costs and increased profitability from 

1997 to 2005 of between $21/ha and $49/ha annually. The net gain to farm income in 2005 

was $161 million. Since glyphosate-tolerant cotton was first commercially produced in 1997, 

the farm income benefit has been $919 million. In terms of added value, the effect on farm 

income in 2005 was equivalent to an increase in production of 3% (151,000 tons). 

N.4.4 Soybean 

A description of herbicide use in conventional soybean is available in USDA petition 

06-178-01P22. 

Conventional herbicide programs typically require a preemergence application with one to two 

herbicides, followed by a postemergence application with one to two herbicides. Conversely, 

glyphosate-tolerant soybean typically require only one timely application of glyphosate at 0.95 

lb ai/A. Consequently, in 2005, growers planting glyphosate-tolerant soybean varieties 

reduced the overall number of herbicide applications by 39.4 million, which translated to cost 

savings of $134 million (Sankula, 2006). On average, glyphosate-tolerant soybean programs 

used 1.03 lb ai/A at a cost of $21.28/A in 2005, compared to growers of conventional soybean 

varieties who used an additional 0.32 lb ai/A or 32% more herbicide active ingredients at an 

additional cost of $18.09/A. When considering herbicide costs and application costs in 2005 

alone, U.S. soybean growers saved $1.17 billion on weed management costs using glyphosate 

based weed-control programs (Sankula, 2006). 

This reduction of herbicide use and the physical properties of glyphosate have secondary 

environmental benefits also. Shipitalo, et al. (2008) investigated the transport loss of 

herbicides from agriculture fields due to runoff. They found that the use of glyphosate-tolerant 

soybean usually, but not always, resulted in substantially less runoff than the residual 

herbicides that glyphosate herbicides replaced. In contrast to the residual herbicides, the 

glyphosate concentrations from runoff in the watershed remained below the allowable limit for 

drinking water. The authors concluded that the impact of herbicide losses in runoff resulting 

from production of growing transgenic, glyphosate-tolerant soybean should be reduced 

(Shipitalo, 2008). 

 

20 http://www.aphis.usda.gov/brs/aphisdocs/04_08601p.pdf 21 2% of the herbicide-tolerant cotton was glufosinate-tolerant cotton. 22 http://www.aphis.usda.gov/brs/aphisdocs/06_17801p.pdf
Monsanto Company 09-SY-201U Page 406 of 471

N.4.5 Sugar beet 

For a description of the weed control practices in conventional sugar beet, please see USDA 

petition 03-323-01p23. 

As of 2007, glyphosate-tolerant sugar beet did not have a history of widespread production, 

although a significant portion of the sugar beet acres is expected to be planted with glyphosatetolerant varieties in 2009. USDA-NASS data are not yet available on adoption rates, impacts 

on herbicide use rates, and other relevant parameters, a predictive ex ante assessment of 

potential impact is included in Appendix A. Coyette et al. (2002) predicted that introduction of 

glyphosate-tolerance technology in sugar beet cultivation in Europe would lead to a reduction 

in herbicide use by as much as 43%. Based on this study and data from other glyphosatetolerant crops, beneficial results in cost savings, pesticide reduction, and weed control are 

expected. 

N.4.6 Bentgrass 

Glyphosate-tolerant creeping bentgrass is currently under review for deregulation and APHIS 

is conducting an environmental impact statement (EIS) to support its decision-making under 

NEPA. 

The stated rationale for developing glyphosate-tolerant creeping bentgrass event ASR368 was 

to benefit current agronomic practices in creeping bentgrass seed production and its use as a 

principal turf on golf course tees, greens, and fairways. Control and management of annual 

and perennial grass, broadleaf, and sedge weed species that invade golf courses has met with 

variable success using a variety of herbicides and plant growth regulators. A discussion of the 

pesticides used on conventional bentgrass is available in USDA petition 03-104-01p, p. 29024. 

The most troublesome of these weeds includes annual bluegrass, rough stalk bluegrass, and 

bermuda grass. Considerable investments in labor, chemicals, water, and time are made to 

manage creeping bentgrass in golf course fairways and putting greens invaded by annual 

bluegrass because of bentgrass’ susceptibility to disease, heat, and drought stress. Grassy 

weeds occurring in creeping bentgrass seed production areas can reduce the purity of harvested 

seed, and can limit the land on which such seed production can profitably occur. Golf courses 

and seed producers who adopt glyphosate-tolerant creeping bentgrass instead of conventional 

creeping bentgrass varieties could use the Roundup family of industrial turf and ornamental 

herbicides as needed (pending U.S. Environmental Protection Agency label change approvals) 

as an effective, new, over-the-top method to control the majority of annual and perennial 

grasses and broadleaf weeds. 

N.4.7 Alfalfa 

For a description of the weed control practices in conventional alfalfa, please see USDA 

petition 04-110-01p25. 

 

23 http://www.aphis.usda.gov/brs/aphisdocs/03_32301p.pdf 24 http://www.aphis.usda.gov/brs/aphisdocs/03_10401p.pdf 25 http://www.aphis.usda.gov/brs/aphisdocs/04_11001p.pdf 
Monsanto Company 09-SY-201U Page 407 of 471

Deregulation of glyphosate-tolerant alfalfa likely would result in significant economic and 

environmental benefits from annual or semi-annual Roundup herbicide applications to 

glyphosate-tolerant alfalfa26. Forage quality is an important factor in the price paid for hay and 

the presence of weeds in the hay will reduce the quality considerably. For example, it is 

estimated that two applications of glyphosate at 0.75 lb a.i./acre would provide effective 

season-long control of troublesome weeds in California alfalfa fields. It is estimated that the 

Roundup herbicide cost would be $15/A, and a seed premium of $5/A would be charged. As 

mentioned previously, glyphosate is well recognized for the broad spectrum of weeds it 

controls. In fact, if a grower were to use a season-long herbicide control program that provides 

the same spectrum and performance as glyphosate, it would require the use of trifluralin, 

EPTC, and imazethapyr. The level of performance provided by these three herbicides would 

be full control of 38 species, partial control of eight species, and no control of two species 

found in California alfalfa seed production fields. This weed control program would require 

4.7 lb. a.i./acre/yr. at a cost of $45/acre. Use of the glyphosate-tolerant alfalfa weed control 

program would provide full control of 42 species, partial control of 4 species, no control of one 

species with undetermined control of the remaining one species (University of California, 

2001). Compared to the three herbicide program described above, the glyphosate weed control 

program would not only provide broader weed control but it would reduce herbicide inputs by 

3.2 lb. a.i./acre and reduce costs by $25/acre. 

The use of glyphosate-tolerant crops has resulted in a drastic reduction in the amount of total 

herbicide applied and a change in the herbicide active ingredient applied to U.S. cropland 

(Fernandez-Cornejo and Caswell, 2006). Deregulation of glyphosate-tolerant alfalfa and 

concomitant in-crop use of glyphosate is expected to reduce the total amount of herbicide use 

in alfalfa production. Therefore, the cumulative impact from deregulation of glyphosatetolerant alfalfa is expected to be a further reduction in the amount of herbicide applied to U.S. 

cropland. 

N.4.8 Other Analyses on Herbicide Use and Impacts from Commercial Use of 

Glyphosate-Tolerant Crops 

Benbrook (2004) analyzed the USDA-NASS data from 1996 to 2004 and concluded that 

herbicide-tolerant crops have increased herbicide use by 138 million pounds with a 5% 

increase in herbicide use across the three major crops (corn, cotton, and soybean). Benbrook 

stated that the reliance on a single herbicide, glyphosate, as the primary method for managing 

weeds on millions of acres planted to herbicide-tolerant varieties remains the primary factor 

that has led to the need to apply more herbicides per acre to achieve the same level of weed 

control. These conclusions by Benbrook are not supported by the findings of Brooks and 

Barfoot (2006), Sankula (2006) or the USDA-ERS (2007). 

USDA-ERS conducted a study entitled, “The First Decade of Genetically Engineered Crops in 

the United States” (Fernandez-Cornejo and Caswell, 2006). In 2001, 2002, and 2003 USDAERS surveys, growers responded that they adopted herbicide-tolerant crops for the following 

reasons: increased yields (60 – 67% of the responses), reduced pesticide input costs (11 – 17% 

of the responses), and decreased management time/ease of operation (15 – 26% of the 

 

26 USDA Petition 04-110-01p, Section D.6., p. 282, http://www.aphis.usda.gov/brs/aphisdocs/04_11001p.pdf 
Monsanto Company 09-SY-201U Page 408 of 471

responses). This analysis found a cumulative reduction in herbicide use coinciding with the 

adoption of herbicide-tolerant crops. In addition, the report stated that glyphosate used on 

glyphosate-tolerant crops is less than one-third as toxic to humans and that glyphosate is not as 

likely to persist in the environment as the herbicides it replaces (Fernandez-Cornejo and 

McBride, 2002).

Based on the adoption rates of other glyphosate-tolerant crops, it is expected that glyphosatetolerant alfalfa would replace a significant portion of conventional alfalfa varieties, especially 

where alfalfa is highly managed (e.g., Western and Southwestern states). As glyphosatetolerant alfalfa is adopted, it is expected that Roundup herbicide will replace other forms of 

weed control currently used in alfalfa, as discussed below. 

Glyphosate-tolerant alfalfa is highly tolerant to glyphosate and shows no yield loss or loss in 

forage quality when treated over multiple seasons over and above maximum glyphosate 

application rates (Pierson and Reyes, 2006). Recommended use rates are provided by 

Monsanto to growers in the Monsanto Technology Use Guide and Regional Technical 

Bulletins (See Appendix I of USDA petition 04-110-01p). In-season maximum application of 

glyphosate is up to 1.5 lbs a.e. per acre at a single application, and total per-year applications is 

4.5 lbs a.e./acre. These application rates were used by Monsanto when conducting residue 

studies and for event selection purposes. In practice, these maximum rates will rarely be used 

since the vast majority of weeds are controlled using recommended rates and it would be 

uneconomical for growers to apply excess herbicide. 

N.5 Impact on Soil Tillage Practices 

Tillage is a farming practice used to prepare the seed bed and to mechanically remove weeds 

from the field. No-till farming, also known as conservation tillage or zero tillage, is defined as 

planting and growing crops without disturbing the soil through tillage. No-till and other 

conservation tillage farming practices improve soil and air quality, minimize surface runoff and 

soil erosion, enhance water quality, and reduce contributions to the greenhouse gases effect, 

particularly carbon dioxide (Fernandez-Cornejo and McBride, 2002; Leep et al., 2003). No-till 

seeding reduces equipment use, which can result in economic benefits of reduced labor and 

fuel cost. In the dryland areas of the northwest U.S., no-till seeding helps conserve soil 

moisture (G. Shewmaker, Extension Forage Specialist, University of Idaho, Personal 

Communications, 2007). 

The main difference between no-till and conventional tillage seed practices is that no tillage is 

used to prepare the seedbed for seeding. When no-till seeding a field, a postemergence or 

burndown herbicide is applied prior to or at seeding to control any emerged weeds in place of 

tillage. Herbicide-tolerant crops facilitate the adoption of no-till farming; consequently, no-till 

farming has been extensively adopted for corn, cotton and soybean, for which herbicidetolerant varieties are readily available. Data available for individual crops are discussed below. 

N.5.1 Corn 

As the adoption and commercial use of glyphosate-tolerant corn has increased, no-till corn 

acreage has steadily and significantly increased in parallel. In 2004 (the most recent year for 

which survey information is available), no-till practices were used on 19.7% of the total corn 
Monsanto Company 09-SY-201U Page 409 of 471

acres, compared to zero no-till acres before glyphosate-tolerant corn was commercially 

available in 1996. The positive impacts from no-till production (such as reduced fuel use, soil 

erosion, runoff of pesticides and water, global warming potential, greenhouse gas emissions, 

and improved wildlife habitat) will increase as the adoption of herbicide-tolerant crops 

continues to increase (Leep et al, 2003; CTIC, 2004). 

N.5.2 Cotton 

The use of glyphosate-tolerant cotton has led to a significant increase in no-till production 

practices (Sankula, 2006). Total no-till cotton acres in 2004 were 2.4 million acres, or 18% of 

the total cotton acres. This is an increase of 371% compared to the no-till acres in 1996. The 

main reason for the increase in no-till cotton acreage since 1996 is the adoption of herbicidetolerant cotton, allowing over-the-top glyphosate applications (Doane, 2002). Other reasons 

include enhanced grower awareness of the benefits of conservation tillage practices, increase in 

fuel prices, access to better no-till equipment, and availability of better herbicides to control 

weeds in no-till fields. Assuming that the entire no-till cotton acreage in 2004 (2.4 million 

acres) was planted to herbicide-tolerant varieties, fuel and labor cost savings were estimated to 

be $48 million (Sankula, 2006). 

N.5.3 Soybean 

In 1995, before the introduction of glyphosate-tolerant soybean, approximately 27% of the 

U.S. soybean acres used no-till production. By 2004, no-till acres increased to 36% of the total 

soybean acres (Sankula, 2006). A few states provide statistics on the adoption of no-till acres 

in their state. For 2007 in Indiana, no-till soybean was planted on 69% of the total soybean 

acres27. In 2006 in Illinois, no-till farming was used on 51% of the soybean acres. The 

University of Illinois Extension Service attributed that figure to the fact that 90% of the state’s 

acres were planted to glyphosate-tolerant varieties, along with other factors, such as high fuel 

prices, improved equipment, higher yields using no-till practices, and better grower awareness 

of the advantages to soil and water quality from no-till farming28. 

N.5.4 Alfalfa 

According to forage specialists, all alfalfa-growing regions currently practice some level of notill seeding (J. Caddel, Forage Specialist, Oklahoma State University, Personal 

Communications, 2007; D. Hancock, Forage Specialist, University of Georgia, Personal 

Communications, 2007; D. Putnam, Extension Forage Specialist, University of California, 

Personal Communications, 2007; G. Shewmaker, Extension Forage Specialist, University of 

Idaho, Personal Communications, 2007; M. Sulc, Extension Forage Specialist, Ohio State 

University, Personal Communications, 2007; and D. Undersander, Extension Forage Specialist, 

University of Idaho, Personal Communications, 2007). All soil management groups (soil class 

 

27 Summed from State of Indiana county level data from at 

http://www.in.gov/isda/files/Soybean_Rank_Percentage.pdf 28 University of Illinois Extension Servive News Release: No-till is now the "Conventional" Tillage System for 

Illinois Farmers; http://web.extension.uiuc.edu/state/newsdetail.cfm?NewsID=4991
Monsanto Company 09-SY-201U Page 410 of 471

1 to 5) and all textures are suitable for no-till establishment and production provided the soils 

are naturally well-drained or tilled (Leep et al., 2003). 

Few peer-reviewed publications are available to quantify the current acreage of no-till seeded 

alfalfa. Therefore, personal communication with local extension forage and weed specialists 

was heavily relied upon to address this question. Pennsylvania is the only state that conducts 

surveys quantifying no-till alfalfa acreage. The highest levels of no-till seeded alfalfa are 

practiced in the North Central, East Central, and Southern regions of the U.S. The amount of 

no-till varies significantly within these regions and states. No-till seeding in the North Central 

region can range from 5 to 20% of the seeded acres, with Minnesota practicing the lowest 

amount of no-till seeding (R. Becker, Weed Specialist, University of Minnesota, Personal 

Communications, 2007; S. Moeschnig, Extension Weed Specialist, South Dakota State 

University, Personal Communications, 2007; P. Murphy, NRCS Wisconsin, Personal 

Communications, 2007; D. Undersander, Extension Forage Specialist, University of Idaho, 

Personal Communications, 2007). Soil compaction plus cold and wet soil conditions in the 

spring are the primary reasons for the low adoption rate in Minnesota. The East Central region 

can range from 10 to 30% no-till29 (Leep, 2003). Ohio reported the highest percentage of notill. The no-till alfalfa acreage in the state of Pennsylvania is 21.4% based on actual survey 

results by the Pennsylvania NASS30. 

As with the other glyphosate-tolerant crops, the ability to use over-the-top treatments of 

glyphosate is expected to facilitate the further adoption of no-till practices. Deregulation of 

glyphosate-tolerant alfalfa, when considering the data from other glyphosate-tolerant crops, is 

expected to further increase the benefits from no-till farming. These benefits include improved 

soil and air quality, reduced surface runoff and soil erosion, enhanced water quality, and 

reduced contributions to the greenhouse gases effect, particularly carbon dioxide. 

N.5.5 Other Crops 

Limited information is available on the use of no-till farming practices in canola; however, a 

survey conducted in 2001 for the Canola Council of Canada (2001) did find that planting of 

herbicide-tolerant canola varieties resulted in an increase in the use of no-till practices. For 

sugar beet, more information should be available after more acres are planted in 2008; 

however, the presence of the herbicide-tolerant trait should allow for adoption of no-till 

practices, just as experienced for corn, cotton, and soybean. 

 N.6 Conclusions

Data show the introduction of glyphosate-tolerant crops has had no impact on the overland 

acres of crops planted in the U.S. The glyphosate-tolerant trait does increase grower flexibility 

in deciding what crop to plant, but has only a minor influence on the specific crop acreage 

planted, compared to other market forces, such as price. The planting of glyphosate-tolerant 

crops has resulted in a substantial decrease in the overall amount of herbicides applied to 

croplands. Use of glyphosate-tolerant crops has facilitated the adoption of no-till practices, 

which improve soil and water quality, reduce fuel use and reduce air pollution. Glyphosate-

 

29 Sulc, M., Extension Forage Specialist, Ohio State University, Personal Communications, 2007. 

30 http://www.nass.usda.gov/Statistics_by_State/Pennsylvania/Publications/Survey_Results/tillage07_feb.pdf 
Monsanto Company 09-SY-201U Page 411 of 471

tolerant crops increase grower flexibility regarding crop rotation but have minimal impacts on 

crop rotation. 

According to USDA-ERS (2006), adoption of currently deregulated glyphosate-tolerant crops 

has resulted in an overall increase in farmer profitability, farmer household income, crop yield, 

soil conservation, and a reduction in herbicide use. Deregulation of other glyphosate-tolerant 

crops is expected to provide similar benefits to growers and the environment by reducing costs, 

reducing herbicide use, and increasing grower planting flexibility in crop choice. 

 
Monsanto Company 09-SY-201U Page 412 of 471

Appendix N References 

Ash, M., J. Livezey, and E. Dohlman. 2006. Soybean backgrounder. U.S. Department of 

Agriculture-Economic Research Service, Washington, D.C. 

Benbrook, C. 2004. Genetically engineered crops and pesticide use in the United States: The 

first nine years. BioTech InfoNet Technical Paper No. 7. www.biotechinfo.net/Full_version_first_nine.pdf [Accessed July 2, 2009]. 

Brookes, G and P.Barfoot. 2006. Global impact of biotech crops: socio-economic and 

environmental effect in the first ten years of commercial use. AgBioForum. 9:139-151. 

CCC. 2001. An agronomic and economic assessment of transgenic canola. Canola Council of 

Canada. http://www.canola-council.org/gmo_toc.aspx [Accessed July 2, 2009]. 

Coyette, G., F. Tencalla, I. Brants, Y. Fichet, and D. Rouchouze. 2002. Effect of introducing 

glyphosate-tolerant sugar beet on pesticide usage in Europe. Pesticide Outlook 13:219-224. 

CTIC. 2004. Crop residue management. Conservation Technology Information Center, West 

Lafayette, Indiana. 

Doane Marketing Research 2002. Conservation tillage study prepared for the National Cotton 

Foundation. http://www.cotton.org/tech/biotech/contill-study.cfm [Accessed July 2, 2009]. 

Fernandez-Cornejo, J. and W.D. McBride. 2002. Adoption of bioengineered crops. 

Agricultural Economic Report AER810, U.S. Department of Agriculture, Washington, D.C. 

Fernandez-Cornejo, J. and M. Caswell. 2006. The first decade of genetically engineered crops 

in the United States. Economic Information Bulleting E1B-11, U.S. Department of Agriculture, 

Agricultural Economic Report, Washington, D.C. 

Leep, R., D. Undersander, P. Peterson, D-H Min, T. Harrigan, and J. Grigar. 2003. Steps to 

successful no-till establishment of forages. Michigan State University, University of Wisconsin, 

University of Minnesota, and Natural Resources Conservation Service, Extension Bulletin E2880. 

Monsanto. 2007. Monsanto biotechnology trait acreage: Fiscal years 1996 to 2007. Monsanto 

Company, St. Louis, Missouri. 

Pierson, P. and C. Reyes. 2006. Roundup Ready alfalfa: Summary of third-party data for yield, 

forage quality and crop safety. National Meeting of the North American Alfalfa Improvement 

Conference. 

Sankula, S. 2006. Quantification of the impacts on US agriculture of biotechnology-derived 

crops planted in 2005. National Center for Food and Agriculture Policy, Washington, D.C. 

Shipitalo, M.J., R.W. Malone, and L.B. Owens. 2008. Impact of glyphosate-tolerant soybean 

and glufosinate-tolerant corn production on herbicide losses in surface runoff. Journal of 

Environmental Quality 37:401-408. 
Monsanto Company 09-SY-201U Page 413 of 471

UC. 2001. Alfalfa: Susceptibility of weeds to herbicide control. University of California Pest 

Management Guidelines. http://www.ipm.ucdavis.edu/PMG/r1700411.html#WINTER 

[Accessed July 2, 2009]. 

USDA-ERS. 2007. Adoption of genetically engineered crops in the U.S. U.S. Department of 

Agriculture, Washington, D.C 

USDA-ERS. 2006. Soybean production costs and returns per planted crop acre, by region, 

excluding government payments for 2006. U.S. Department of Agriculture-Economic Research 

Service. (http://www.ers.usda.gov/data/costsandreturns/Soy_all.xls) [Accessed June 26, 2009]. 

USDA-NASS. 2006. Crop production 2005 summary. U.S. Department of Agriculture 

National Agricultural Statistics Service. 

USDA-NASS. 2007. Crop production historical track records. 

http://usda.mannlib.cornell.edu/usda/current/htrcp/ [Accessed July 16, 2009] 

Vesterby, M. and K.S. Krupa. 1997. Major uses of land in the United States. Statistical Bulletin 

973. U.S. Department of Agriculture, Washington, D.C 
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Appendix O. Potential Impact of Glyphosate on Human Health and the Environment 

O.1 Overview 

Glyphosate is a herbicide approved for use (registered) by the U.S. Environmental Protection 

Agency (EPA or the agency) for the control of weeds that would interfere with the growth of 

many food and non-food crops, including biotechnology-derived crops, as well as for control 

of weeds growing in non-crop areas. In 2001, EPA identified glyphosate as the most widely 

used conventional agricultural pesticide in the U.S. (Kiely et al., 2004). Glyphosate has been 

registered, and food and feed tolerances have been established in the U.S. for its residues, since 

1979. Glyphosate has also successfully completed the reregistration process, as required for 

the continued registration of all pesticides originally registered before 1984. 

Glyphosate has a complete and comprehensive regulatory data base (toxicity, environmental 

fate, and ecological toxicity) that has been evaluated by EPA to support all currently approved 

uses including glyphosate-tolerant alfalfa. EPA has stated that it has a high level of confidence 

in the quality of the existing studies and the reliability of the toxicity endpoints that are the 

basis for risk assessment (EPA, 2006a,b,c). In establishing food and feed tolerances to support 

the use of glyphosate on animal feed and forage crops (the group tolerance that supports the 

use of glyphosate in conventional and glyphosate-tolerant alfalfa), EPA noted that it had 

conducted “a complete and thorough review of the available data for glyphosate,” and 

determined that “glyphosate will not pose unreasonable risks or adverse effects to humans or 

the environment” (EPA, 200231). 

The following discussion provides an overview of the regulatory and risk assessment processes 

applicable to glyphosate and all other agricultural use pesticides. Glyphosate has been 

approved by the EPA for a large number of food and feed uses, including uses associated with 

glyphosate-tolerant crops. Over 180 food and feed tolerances (40 CFR § 180.364) have been 

established for glyphosate in support of these uses. A complete listing of all U.S. glyphosate 

tolerances is provided in Attachment 1. 

O.1.1 Pesticide Registration and Tolerance Setting 

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) requires that before sale or 

distribution of a pesticide in the U.S., a person or company must obtain a registration, or 

license, from EPA. Before registering a new pesticide or a new use for a previously registered 

pesticide, EPA must first ensure that the pesticide, when used according to its label directions, 

will not cause unreasonable adverse effects on the environment. In order to address this 

standard, EPA must evaluate potential risks to humans and the environment, and may require 

applicants to submit more than 100 different scientific studies and tests conducted according to 

EPA guidelines. According to EPA, glyphosate is one of more than 1055 active ingredients 

currently registered as pesticides, which are formulated into many thousands of pesticide 

products that are available in the marketplace. 

The process of registering a pesticide is a scientific, legal, and administrative procedure 

through which EPA examines the ingredients of the pesticide; the particular site or crop on 

 

31 Prior to 2002, separate alfalfa forage and alfalfa hay tolerances were established for glyphosate.
Monsanto Company 09-SY-201U Page 415 of 471

which it is to be used; the amount, frequency, method and timing of application, and other 

conditions of its use; and storage and disposal practices. In evaluating a pesticide registration 

application, EPA assesses a wide variety of potential human health and environmental effects 

associated with use of the product. 

The data required by EPA are used to evaluate whether a pesticide has the potential to cause 

adverse effects on humans, wildlife, fish, and plants (including endangered species and NTOs, 

organisms that the pesticide is not intended to act against). The registration applicant must also 

supply data addressing the pesticide’s potential impact on surface water or ground water 

(which might result from leaching or runoff, for example). Potential human health and safety 

risks range from short-term toxicity to long-term effects such as cancer and reproductive 

system disorders. 

EPA also must approve the language that appears on each pesticide label. A pesticide product 

can only be used legally according to the directions for use on the labeling accompanying it at 

the time of sale. Following these directions carefully and precisely is necessary to ensure safe 

use. 

A pesticide’s registration is not the only opportunity EPA has to evaluate that product’s safety. 

For example, EPA is currently completing a one-time program to review older pesticides 

(those initially registered before November 1984) under FIFRA to ensure that they meet 

current scientific and regulatory standards. This process, called reregistration, considers the 

human health and ecological effects of pesticides and results in actions to reduce risks that are 

of concern. EPA concluded its reregistration evaluation of glyphosate in 1993. At that time, 

the EPA produced a 291-page Reregistration Eligibility Decision document (RED) on 

glyphosate, setting forth the data on which it made a decision to reregister all then-existing 

uses of the pesticide, based on the pesticide having met the no unreasonable adverse effects 

standard found in FIFRA. 

Where pesticides may be used on food or feed crops, EPA also sets tolerances (maximum 

pesticide residue levels) for the amount of the pesticide that can legally remain in or on foods. 

EPA undertakes this analysis under the authority of the Federal Food, Drug, and Cosmetic Act 

(FFDCA). Under the FFDCA, EPA must find that such tolerances will be safe, meaning that 

there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide 

chemical residue. This finding must be made and the appropriate tolerance established before 

a pesticide can be registered for use on the particular food or feed crop in question. Several 

factors must be addressed before a tolerance can be established, including: 

• the aggregate, non-occupational exposure from the pesticide (exposure through diet, 

from using pesticides in and around the home, and from drinking water); 

• the cumulative effects from exposure to different pesticides that produce similar effects 

in the human body; 

• whether there is increased susceptibility to infants and children, or other sensitive 

subpopulations, from exposure to the pesticide; and 

• whether the pesticide produces an effect in humans similar to an effect produced by a 

naturally occurring estrogen or produces other endocrine-disruption effects. 
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O.1.2 Pesticide Risk Assessment 

The process EPA uses for evaluating the health impacts of a pesticide, under either FIFRA or 

the FFDCA, is called risk assessment. EPA uses the National Research Council’s four-step 

process for human health risk assessment, which involves hazard identification, dose-response 

assessment, exposure assessment and risk characterization. Each of these steps is discussed 

below: 

The first step in the risk assessment process is to identify potential health effects, or hazards 

that may occur from different types of pesticide exposure. EPA considers the full spectrum of 

a pesticide’s potential health effects. Hazards are identified through a battery of studies that 

examine the potential toxicity of the pesticide in various tests including, where appropriate, 

tests with laboratory animals. 

Generally, for human health risk assessments, many toxicity studies are conducted, based on 

EPA guidelines, by pesticide companies in independent laboratories following the Good 

Laboratory Practice (GLP) standards, and evaluated for acceptability by EPA scientists. EPA 

evaluates pesticides for a wide range of effects, from eye and skin irritation to cancer and birth 

defects. EPA may also consult the public literature or other sources of information on any 

aspect of the chemical. 

The next step of the risk assessment considers the levels at which the pesticide produces 

adverse effects. Dose-response assessment involves considering the dose levels at which 

adverse effects were observed in test animals, and using these dose levels to calculate an equal 

dose in humans. 

Step three of the process involves an exposure assessment. People can be exposed to 

pesticides in three ways: 

1. Inhaling pesticides (inhalation exposure), 

2. Absorbing pesticides through the skin (dermal exposure), and 

3. Ingesting pesticides (oral exposure). 

Depending on the situation, pesticides could enter the body by any one or all of these routes. 

Typical sources of pesticide exposure include agricultural (food); home and personal use 

pesticides; pesticides applied to lands that make their way into the drinking water; or 

occupational exposure for agricultural workers or pesticide applicators. 

Risk characterization is the final step in assessing human health risks from pesticides. It is the 

process of combining the hazard, dose-response and exposure assessments to describe the 

overall risk from the use of a pesticide. It explains the assumptions used in assessing exposure 

as well as the uncertainties that are built into the dose-response assessment. The strength of the 

overall database is considered, and broad conclusions are made. EPA’s role is to evaluate both 

toxicity and exposure and to determine the risk associated with use of the pesticide. 

The risk to human health from pesticide exposure depends on both the toxicity of the pesticide 

and the likelihood of people coming into contact with it (exposure). At least some exposure 

and some toxicity are required to result in a risk. For example, if the pesticide is found to have 

a high level of toxicity, but people are not exposed to the pesticide, there is no risk. Likewise, 
Monsanto Company 09-SY-201U Page 417 of 471

if there is ample exposure but the pesticide is nontoxic, there is no risk. However, usually 

when pesticides are used, there is some toxicity and exposure, which results in a potential risk. 

EPA recognizes that effects vary between animals of different species and from person to 

person. To account for this variability, a 100-fold uncertainty factor is built into the risk 

assessment. This uncertainty factor creates an additional margin of safety for protecting people 

who may be exposed to the pesticides. FQPA requires EPA to use an extra 10-fold safety 

factor, if necessary, to protect infants and children from effects of the pesticide. 

Once EPA completes the risk assessment process for a pesticide, the Agency uses this 

information to determine if (when used according to label directions), there is a reasonable 

certainty that the pesticide will not harm a person’s health. 

Using the conclusions of a risk assessment, EPA can then make a more informed decision 

regarding whether to approve a pesticide chemical or use, as proposed, or whether additional 

protective measures are necessary to limit occupational or non-occupational exposure to a 

pesticide. For example, EPA may prohibit a pesticide from being used on certain crops 

because consuming that commodity treated with the pesticide may result in an unacceptable 

risk to consumers. Another example of protective measures is requiring workers to wear 

personal protective equipment (PPE) such as a respirator or chemical resistant gloves, or not 

allowing workers to enter treated crop fields until a specific period of time has elapsed. 

If, after considering all appropriate risk reduction measures, the pesticide still does not meet 

EPA’s safety standard, the Agency will not allow the proposed chemical or use. Regardless of 

the specific measures enforced, EPA’s primary goal is to ensure that legal uses of the pesticide 

are protective of human health, especially the health of children, and the environment. 

O.2 Potential Impact of Glyphosate on Human Health 

O.2.1 Glyphosate Safety Evaluations 

Glyphosate presently has 186 established food and feed tolerances in the U.S (see Attachment 

1). Each time EPA reviews an application to add a new food or feed use to the glyphosate 

label the Agency is required by FFDCA to conduct an aggregate risk assessment, considering 

all non-occupational sources of human exposure to the pesticide, and find that aggregate 

exposure to the pesticide will be safe as defined by the statute and regulations. Issues 

associated with potential occupational exposure for each new use are considered under 

FIFRA’s unreasonable risk standard. 

Over the course of these numerous reviews, the toxicology of glyphosate has been extensively 

studied. Comprehensive toxicological studies in animals have demonstrated that glyphosate 

does not cause cancer, birth defects, mutagenic effects, nervous system effects or reproductive 

problems32 (EPA, 1993; WHO/FAO, 2004). In fact, after a thorough review of all available 

 

32 European Commission (EC ). 2002. Report for the Active Substance Glyphosate, Directive 6511/VI/99, January 21. 

http://europa.eu.int/comm/food/fs/ph_ps/pro/eva/existing/list1_en.htm. 
Monsanto Company 09-SY-201U Page 418 of 471

toxicology data, the EPA concluded that glyphosate should be classified in Group E - Evidence 

of Non-carcinogenicity in Humans, the most favorable category possible (EPA, 1993). 

Despite this extensive safety data, glyphosate safety is reviewed with every new use for which 

registration is sought, including, where necessary, uses associated with glyphosate-tolerant 

crops developed through biotechnology. As discussed above, prior to the approval of any new 

use of an existing registered pesticide, EPA must consider the potential human health effects 

from the aggregate (total combined) human exposure to that pesticide, combining the potential 

exposure from the proposed new use with all other existing exposures to the pesticide. Dietary 

exposure is considered, which addresses pesticide residues that may remain on food from crops 

on which the pesticide is applied (pre- or postemergence), as well as any residue that could be 

found in drinking water as a result of pesticide use. Non-dietary exposure is also included in 

this assessment, which includes exposure to the pesticide through residential use, such as on 

lawns or in flower beds, as well as exposure in a recreational context, such as from a golf 

course or sports field. Based on these data, EPA must be able to make a determination of 

reasonable certainty of no harm to human health as required by the FFDCA. 

EPA does not conduct an acute dietary risk assessment for glyphosate because no acute effects 

have ever been identified in the toxicological studies conducted for glyphosate. Accordingly, 

EPA does not expect glyphosate to pose an acute risk (EPA, 2006d). EPA does conduct a 

chronic dietary (food and water) risk assessment for glyphosate based on a theoretical worst 

case exposure estimate. For food, this estimate assumes that glyphosate is used on 100 percent 

of all the crops on which the pesticide is currently approved for use. It further assumes that the 

resulting pesticide residues found on all harvested food crops are at the level of the legally 

established tolerance (i.e., the maximum allowable pesticide residue level). For water, EPA 

assumes that glyphosate is used to control weeds in water bodies by direct application to the 

water at the maximum application rate, without taking into account degradation in the water 

body (EPA, 2006b,c). 

Applying this unrealistic, theoretical maximum exposure estimate, EPA determines how much 

of the established Reference Dose (RfD) would be utilized by all currently approved product 

uses. The RfD is an estimate of the amount of daily pesticide exposure to the human 

population that can occur over a lifetime with a reasonable certainty of no harm to human 

health.33 For glyphosate, the RfD is 1.75 mg per kg body weight per day (mg/kg/day) (EPA, 

2002). Provided that the utilization of the RfD from food and drinking water does not exceed 

the EPA level of concern of 100 percent 34 , 35 , EPA will then conduct an aggregate risk 

assessment (chronic and short/intermediate-term) to consider additional exposures from nondietary exposure routes (residential and recreational). 

If the aggregate risk assessment shows that utilization of the RfD does not exceed the EPA 

level of concern, then EPA will conclude that the new use does not pose an unreasonable risk 

to human health. EPA will then establish or revise, as needed, any food or animal feed crop 

 

33 RfD is the current terminology used by EPA; however earlier EPA risk assessment terminology used the term Allowable 

Daily Intake (ADI). RfD and ADI are synonymous.

34 EPA has reviewed state monitoring data on the occurrence of glyphosate in public drinking water systems and found that 

these data “reinforce the Agency’s conclusion that aggregate exposure to glyphosate via all exposure routes, including drinking

water, will not exceed the Agency’s level of concern” (U.S. EPA, 2002).

35 For glyphosate, the EPA’s level of concern for chronic dietary exposure is 100 (U.S. EPA , 2006b).
Monsanto Company 09-SY-201U Page 419 of 471

tolerances to allow for the presence of glyphosate residue on that crop. EPA publishes these 

new tolerances in the Federal Register, along with a summary of the risk assessment and 

approves pesticide labeling for the new use. In issuing the final tolerance rule, EPA considers 

and discusses any comments received in response to the original notice regarding EPA’s 

intention to establish tolerances that was published in the Federal Register. 

Despite the large number of approved food and feed uses of glyphosate, including uses 

associated with glyphosate-tolerant crops, a large margin of safety exists for glyphosate. While 

use of glyphosate has increased in the decade since the introduction of glyphosate-tolerant 

crops, the associated risk to human health as a result of the increased human exposure to 

glyphosate remains low, due to the low mammalian toxicity of glyphosate and the relatively 

low dietary exposure associated with the herbicide’s approved uses. 

Prior to the first approval of a glyphosate-tolerant crop (soybean) in 1996, theoretical dietary 

exposure for all registered conventional uses of glyphosate utilized approximately 2.9% of the 

glyphosate RfD for the most sensitive subpopulation of non-nursing infants less than one year 

old (EPA, 1993). Incremental theoretical dietary exposure from the additional uses of 

glyphosate in glyphosate-tolerant soybean, cotton, corn, canola, alfalfa, sugar beet and 

creeping bentgrass combined with all other conventional crop uses of glyphosate approved 

between 1996 and 2006, account for only an additional 1% utilization of the glyphosate RfD 

for the subpopulation of infants less than one year old (EPA, 2004d). The total combined 

chronic aggregate exposure (dietary and non-dietary) from all uses of glyphosate utilizes well 

below 100% of the glyphosate RfD, only 9% for the most sensitive subpopulation of nonnursing infants less than one year old (EPA, 2006b). The total combined short/intermediate 

term aggregate exposure (dietary and non-dietary) for all current registered uses of glyphosate 

utilizes only 11% of the glyphosate RfD for the most sensitive subpopulation of non-nursing 

infants less than one year old (EPA, 2006b). The utilization of the glyphosate RfD, which is 

well below 100 percent, has allowed EPA to continue to make the conclusion of reasonable 

certainty of no harm to human health for each glyphosate use, including glyphosate-tolerant 

alfalfa and other new glyphosate-tolerant crop uses. 

These figures are supported by the data provided in the tables below. Table O-1 summarizes 

the established food and feed tolerances supporting the use of glyphosate in the conventional 

crops of alfalfa, cotton, sugar beet and soybean prior to the first glyphosate-tolerant crop in 

1996. A summary of the regulatory approvals, including new or modified food and feed 

tolerances, and associated dietary exposure assessments for approved glyphosate-tolerant crops 

is provided in Table O-2. Table O-3 summarizes the most recent chronic and 

short/intermediate-term aggregate risk assessments for glyphosate. 

 
Monsanto Company 09-SY-201U Page 420 of 471

Table O-1. Established Glyphosate Tolerances Prior to Glyphosate-tolerant Crops 

(1993) 

Crop Established Food/Feed Tolerances Publication % of Reference Dose (RfD) 

Soybean • Seed – 20 ppm 

• forage & hay – 15 ppm 

• hulls – 100 ppm 

Glyphosate Reregistration 

Eligibility Decision 

Document 

September 1993 

(EPA, 1993) 

General Population - 1.2 

Non-nursing infants <1 year old - 2.9 

Alfalfa 200 ppm 

Cotton forage, hay, & seed – 15 ppm 

Sugar beet Roots – 0.2 ppm 
Monsanto Company 09-SY-201U Page 421 of 471

Table O-2 (continued). Summary of EPA Approvals for Glyphosate Use in Glyphosatetolerant Crops 

 Commercial 

Introduction 

Year 

Required Changes in Food/Feed 

Tolerances 

Federal Register 

Publication 

Establishing New or 

Modified Tolerance 

% of Reference Dose (RfD) 

Dietary Exposure Only 

(Food + Water) 

Roundup Ready 

soybean 

1996 • Increase soybean forage to 100 

ppm. 

• Increase soybean hay to 200 

ppm. 

• Establish new tolerance for 

aspirated grain fractions at 50 

ppm. 

61 FR 15192 

Petition No. 4F4369 

Apr. 1996 

(EPA, 1996b) 

General Population – 1 

Non-nursing infants- 2.5 

Roundup Ready 

cotton 

1997 Establish new tolerance for gin 

byproduct at 100 ppm. 

61 FR 7729 

Petition No. 5F4493 

Feb. 1996 

(EPA, 1996a) 

General Population - 1 

Non-nursing infants - 2.4 

Roundup Ready 

corn 

1998 Establish new tolerance for corn 

forage at 1 ppm. 

62 FR 17723 

Petition No. 5F4555 

Apr. 1997 

(EPA, 1997) 

General Population - 1 

Non-nursing infants < 1 year old 

- 3 

Roundup Ready 

canola 

1999 Establish new tolerances for 

canola. 

• seed at 10 ppm 

• meal at 15 ppm 

64 FR 18360 

Petition No. 2E4118 

Apr. 1999 

(EPA, 1999) 

General Population - 1.5 

Non-nursing infants <1 year old 

- 3.3 

Roundup Ready 

sugar beet

2008 Establish new tolerances for sugar 

beet. 

• roots at 10 ppm 

• tops at 10 ppm 

• pulp (dried) at 25 ppm 

Roundup Ready 

corn 2

2004 Increased tolerance for corn forage 

to 6 ppm. 

68 FR 36472 

Jun. 2003 

(EPA, 2003) 

Change in forage tolerance did 

not affect estimated dietary 

exposure from animal products; 

therefore no dietary risk 

assessment was conducted. 

Roundup Ready 

Flex cotton

2006 • Increase tolerance for gin 

byproduct to 175 ppm. 

• Increase tolerance for 

cottonseed to 35 ppm. 

69 FR 65081 

Petition No. 3F6570 

Nov. 2004 

(EPA, 2004d) 

General Population - 2.2 

All infants < 1 year old - -3.9 

Roundup Ready 

alfalfa

2006 Establish new tolerances for alfalfa 

seed at 0.5 ppm. 

Existing tolerances for alfalfa forage 

(400 ppm) and hay (200 ppm) were 

sufficient to cover new in-crop uses. 

70 FR 7861 

Petition No. 2F6487 

Feb. 2005 

(EPA, 2005)1

Dietary exposure insignificant, 

did not conduct new risk 

assessment. Deferred to 

assessment conducted for flex 

cotton as published in 69 FR 

65081. 

1

U.S. Environmental Protection Agency. 2005. Final Rule. Federal Register Vol. 70, No. 31: 7861. Feb. 2005. 
Monsanto Company 09-SY-201U Page 422 of 471

Table O-3. Aggregate Exposure Assessment for Glyphosate 

Population 

Subgroup 

Acute 

Aggregate2

RfD 

(mg/kg/day) 2

Chronic 

Aggregate1,2 

Short/Intermediate Term 

Aggregate,2

Exposur

e 

(mg/kg/

day) 

% RfD Exposure 

(mg/kg/day) 

% RfD 

General U.S. 

population 

Not 

applicable 

1.75 

0.041 2 - - 

All infants (<1 

year) 

0.127 7 0.157 9 

Non-nursing 

infants (<1 year) 

0.158 9 0.188 11 

Children 1-2 

years 

0.095 5 0.125 7 

Children 3-5 

years 

0.088 5 0.118 7 

Children 6-12 

years 

0.059 3 0.089 5 

Youth 13-19 

years 

0.037 2 - - 

Adults 20-49 

years 

0.033 2 0.063 4 

Adults 50+ years 0.028 2 - - 

Females 13-49 

years 

0.031 2 - - 

1

Chronic aggregate exposure is the same as chronic dietary exposure because chronic non-dietary 

exposure is not expected based upon the current registered non-crop uses of glyphosate. 

2

EPA OPPTS. Glyphosate Human Health Risk Assessment for Proposed Uses on Safflower and 

Sunflower. Petition No. 4E6878. Sept. 5, 2006.

O.2.2 Glyphosate Safety Evaluation for Applicator and Bystander Exposure 

Another potential impact of the use of glyphosate on human health that EPA considers in 

its human health analysis is applicator and bystander exposure resulting from increased 

glyphosate use. Based on the toxicity of glyphosate and its registered uses, including use 

on glyphosate-tolerant crops, EPA has concluded that occupational exposures (short-term 

dermal and inhalation) to glyphosate are not of concern because no short-term dermal or 

inhalation toxicity endpoints have been identified for glyphosate (EPA, 2006b; EPA, 

2006c). 

Additional evidence to support the EPA conclusion can be found in the Farm Family 

Exposure Study (Acquavella et al., 2004), a biomonitoring study of pesticide applicators 

conducted by independent investigators. This biomonitoring study determined that the 

highest estimated bodily adsorption of glyphosate as the result of routine labeled 

applications of registered glyphosate-based agricultural herbicides to crops, including 

glyphosate-tolerant crops, was approximately 400 times lower than the RfD established 

for glyphosate. Furthermore, investigators determined that 40% of applicators did not 

have detectable exposure on the day of application, and 54% of the applicators had an 
Monsanto Company 09-SY-201U Page 423 of 471

estimated bodily adsorption of glyphosate more than 1000 times lower than the RfD 

(Acquavella et al., 2004). 

The biomonitoring study also found little evidence of detectable exposure to individuals 

on the farm who were not actively involved in or located in the immediate vicinity of 

labeled applications of glyphosate-based agricultural herbicides to crops. Considering 

the similarity of the use pattern and application rates of the glyphosate products in this 

study compared to those registered for use on glyphosate-tolerant alfalfa and glyphosatetolerant crops in general, bystander exposure attributed to the use of glyphosate on 

glyphosate-tolerant crops is expected to be negligible. 

O.3 Potential Impact of Glyphosate on the Environment 

Potential environmental effects are carefully considered as a part of the FIFRA pesticide 

registration process. Prior to the approval of a new pesticide or a new use (including a 

change in pesticide application rates and/or timing) and before reregistering an existing 

pesticide, EPA must consider the potential for environmental effects and make a 

determination that no unreasonable adverse effects to the environment will be caused by 

the new pesticide, new use or continued use. 

To make this determination, EPA requires a comprehensive set of environmental fate and 

ecotoxicology data on the pesticide’s active ingredient (40 CFR Part 158). EPA uses 

these data to assess the pesticide’s potential environmental risk (hazard x exposure). The 

required data include both short and long-term hazard data on representative organisms 

that are used to predict hazards to terrestrial animals (birds, nontarget insects, and small 

mammals), aquatic animals (freshwater fish and invertebrates, estuarine and marine 

organisms), and nontarget plants (terrestrial and aquatic). 

EPA reevaluated the environmental safety of glyphosate in 1993 as part of the FIFRArequired reregistration of all pesticides. At the end of this evaluation, EPA concluded 

that all registered uses of glyphosate were eligible for reregistration, including terrestrial 

(i.e., land-based) applications up to 7.5 pounds glyphosate acid equivalents (a.e.) per 

acre. 

Since the reregistration evaluation in 1993, EPA has reviewed and approved a significant 

number of new glyphosate uses: conventional crops such as legume vegetables and 

sunflower/safflower seed, glyphosate-tolerant crops such as corn, cotton, canola and 

soybean, and non-crop areas. In each case, EPA concluded that the new use, including 

any incremental environmental exposure to glyphosate caused by that new use, did not 

pose an unreasonable risk to the environment, and approved pesticide labeling for the 

new use. 

The studies and data collected by Monsanto, both for the initial EPA registration and 

reregistration of glyphosate, as well as data developed by independent academics, present 

a well-established safety profile for glyphosate. The following sections provide greater 

detail regarding some of the key findings from these studies. 
Monsanto Company 09-SY-201U Page 424 of 471

O.3.1 Persistence of Glyphosate in the Soil 

Persistence of agricultural chemicals in the soil is widely regarded as an undesirable 

environmental characteristic. Glyphosate has been shown to rapidly dissipate from most 

agricultural ecosystems across a wide range of soil and climatic conditions, with a 

median soil half-life (the time it takes for half of the glyphosate to dissipate in the soil) of 

13 days (Giesy et al., 2000). The potential for glyphosate to accumulate in soil following 

repeated applications has been studied both in the laboratory and the field. 

A laboratory study was conducted on two soil samples, with each sample receiving up to 

three sequential applications of 5 pounds glyphosate a.e. per acre over a 6-week period, at 

two-week intervals. The concentration of glyphosate in soil 24 weeks following 

application had declined to 1-5% of the concentration immediately after application, 

regardless of whether it was the first, second or third application. 

Glyphosate degradation in the soil following multiple glyphosate applications was also 

shown under field conditions. Soil was collected from pesticide efficacy and tolerance 

trials in orchards and vineyards that received repeated applications of glyphosate over a 

one- to six-year period, at cumulative rates of 6 to 120 pounds glyphosate a.e. per acre. 

These soil samples did not show any accumulation of glyphosate residues, even at the 

exaggerated rate of three sequential applications of eight pounds glyphosate a.e. per acre 

within a three-month interval for five out of six sequential years. Glyphosate degradation 

continued after multiple applications, and less than 10 percent of the total applied 

glyphosate remained in the soil one year after the last glyphosate application. 

A typical agronomic (annual) use pattern for glyphosate on glyphosate-tolerant crops is a 

preemergence burn down application of 1.5 pounds glyphosate acid per acre followed by 

one to two postemergence applications ranging from 0.77 to of 1.5 pounds glyphosate 

a.e. per acre, and where sequential application intervals are longer than two to four 

weeks. The maximum labeled rates and typical use patterns of glyphosate on glyphosatetolerant crops are well within the rates and frequencies used in the studies above. As a 

result, glyphosate is not expected to accumulate in soil as a result of labeled uses in 

glyphosate-tolerant crop. 

O.3.2 Persistence of Surfactant in the Soil 

Pesticide products approved for application to emerged weeds normally are applied with 

surfactants. Glyphosate products are formulated with surfactants to increase the 

permeability of the cuticle wax of the weed foliage to increase the foliar uptake of 

glyphosate. In other words, the surfactant acts to break down the plant’s natural 

protective wax coating, allowing the plant to better absorb the glyphosate, thereby 

improving the efficacy of the herbicide. 

The predominant type of surfactant used in formulated glyphosate products worldwide is 

polyethoxylated alkyl amine (POEA). When degradation of POEA was investigated in 

three types of soil (silt loam, silty clay loam, and sandy loam), microbial degradation was 

determined to be the primary degradation route, with minimal degradation occurring 

under sterile conditions. Approximately 25-30% of applied 14C-POEA was mineralized to 

14CO2 within seven weeks. The estimated degradation half-life for parent POEA was less 
Monsanto Company 09-SY-201U Page 425 of 471

than one week and possibly as short as one to two days. Because limited data are 

available for POEA dissipation, a conservative estimate of half-life values for POEA in 

soil would be 7-14 days36 (Giesy et al., 2000). Glyphosate and the POEA surfactant have 

similar soil dissipation rates and the same primary route of dissipation, i.e., microbial 

degradation. Therefore, it is reasonable to assume that the POEA surfactant will behave 

similarly to glyphosate in field soil, and an increase in residual soil concentrations 

(accumulation) of the POEA surfactant is not anticipated as a result of increased use of 

glyphosate associated with the planting of glyphosate-tolerant crops in general. 

O.3.3 Surface Water and Groundwater 

Glyphosate binds strongly to soil and has a low potential to move offsite to surface water 

or leach to groundwater (EPA, 1993). The EPA has estimated glyphosate levels that 

could occur in surface water based on presently approved use patterns. Relying on 

toxicological data from acute and chronic tests on fish and other aquatic organisms, EPA 

has determined that “the potential for environmental effects of glyphosate in surface 

water is minimal” (EPA, 2002). 

O.3.4 Wildlife 

1. Animals: As a part of the reregistration evaluation under FIFRA, EPA conducted an 

ecological assessment for glyphosate. This assessment compared the results from 

toxicity tests with glyphosate conducted with various plant and animal species to a 

conservative estimate of the concentration of glyphosate to which an organism might be 

exposed in the environment. This estimate, called the Estimated Exposure Concentration 

(EEC), is a point estimate for exposure that does not take into account normal 

environmental dilution or dissipation, or the frequency of exposure to the pesticide by 

wildlife. In the Reregistration Eligibility Decision (RED) for glyphosate (EPA, 1993), 

the exposure estimates were determined assuming an application rate of 5.0625 lb a.e., 

which exceeds the maximum labeled use rate for agricultural purposes. When the EECs 

were calculated for aquatic plants and animals, the direct application of this rate to water 

was assumed. Based on this assessment, EPA concluded that effects to birds, mammals, 

fish and invertebrates are minimal based on available data (EPA, 1993). 

Glyphosate is practically nontoxic to honey bees (which are used to assess effects on 

nontarget insects in general) and practically nontoxic to slightly toxic to birds, freshwater 

fish, marine and estuarine species, aquatic invertebrates and mammals (EPA, 1993). 

Glyphosate has a low octanol-water coefficient, indicating that it has a tendency to 

remain in the water phase rather than move from the water phase into fatty substances; 

therefore, it is not expected to accumulate in fish or other animal tissues. 

 

36 Marvel, J.T., Brightwell, B.B. and Suba, L.A. 1974. G3780A Surfactant: Biodegradation, plant uptake and 14C Distribution. Monsanto Company Report No. 321 
Monsanto Company 09-SY-201U Page 426 of 471

The glyphosate end-use products used in agriculture contain a surfactant to facilitate the 

uptake of glyphosate into the plant (Ashton and Crafts, 1981). Depending on the 

surfactant used, the toxicity of the end-use product may range from practically nontoxic 

to moderately toxic to fish and aquatic invertebrates (EPA, 1993). For this reason, the 

1993 Glyphosate RED stated that some formulated end-use products of glyphosate 

needed to be labeled as “Toxic to fish” if they were labeled for direct application to water 

bodies. Due to the associated hazard to fish and other aquatic organisms, glyphosate enduse products that are labeled for applications to water bodies generally do not contain 

surfactant. 

2. Plants: Glyphosate is a non-selective herbicide with activity on essentially all annual 

and perennial plants. As such, exposure to glyphosate could put aquatic and terrestrial 

nontarget plants as well as threatened and endangered plants at risk (EPA, 1993). 

Nontarget plants may potentially be at risk from applications of glyphosate as a result of 

spray drift. As discussed earlier, glyphosate binds tightly to soil and does not move 

offsite. Moreover, glyphosate is not taken up from agricultural soils by plants. Therefore 

risks to nontarget plants are only attributed to the spray drift of the pesticide. Pesticide 

labels include specific risk management measures to manage spray drift, including 

mandatory requirements for aerial applications. 

During the reregistration process in 1993, additional data on terrestrial nontarget plants 

were requested by the EPA. These additional data have been utilized in conjunction with 

an exposure assessment to further understand the potential risk to threatened and 

endangered plants from the use of glyphosate37 (Mortensen et al., 2008).

O.3.5 Endangered and Threatened Species 

The EPA Endangered Species Protection Program web site, http://www.epa.gov/espp/, 

describes the EPA assessment process for endangered species. The essential elements of 

that process, generally taken from the web site, are summarized below. 

The Endangered Species Act (ESA) was intended to protect and promote the recovery of 

animals and plants that are in danger of becoming extinct. All federal agencies are 

required under the ESA to ensure that their regulatory actions, including EPA’s 

registration of pesticides in the U.S., are not likely to jeopardize the continued existence 

of threatened or endangered species (“listed” species) or destroy or adversely modify 

their critical habitat. 

EPA's Endangered Species Protection Program (ESPP) helps promote the recovery of 

listed species. The ESPP is a program designed to determine whether pesticide use in a 

certain geographic area may affect any listed species. 

When registering a pesticide or reassessing the potential ecological risks from use of a 

currently registered pesticide, EPA evaluates extensive toxicity and ecological effects 

 

37 Mortensen, SR, Carr, KH, and Honegger, JL. 2008. Tier I Endangered Species Assessment for Agricultural Uses of 

Glyphosate and Glyphosate-Containing Herbicides. Monsanto Study Number RPN-2007-227. January 2008. 
Monsanto Company 09-SY-201U Page 427 of 471

data to determine how a pesticide will move through and break down in the environment. 

Risks to birds, fish, invertebrates, mammals and plants are routinely assessed and used in 

EPA’s determinations of whether a pesticide may be licensed for use in the U.S. 

EPA’s core pesticide risk assessment and regulatory processes ensure that protections are 

in place for all populations of nontarget species. Because endangered species may need 

specific protection, EPA has developed risk assessment procedures described in the 

Overview of the Ecological Risk Assessment Process (EPA, 2004a) to determine whether 

individuals of a listed species have the potential to be harmed by a pesticide, and if so, 

what specific protections may be appropriate. EPA’s conclusion regarding the potential 

risks a pesticide may pose to a listed species and any designated critical habitat for the 

species, after conducting a thorough ecological risk assessment, results in an "effects 

determination." 

An assessment of the effects of glyphosate use on all types of threatened and endangered 

species was conducted by Monsanto. This assessment generally followed the procedures 

described in the Overview of the Ecological Risk Assessment Process (EPA, 2004a), as 

summarized in Figure O-1. 
Monsanto Company 09-SY-201U Page 428 of 471

Figure O-1. Tier I Endangered Species Assessment 

Risk quotients (RQ’s) were calculated as the quotient of the EEC and the relevant toxicity 

endpoint for the most sensitive species for a given taxon (class of species). For acute 

studies of a few days duration, the concentration calculated to result in 50% mortality 

(LC50) or 50% designated effect (EC50) on the test species was utilized in the RQ 

calculation. For chronic studies, representing a significant portion of the species lifecycle, the highest concentration at which no effects were observed (No Observed Effect 

Concentration, NOEC) was used in the RQ calculation. 

Toxicity values (effects endpoints) for most categories of species were taken from the 

EPA assessment for new glyphosate uses on bentgrass (EPA, 2006a), or from EPA 

guideline studies conducted by Monsanto if these endpoints were lower. Studies from the 

literature were considered when the study design was appropriate for the assessment 

being made and where sufficient information regarding glyphosate or formulation test 

concentrations was available. Exposure estimates were based on standard EPA methods 

for calculating exposure (EPA, 2004a). For aquatic organisms, the model GENEEC2 

(EPA, 2004a), which calculates high-end estimates of surface water concentrations of 

pesticides in a generic farm pond, was utilized. When formulation toxicity was 

Exposure Estimate: 

Worst Case 

Estimated Environmental Concentration 

(EEC) Using Standard EPA Models or 

Analysis Methods

Toxicity Endpoint (TE): 

Lowest Acute or Chronic Value 

Meeting EPA Quality Standards 

RQ ≥ Level of Concern 

(LOC)? 

Further Analysis 

Determine Risk Quotient 

(RQ = EEC/TE) 

Yes

For Each 

Taxon 

Risk to Taxon Is 

Expected to be Low 

No 
Monsanto Company 09-SY-201U Page 429 of 471

considered, default drift values and the EPA standard pond38 were utilized for estimation 

of aquatic exposure. For terrestrial animals, the T-Rex model39 was utilized to calculate 

estimated dietary exposure and risk. For terrestrial and semi-aquatic plants, only the drift 

component of the TerrPlant model (EPA, 2004a) was used to determine exposure levels 

(the runoff component was disregarded). Runoff was not considered to contribute to 

exposure, since glyphosate binds very tightly to agricultural soils and does not have 

herbicidal properties when bound to soil (EPA, 2006a). 

The conclusion from this assessment, submitted to USDA is that threatened or 

endangered terrestrial or semi-aquatic plant species are not at risk from ground 

applications of glyphosate at rates less than 3.5 lb glyphosate (a.e.) per acre, or from 

aerial applications at rates less than 0.70 lb a.e. per acre. However, these species may be 

at risk when rates exceed these levels. Since the maximum single application rate before 

or after crop emergence in glyphosate-tolerant alfalfa is 1.55 lb a.e. per acre, no listed 

plant species are predicted to be at risk from ground application of glyphosate to 

glyphosate-tolerant alfalfa. For other glyphosate-tolerant crops, in-crop application rates 

are typically less than 3.5 lb a.e. per acre, resulting in a prediction of no risk to listed 

plant species. Rates that exceed 3.5 lb a.e. per acre are generally for control of perennial 

species prior to crop emergence or prior to harvest. 

The same assessment determined that other taxa (including birds, mammals, insects, fish, 

amphibians, aquatic invertebrates, and aquatic plants) were not at risk from the use of 

glyphosate herbicides in alfalfa production or in the production of other crops. 

Furthermore, this assessment determined that these other taxa were not at risk from 

indirect effects resulting from habitat alteration from the use of glyphosate, since nonendangered terrestrial or semi-aquatic plants were not considered to be at risk of direct 

effects. 

Based on Monsanto’s determination that threatened and endangered plant species may be 

at risk from certain uses of glyphosate in crop production (e.g., aerial application), a more 

detailed evaluation of the locations of threatened and endangered plant species relative to 

areas of crop production has been undertaken. The first crop to be assessed was alfalfa. 

The assessment process was divided into three phases, as outlined below. 

o First, the co-occurrence of observations of threatened and endangered plant 

species and the presence of alfalfa production was determined at the county 

level40,41. (Phase 1) This assessment considered the 2663 counties in which 

 

38 A water body with a depth of 2 m and a volume of 20,000 liters. 

39 U.S. Environmental Protection Agency. 2006. T-REX (Terrestrial Residue EXposure Model). Version 1.3.1. 

40 Priester T, Kemman R, Rives Frank A, Turner L, McGaughey B, Howes D, Giddings J, Dressel S. 2007. An 

Analysis of Possible Risk to Threatened and Endangered Plant Species Associated with Glyphosate Use in Alfalfa: A 

County-Level Analysis. Monsanto Study Number CS-2005-125. 

41 Priester T, Kemman R, Rives Frank A, Turner L, McGaughey B, Howes D, Giddings J, Dressel S. 2008. An 

Analysis of Possible Risk to Threatened and Endangered Plant Species Associated with Glyphosate Use in Alfalfa: 

A County-Level Analysis (Supplement). Monsanto Study Number CS-2007-229. 

 
Monsanto Company 09-SY-201U Page 430 of 471

alfalfa is grown, which comprise 85% of the 3141 counties and equivalent areas42

in the 50 states of the U.S. 

o Next, in counties with both threatened or endangered plant species observations 

and alfalfa production, the possible exposure of threatened and endangered plant 

species to glyphosate was assessed at the sub-county level. (Phase 2) This 

assessment used information available at the sub-county level for threatened and 

endangered plant species locations and for land use. Land uses considered in this 

assessment are identified as Pasture/Hay and Cultivated Crops.43

o Finally, in subcounty areas where, under certain application conditions, the 

potential for threatened and endangered plant species to be at risk from exposure 

to glyphosate could not be excluded, these areas have been defined so that grower 

practices can be implemented to limit glyphosate exposure. Measures to limit 

glyphosate exposure in these areas will be proposed. (Phase 3) These measures 

include (1) limiting ground application rates to less than 3.5 lb glyphosate a.e. per 

acre in areas identified for potential use limitation when the potential habitat for 

the threatened or endangered species is present, and (2) for aerial applications 

implementing an unsprayed buffer between the potential habitat for the listed 

species and the application area. Buffers are proposed to be based on application 

rate, droplet size and wind direction. 

This analysis has been completed for the 2663 U.S. counties in which alfalfa is grown. 

The evaluation of the remaining 478 counties of the U.S. is currently in progress, based 

on the presence of threatened or endangered plant species, the production of other crops 

in those counties, and the cultivated crop or pasture/hay land use. In counties that have 

observations of threatened and endangered plant species and agricultural land uses, a 

more detailed sub-county analysis will be conducted. 

Of the 2663 U.S. counties where alfalfa is produced, less than 11% of counties (284 

counties) have required the definition of potential areas for use limitations. In the other 

2379 counties, either there are no threatened or endangered plant species present, or the 

species present are either excluded from concern (based on habitat or proximity 

information), have existing protections, or are not in proximity to potential areas of 

alfalfa production that are not already excluded or protected in some way. 

The Roundup Ready alfalfa assessment considered all land that could potentially be used 

for agricultural crop production (in counties with reported alfalfa farms) in the 

assessment of proximity to observations of threatened or endangered species. Thus, the 

identification of potential use limitation areas also applies to other crops in those 

counties. Because alfalfa is grown in such a large number of counties, the endangered 

plant species assessment conducted for alfalfa covers more than 90% of the acres where 

corn, cotton, soybean, sugar beets and canola are grown. 

 

42 Equivalent areas include independent cities that are not within the boundaries of a county. 43 Land use was based on the National land Cover Database (2001) for the continental U.S. and on the NOAA (National 

Oceanic and Atmospheric Administration) Coastal Services Center land cover data for Hawaii.
Monsanto Company 09-SY-201U Page 431 of 471

O.3.6 Potential Effects on Endangered Animal Species Identified by EPA or in 

Litigation 

As previously discussed, no indirect effects on threatened or endangered animal species 

are predicted, since no significant direct effects due to pesticide drift onto nonendangered plant species are predicted. In the Glyphosate Reregistration Eligibility 

Decision (RED) (EPA, 1993), EPA suggested that glyphosate may have effects on the 

habitat of the Houston Toad. After the issuance of the 1993 Glyphosate RED, Monsanto 

conducted a vegetative vigor study. When relevant effects data from that study are 

considered, it can be determined that the amount of glyphosate per unit area predicted to 

drift away from the site of an agricultural application is less than the amount per unit area 

observed to have a 25% effect on plant dry weight or growth of the most sensitive of ten 

species tested in the study. Thus, the habitat of the toad is not likely to be significantly 

affected by glyphosate drift, and hence the toad is not likely to be at risk from the 

agricultural use of glyphosate. Similar conclusions can be drawn with respect to the 

habitat of other endangered animal species, such as the California red-legged frog (which 

has been the subject of years-long litigation between public interest groups and the 

federal government44). The assessment conducted also indicates that the frog itself is not 

at risk from aquatic exposures to glyphosate used in agriculture because estimated 

exposure concentrations are much lower than concentrations at which effects on 

amphibians have been observed. 

The EPA also has evaluated the potential effect of glyphosate on salmon in eleven areas 

in California and Southern Oregon45 in response to the consent agreement reached in 

another lawsuit46). The conclusion of the EPA’s risk assessment is as follows: 

“For all uses with application rates of 5 lb a.i. per A or below, the Agency has determined 

that glyphosate will have No Effect on the subject listed species.” (EPA, 2004b,c). All 

glyphosate use rates for agricultural uses are 5 lb a.i. per acre (3.75 lb glyphosate a.e. per 

acre) or below, so no risk to salmon is anticipated from these uses. 

O.3.7 Other Potential Environmental Impacts Associated with Glyphosate Use in 

Glyphosate-tolerant Crops 

As discussed more fully below, the potential impacts to soil attributable to the change in 

production (cultivation) practices associated with the deregulation of glyphosate-tolerant 

crops have been assessed. The adoption of glyphosate-tolerant crops and the ability to use 

glyphosate-based agricultural herbicides is not expected to significantly change 

agricultural practices, except to enable the adoption of no-till seeding practices. 

1. No-Till Practices: No-till production is the practice of establishing an agricultural 

seed bed and controlling weeds without mechanically tilling the soil. Instead, the only 

tillage of the soil is done at the time of planting, with the crop being seeded directly into 

the previous year’s crop residue. Among other environmental benefits, no-till production 

 

44 Center For Biological Diversity v. Leavitt, 2005 WL 2277030 (N.D.Cal., September 19, 2005). 45 These areas are call Evolutionarily Significant Units based on the salmonid populations present in these areas. 46 , Washington Toxics Coalition v. Environmental Protection Agency, 413 F.3d 1024 (9th Cir. 2005).
Monsanto Company 09-SY-201U Page 432 of 471

reduces soil erosion and the use of petroleum-based fuels for tractors. The practice has 

been shown to minimize surface water runoff and soil erosion and to improve soil quality 

by increasing the soil organic matter that helps bind soil nutrients and prevent their loss 

to runoff, erosion and leaching (Leep et al., 2003). Less soil erosion into surface waters 

would positively impact stream dynamics (McVay et al., 2005). 

No-till agriculture can provide benefits to water bodies, as well. No-till practices reduce 

soil erosion to surface water bodies, decreasing the amount of sediment in rivers and 

streams. Sedimentation increases the turbidity (cloudiness) of surface water bodies, 

reducing light penetration, impairing photosynthesis and altering oxygen levels, which 

cause a reduction of food sources for some aquatic organisms. Sediment can also cover 

spawning beds and impact fish populations. Phosphorus (a major component of fertilizer) 

bound to soil particles can be transferred to rivers and lakes via soil erosion, giving rise to 

high levels of phosphorus in surface waters, which may lead to algae blooms that can 

impact desirable fish populations (Hill et al., 1995). 

2. Soil Microorganisms: Soybean is a legume that forms a symbiotic relationship with 

the nitrogen-fixing bacterium Bradyrhizobiacea japonicum (see Section VIII.D.4). The 

effects of glyphosate and glyphosate-based formulations on soil microorganisms have 

been extensively investigated (Sullivan and Sullivan, 2000). Results of standardized tests 

with glyphosate formulations performed for submission to regulatory agencies indicate 

no long-term effects on microorganisms in soil even at rates that exceed maximum use 

rates (up to five times the labeled rate). In addition, independent researchers have 

reviewed numerous laboratory and field studies, investigating the effects of glyphosate on 

soil bacteria and fungi47 (Giesy et al., 2000). Although some laboratory tests have shown 

effects on nitrogen-fixing bacteria (Moorman et al., 1992; Santos and Flores, 1995) and 

soil fungi (Estok et al., 1989; Busse et al., 2001), effects are typically observed only 

under artificial laboratory conditions and at glyphosate concentrations well above normal 

field application rates. Several researchers have concluded that it is difficult to 

extrapolate results from the laboratory to the natural soil environment (Estok et al., 1989; 

Wan et al., 1998; Busse et al., 2001). 

In studying microorganisms from soil in pine plantations, Busse et al. (2001) state: “Our 

findings suggest that artificial media assays are of limited relevance in predicting 

glyphosate toxicity to soil organisms and that field rate applications of glyphosate should 

have little or no affect on soil microbial communities in ponderosa pine plantations.” 

Long-term studies following repeated applications of Roundup agricultural herbicides in 

the field for six (Olson and Lindwall, 1991) or over 10 years (Hart and Brookes, 1996; 

Biederbeck et al., 1997) have shown no detectable adverse effects on soil microbes. 

Investigations by Haney et al. (2000, 2002) related to the increased use of glyphosatetolerant crops indicate that glyphosate was degraded over time by soil microbes, even at 

high application rates, without adversely impacting the soil microbial community. In 

 

47 Felsot, A.S. 2001. Herbicide tolerant genes, Part 4: Withering wildlife? Agric. & Environ News, No. 178. 

http://www.aenews.wsu.edu/Feb01AENews/Feb01AENews.htm. 
Monsanto Company 09-SY-201U Page 433 of 471

addition, results from field studies that have evaluated the fungal component of the soil 

microbial community indicate that glyphosate treatment had no deleterious effects on 

beneficial soil fungi (Araujo et al., 2003; Biederbeck et al., 1997; Busse et al., 2001; 

Wardle and Parkinson, 1990a,b). Moreover, the history of safe use and yield data 

obtained for nearly 10 years of glyphosate-tolerant crop production, combined with incrop applications of glyphosate-based agricultural herbicides, reinforce the findings that 

soil microbes and microbially mediated processes are not adversely impacted by fieldrate applications of glyphosate. 

3. The Potential for Glyphosate Metal Chelation to Affect Soil Fertility: Plants are 

dependent on the uptake of a number of different metal cations from the soil for optimal 

growth. Glyphosate is known to chelate, or tightly bind, to several di- and trivalent metal 

cations such as Fe3+, Cu2+, Mn2+, Al3+, and Ca2+ that are needed by plants (Madsen et 

al., 1978; Glass, 1984). Cations that chelate glyphosate have been shown to reduce the 

efficacy of glyphosate when present in sufficient amounts in the tank mix spray 

solution48. In the spray solution, there is a simple interaction between glyphosate and 

metal cations, which reduces the herbicidal activity of glyphosate. However, in the soil 

environment, the interactions between metals and chelators are much more complex 

(Parker et al., 2005). Glyphosate can interact with metals that are present on the surface 

of soil particles, as well as with dissolved metal ions in the water soil solution. In 

addition to glyphosate, many other potential ligands or chelators are present in soil that 

can also interact with metals. As a result, there is a complex multi-component 

equilibrium between glyphosate, other ligands or chelators, and numerous metals present 

in soil. Glyphosate is only one factor in this system. Numerous compositional analysis 

studies have demonstrated a lack of any significant immobilization of mineral nutrients 

by glyphosate in soil that results in reduced uptake by plants. These studies have shown 

that glyphosate-tolerant crops that have been sprayed with glyphosate do not have 

decreased micronutrient levels compared to untreated controls (McCann et al., 2006; 

Obert et al., 2004; Ridley et al., 2002). 

4. Nitrogen Fixation: Nitrogen fixation is the process by which inorganic nitrogen is 

fixed into an organic form necessary for plant growth. It occurs in root nodules that are 

an integral part of the legume root system. The process of nitrogen fixation is subject to 

subtle variations depending on soil type and environmental conditions. While some 

laboratory investigations have indicated that glyphosate may inhibit pure cultures of 

nitrogen-fixing bacteria (Moorman et al.1992; Santos and Flores, 1995), effects were 

only observed at glyphosate concentrations above normal field application rates. 

Several researchers (King et al., 2001; Hoagland et al., 1999; Goos et al., 2002; 

Zablotowicz and Reddy, 2004) have investigated potential effects of glyphosate 

formulated herbicides on nitrogen-fixing bacteria associated with glyphosate-tolerant 

 

48 de Ruiter, H., R.A. Downer, A.J.M. Uffing, T.A. Ebert, P.J.J. Pikaar and F.R. Hall. 2001. The influence of inorganic 

cations on glyphosate activity - review and perspectives. American Society for Testing and Materials, 

West Conshohocken. 
Monsanto Company 09-SY-201U Page 434 of 471

soybean. In general, any effects observed on the formation of nodules or nitrogen 

fixation were not observed uniformly and were noted to be transient in nature. Hoagland 

et al. (1999) reported some reduction in nodulation in Roundup Ready soybean, but noted 

that effects were of minimal consequence due to the soybean’s ability to compensate for 

short durations of stress caused by environmental factors such as high or low 

temperature, water availability, or nutrient status. King et al. (2001) reported that 

application of Roundup Ultra® herbicide delayed nitrogen fixation and decreased 

nitrogen accumulation in some glyphosate-tolerant soybean cultivars. However, effects 

were only observed under drought conditions and at rates of glyphosate above the 

recommended label rates. The soybean yield was not affected. In a study which included 

four soybean varieties and four sites, Goos et al. (2002) reported that there was no 

indication Roundup Ultra herbicide inhibited nitrogen fixation, except that in one 

soybean variety at one site there was a small reduction in ureides, the principal products 

of nitrogen fixation in shoots of soybean. At recommended use rates, the application of 

glyphosate-based herbicides in the glyphosate-tolerant soybean production system is not 

expected to negatively affect soil fertility, nodule formation, or nitrogen fixation. 

5. Transport through the Soil – Surfactant: Available data also suggest that the POEA 

surfactant used in Roundup agricultural herbicides binds strongly to soil (estimated soil 

organic carbon-water partition coefficient (Koc) values range from 2500 to 960049) and 

undergoes microbial degradation with an estimated half-life of less than 14 days 50 . 

POEA is rapidly partitioned (half-life of 13 to 18 hours) from water to sediment in a 

water / sediment study (Wang et al., 2005). The rapid partitioning of the POEA 

surfactant to soil sediment combined with the high Koc values indicates that the 

surfactant will be tightly bound to the soil. The Groundwater Ubiquity Score, GUS, is an 

index that indicates the potential for compounds to leach from soil into groundwater, 

based on their half-life and Koc (Gustafson, 1989). Using an estimated half-life of 14 

days and a Koc of 2500 as conservative estimates of the rate of degradation and binding 

to soil, the GUS index for the POEA surfactant is 0.69. According to the GUS movement 

ranking, this GUS index indicates that POEA has a very low potential to leach to 

groundwater. 

 

 

® Roundup Ultra is a resistered trademark of Monsanto Technology LLC. 49 Estimated from the partition ratio between water and sterile soil as reported in the POEA soil degradation study, 

Marvel et al., 1974. 50 Marvel, J.T., Brightwell, B.B. and Suba, L.A. 1974. G3780A Surfactant: Biodegradation, plant uptake and 14C 

Distribution. Monsanto Company Report No. 321 
Monsanto Company 09-SY-201U Page 435 of 471

Attachment 1. Appendix O. U.S. Glyphosate Tolerances for Food & Feed 

Commodities (40 CFR § 180.364) 

Commodity Parts/million Commodity Partss/million 

Acerola 0.2 Durian 0.2 

Alfalfa, seed 0.5 Egg 0.05 

Almond, hulls 25 Epazote 1.3 

Aloe vera 0.5 Feijoa 0.2 

Ambarella 0.2 Fig 0.2 

Animal feed, nongrass, 

group 18 400 Fish 0.25 

Artichoke, globe 0.2 Flax, meal 8.0 

Asparagus 0.5 Flax, seed 4.0 

Atemoya 0.2 Fruit, citrus, group 10 0.5 

Avocado 0.2 Fruit, pome, group 11 0.2 

Bamboo, shoots 0.2 Fruit, stone, group 12 0.2 

Banana 0.2 Galangal, roots 0.2 

Barley, bran 30 Ginger, white, flower 0.2 

Barley, grain 20 Goat, kidney 4.0 

Beet, sugar, dried pulp 25 Goat, liver 0.5 

Beet, sugar, roots 10 Gourd, buffalo, seed 0.1 

Beet, sugar, tops 10 Governor's plum 0.2 

Berry group 13 0.2 Gow kee, leaves 0.2 

Betelnut 1.0 Grain, aspirated fractions 100 

Biriba 0.2 Grain, cereal, forage, fodder and straw, 

group 16, except corn forage 100 

Blimbe 0.2 Grain, cereal, group 15, except barley, 

field corn, grain sorghum, oat and wheat 0.1 

Borage, seed 0.1 Grape 0.2 

Breadfruit 0.2 Grass, forage, fodder and hay, group 17 300 

Cacao bean 0.2 Guava 0.2 

Cactus, fruit 0.5 Herbs subgroup 19A 0.2 

Cactus, pads 0.5 Hog, kidney 4.0 

Canistel 0.2 Hog, liver 0.5 

Canola, meal 15 Hop, dried cones 7.0 

Canola, seed 10 Horse, kidney 4.0 

Cattle, kidney 4.0 Horse, liver 0.5 

Cattle, liver 0.5 Ilama 0.2 

Chaya 1.0 Imbe 0.2 

Cherimoya 0.2 Imbu 0.2 

Citrus, dried pulp 1.5 Jackfruit 0.2 

Coconut 0.1 Jaboticaba 0.2 

Coffee, bean 1.0 Jojoba, seed 0.1 

Corn, field, forage 6.0 Juneberry 0.2 

Corn, field, grain 1.0 Kava, roots 0.2 

Cotton, gin byproducts 175 Kenaf, forage 200 

Cotton, undelinted seed 35 Kiwifruit 0.2 

Cranberry 0.2 Lesquerella, seed 0.1 

Crambe, seed 0.1 Leucaena, forage 200 

Custard apple 0.2 Lingonberry 0.2 

 
Monsanto Company 09-SY-201U Page 436 of 471

Commodity Parts/million Commodity Parts/million 

Date 0.2 Longan 0.2 

Dokudami 2.0 Lychee 0.2 

Mamey apple 0.2 Sapote, black 0.2 

Mango 0.2 Sapote, mamey 0.2 

Mangosteen 0.2 Sapote, white 0.2 

Marmaladebox 0.2 Sesame, seed 0.1 

Meadowfoam, seed 0.1 Sheep, kidney 4.0 

Mioga, flower 0.2 Sheep, liver 0.5 

Mustard, seed 0.1 Shellfish 3.0 

Noni 0.20 Sorghum, grain, grain 15 

Nut, pine 1.0 Soursop 0.2 

Nut, tree, group 14 1.0 Soybean, forage 100 

Oat, grain 20 Soybean, hay 200 

Okra 0.5 Soybean, hulls 100 

Olive 0.2 Soybean, seed 20 

Oregano, Mexican, leaves 2.0 Spanish lime 0.2 

Palm heart 0.2 Spearmint, tops 200 

Palm heart, leaves 0.2 Spice subgroup 19B 7.0 

Palm, oil 0.1 Star apple 0.2 

Papaya 0.2 Starfruit 0.2 

Papaya, mountain 0.2 Stevia, dried leaves 1.0 

Passionfruit 0.2 Strawberry 0.2 

Pawpaw 0.2 Sugar apple 0.2 

Pea, dry 8.0 Sugarcane, cane 2.0 

Peanut 0.1 Sugarcane, molasses 30 

Peanut, hay 0.5 Sunflower 85 

Pepper leaf, fresh leaves 0.2 Sunflower, seed 0.1 

Peppermint, tops 200 Surinam cherry 0.2 

Perilla, tops 1.8 Tamarind 0.2 

Persimmon 0.2 Tea, dried 1.0 

Pineapple 0.1 Tea, instant 7.0 

Pistachio 1.0 Teff, grain 5.0 

Pomegranate 0.2 Ti, leaves 0.2 

Poultry, meat 0.1 Ti, roots 0.2 

Poultry, meat byproducts 1.0 Ugli fruit 0.5 

Pulasan 0.2 Vegetable, leafy, brassica, group 5 0.2 

Quinoa, grain 5.0 Vegetable, bulb, group 3 0.2 

Rambutan 0.2 Vegetable, cucurbit, group 9 0.5 

Rapeseed, meal 15 Vegetable, foliage of legume, 

except soybean, subgroup 7A 0.2 

Rapeseed, seed 10 Vegetable, fruiting, group 8 0.1 

Rose apple 0.2 Vegetable, leafy, except brassica, 

group 4 0.2 

Safflower 85 Vegetable, leaves of root and 

tuber, group 2, except sugar beet 0.2 

Safflower, seed 0.1 Vegetable, legume, group 6, 

except soybean 5.0 

Salal 0.2 Vegetable, legume, group 6 except 

soybean and pea,dry 5.0 
Monsanto Company 09-SY-201U Page 437 of 471

Commodity Parts/million Commodity Parts/million 

Sapodilla 0.2 Vegetable, root and tuber, group 

1, except sugar beet 0.2 

Wasabi, roots 0.2 Wheat, grain 5.0 

Water spinach, tops 0.2 Wheat, middlings 20 

Watercress, upland 0.2 Wheat, shorts 20 

Wax jambu 0.2 Yacon, tuber 0.2 

Wheat, bran 20 

 
Monsanto Company 09-SY-201U Page 438 of 471

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Araujo, A.S.F., R.T.R. Monteiro, and R.B. Abarkeli8. 2003. Effect of glyphosate on the 

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on soil microbial activity and biomass. Weed Science 48:89-93. 

Haney, R.L., S.A. Senseman, and F.M. Hons. 2002. Effect of Roundup Ultra on 

microbial activity and biomass from selected soils. Journal of Environmental Quality 

31(3):730-735. 

Hart, M.R. and P.C. Brookes. 1996. Soil microbial biomass and mineralization of soil 

organic matter after 19 years of cumulative field applications of pesticides. Soil Biology 

and Biochemistry 28:1641-1649. 

Hill, P.R. and J.V. Mannering. 1995. Conservation tillage and water quality. Purdue 

University Cooperative Extension Service Publication WQ20. 

http://www.ces.purdue.edu/extmedia/WQ/WQ-20.html [Accessed July 2, 2009]. 

Hoagland, R.E., K.N. Reddy, and R.M. Zablotowicz. 1999. Effects of glyphosate on 

Bradyrhizobium japonicum interactions in Roundup Ready soybeans. Annual Meeting of 

the Weed Science Society of American, Vol. 39. 
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Kiely, T., D. Donaldson, and A. Grube. 2004. Pesticide industry sales and usage: 2000 

and 2001 market estimates. Biological and Economic Analysis Division, Office of 

Pesticide Programs, U.S. Environmental Protection Agency, Washington, D.C. 

King, C.A., L.C. Purcel, and E.D. Vories. 2001. Plant growth and nitrogenase activity of 

glyphosate-tolerant soybeans in response to foliar glyphosate applications. Agronomy 

Journal 93:179-186. 

Leep, R., D. Undersander, P. Peterson, D-H Min, T. Harrigan, and J. Grigar. 2003. Steps 

to successful no-till establishment of forages. Michigan State University, University of 

Wisconsin, University of Minnesota, and Natural Resources Conservation Service, 

Extension Bulletin E-2880. 

Madsen, H.E.L., H.H. Christensen, and C. Gottlieb-Petersen. 1978. Stability constants of 

copper (II), zinc, manganese (II), calcium, and magnesium complexes of N-

(phosphonomethyl) Glycine (glyphosate). Acta Chemica Scandinavica A 32:79-83. 

McCann, M.C., G.J. Rogan, S. Fitzpatrick, W.A. Trujillo, R. Sorbet, G.F. Hartnell, S.G. 

Riordan, and M.A. Nemeth. 2006. Glyphosate-tolerant alfalfa is compositionally 

equivalent to conventional alfalfa (Medicago sativa L.). Journal of Agriculture and Food 

Chemistry 54:7187-7192. 

McVay, K.A., D.L. Levlin, and J. Neel. 2005. Effects of conservation practices on water 

quality: Sediment. Kansas State University Agricultural Experiment Station and 

Cooperative Extension Service MF2682. 

Moorman, T.B., J.M. Becerril, J. Lydon, and S.O. Duke. 1992. Production of 

hydroxybenzoic acids by Bradyrhizobium japonicum strains after treatment with 

glyphosate. Journal of Agricultural and Food Chemistry 40:289-293. 

Obert, J.C., W.P. Ridley, R.W. Schneider, S.G. Riordan, M.A. Nemeth, W.A. Trujillo, 

M.L. Breeze, R. Sorbet, and J.C. Astwood. 2004. The composition of grain and forage 

from glyphosate-tolerant wheat MON 71800 is equivalent to that of conventional wheat 

(Triticum aestivum L.). Journal of Agricultural and Food Chemistry 53:1375-1384. 

Olson, B.M. and C.W. Lindwall. 1991. Soil microbial activity under chemical fallow 

conditions: Effects of 2,4-D and glyphosate. Soil Biology and Biochemistry 23(11):1071-

1075. 

Parker, D.R., S.M. Reichman, and D.E. Crowley. 2005. Metal chelation in the 

rhizosphere. Pages 57-93 in Roots and Soil Management: Interactions Between Roots 

and the Soil. American Society of Agronomy, Madison, Wisconsin. 

Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. 

Comparison of the nutritional profile of glyphosate-tolerant corn event NK603 with that 

of conventional corn (Zea mays L.). Journal of Agricultural and Food Chemistry 

50:7235-7243. 

Santos, A. and M. Flores. 1995. Effects of glyphosate on nitrogen fixation of free-living 

heterotrophic bacterial. Letters in Applied Microbiology 20:349-352. 

Sullivan, D.S. and T.P. Sullivan. 2000. Non-target impacts of the herbicide glyphosate: A 

compendium of references and abstracts. Applied Mammal Research Institute, 

Summerland, British Columbia. 
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Wan, M.T., J.E. Rahe, and R.G. Watts. 1998. A new technique for determining the 

sublethal toxicity of pesticides to the vesicular-arbuscular mycorrhizal fungus Glomus 

intraradices. Environmental Toxicology Chemistry 17(7):14-21. 

Wang, N., J.M. Besser, D.R. Buckler, J.L. Honegger, C.G. Ingersoll, B.T. Johnson, M.L. 

Kurtzweil, J. MacGregor, and M.J. McKee. 2005. Influence of sediment on the fate and 

toxicity of a polyethoxylated tallowamine surfactant system (MON 0818) in aquatic 

microcosms. Chemosphere 59:545-551. 

Wardle, D.A. and D. Parkinson. 1990a. Influence of the herbicide glyphosate on soil 

microbial community structure. Plant and Soil 122:21-28. 

Wardle, D.A. and D. Parkinson. 1990b. Effects of three herbicides on soil microbial 

biomass and activity. Plant and Soil 122:29-37. 

WHO/FAO. 2004. Pesticide residues in food — 2004. Report for the Joint Meeting of the 

FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO 

Core Assessment Group on Pesticide Residues (JMPR). Rome, September 20-29, 2004. 

FAO Plant Production and Protection Paper 178. World Health Organization and United 

Nations Food and Agricultural Organization, Rome. 

Zablotowicz, R.M. and K.N. Reddy. 2004. Impact of glyphosate on the Bradyrhizobium 

japonicum symbiosis with glyphosate-resistant transgenic soybean: A Minireview. 

Journal of Environmental Quallity 33:825-831. 

 
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Appendix P. Petitioner’s Environmental Assessment 

P.A. Background 

USDA-AHPIS has responsibility, under the Plant Protection Act (7 U.S.C. § 7701-7772), 

to prevent the introduction and dissemination of plant pests into the U.S. Additionally, 

USDA-APHIS must comply with the National Environmental Policy Act (NEPA) when 

making the decision whether to grant deregulated status to MON 87705. Numerous field 

trials conducted in the U.S. under APHIS notifications and permits since 2005 have 

included MON 87705 as test material. Information has been collected from these field 

trials, other tests, and the literature to assess whether the improved fatty acid profile 

through suppression of FATB and FAD2 RNAs, expression of the CP4 EPSPS enzyme to 

confer glyphosate tolerance and/or the plant transformation process has altered 

MON 87705 in any way that would impart plant pest characteristics or cause significant 

environmental impacts, including cumulative impacts. The purpose of this section is to 

provide relevant, trait-specific information regarding the potential for reasonably 

foreseeable, significant environmental impacts. 

An analysis of the potential impact of deregulation of MON 87705 on current soybean 

agronomic production systems, and related activities such as soybean processing, food 

and feed uses as well as marketing of soybean and soybean products is presented in this 

section. Factors evaluated as part of the assessment include potential impacts to: 

• land use patterns, non-agricultural lands, farming practices, commodity and 

specialty soybean production, 

• marketability of soybean seed for planting and seed for specialty and commodity 

markets, and 

• public health, non-target organisms, threatened or endangered species, and 

biodiversity. 

The analysis conducted considers current conditions, the potential for deregulation of 

MON 87705 to impact these conditions, and potential cumulative impacts. In most cases, 

there are no impacts to current conditions (e.g., no differences between deregulation of 

MON 87705 versus continuing to regulate). Where differences were noted, these 

differences are described and their significance evaluated. 

P.B. Purpose and Need 

Monsanto Company (Monsanto) is submitting to APHIS this petition for the 

determination of nonregulated status for MON 87705 plants genetically enhanced to 

suppress the endogenous FATB and FAD2 RNA in the developing soybean seed; thereby 

improving the fatty acid profile to contain lower saturated fat, higher oleic, and lower 

polyunsaturated fatty acid levels. MON 87705 soybean oil also has improved heat and 

oxidative stability and, due to the lower levels of saturated fats, a healthier profile relative 

to conventional soybean oil. MON 87705 also expresses CP4 EPSPS protein throughout 

the plant conferring tolerance to glyphosate, which is the active ingredient in the Roundup 

family of agricultural herbicides. 

Initially the U.S. market for soybean oil was severely limited by the presence of 

undesirable off-flavors and odors. The double bonds in the polyunsaturated fatty acids 
Monsanto Company 09-SY-201U Page 443 of 471

(PUFAs), particularly linolenic acid, are susceptible to oxidation resulting in the 

formation of “fishy” or acrid flavors and odors in soybean oil (Dutton et al., 1951). As a 

result, chemical hydrogenation was adopted to reduce the content of polyunsaturated fatty 

acids (Dutton, 1963; Okkerse et al., 1967). At first, hydrogenated vegetable oils, 

including hydrogenated soybean oil, were viewed as viable alternatives to animal fats and 

the high-in-saturated-fats tropical oils such as palm oil and coconut oil. However, in the 

1990s, nutrition research showed that the trans fatty acids in hydrogenated oils created by 

the hydrogenation process had negative health consequences (Judd et al., 1994; Mensink 

and Katan, 1990; Zock and Katan, 1992). By 2006, the U.S. FDA had issued a regulation 

obligating food manufacturers to declare the trans fatty acid content of their product on 

nutrition labeling (FDA, 2006). As a result, soybean oil per capita consumption in U.S. 

has been declining since 2006 (Soyatech, 2008). 

MON 87705 soybean oil fatty acid profile provides important new formulation options 

for food companies interested in the development of lower saturated fat food products to 

support heart health. Because MON 87705 soybean oil has a reduced level of PUFAs, it 

has higher oxidative stability without the need for hydrogenation, and has lower levels of 

saturated fats compard to commodity soybean. Saturated fats, notably palmitic acid 

(16:0), contribute to cardiovascular disease and other chronic diseases in epidemiological 

trials (Hu et al., 1997). As a result, reducing saturated fat levels in soybean oil can 

positively impact the goal of keeping human dietary consumption of saturated fats below 

10% of their total energy intake (USHHS, 2005). 

As mentioned, a reduction in saturated fats and increased oxidative stability in 

MON 87705 soybean oil contribute to an important new formulation option for food 

companies. The uses of soybean oil for biodiesel and other industrial applications is also 

enhanced by virtue of a modified oil profile similar to that found in MON 87705 soybean 

oil. Low saturated fats and high (>70%) oleic acid levels are key attributes for vegetable 

oils targeted for biodiesel and industrial uses because these characteristics are vital to 

improved cold weather performance, improved stability, and reduced nitrous oxide 

emissions (Knothe, 2005; Graef et al., 2009). 

Therefore, the decrease in saturated fats and PUFAs (17% vs. 60% FA), in MON 87705 

soybean oil provides important options for food companies to develop lower saturated fat 

foods with greater food functionality. In addition, MON 87705 soybean oil attributes 

provide key enhancements for biodiesel and industrial applications. 

P.C. Soybean Production 

The following section describes the setting for the proposed deregulation and provides 

the context for evaluating the intensity of the impact due to USDA-APHIS granting 

deregulated status to MON 87705. The proposed deregulation would be relevant to the 

production of an intensively cultivated row crop - soybean. Soybean is grown as a 

commercial crop in over 35 countries. In the United States, it is generally grown on 

greater than 70 million acres in at least 27 states with over a million acres grown in each 

of the following states: IA, IL, MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, 

NC, KY, TN (USDA-NASS, 2006). 

In recent years, there has been an increased demand by consumers and food processers 

for soybean and other oilseed crops that have specific physical or chemical characteristics 
Monsanto Company 09-SY-201U Page 444 of 471

to meet specific food or feed needs. Approximately 12% of soybean grown is specialty 

soybean produced for a specific market or use (United Soybean Board at 

http://www.unitedsoybean.org), including high protein, tofu, nonbiotechnology-derived, 

organic and high oleic and low linolenic acid content. The specialty, value-added 

product may be the whole bean or a fraction such as the oil. The vast majority of soybean 

in the U.S. is grown for animal feed and is fed as soybean meal. During processing, 

soybean oil is extracted to produce crude soybean oil and defatted soybean meal. 

According to a 1999 American Soybean Association sponsored study the oil content of 

soybean meal after extraction is relatively low, between 1-3%. 

(http://www.soymeal.org/worldlitarticles_new/globalsmsampling.html). 

Specialty soybean varieties are specified by buyers and end-users of soybean for 

production, and premiums are paid for delivering a product that meets purity and quality 

standards for the soybean variety. Product differentiation and market segmentation in the 

specialty soybean industry includes mechanisms to keep track of the soybean 

(traceability), methods for identity preservation (IDP), such as quality assurance 

processes (e.g., ISO9001-2000 certification), as well as contracts between growers and 

buyers that specify delivery agreements. Ultimately, the amount of planted acreage will 

be driven by consumer preferences based on the demand for the oil produced by 

MON 87705. On the basis of predicted demand for food and feed applications, and the 

additional health benefits and functionality of MON 87705 soybean oil, it is initially 

expected to be a specialty soybean produced for its premium oil. This premium is 

determined by the increased cost of production and distribution throughout the supply 

chain (mostly IDP costs) and market demand for the enhanced composition, which in the 

case of MON 87705 soybean oil is an improved fatty acid profile that is higher in oleic 

acid and lower in saturated fats than commodity soybean oil 

Data in Section VII of this petition show that, aside from the improved fatty acid profile 

present in the oil, the other processed fractions (meal, lecithin, and protein isolate) from 

MON 87705 seed are compositionally and nutritionally equivalent to nonbiotechnologyderived conventional processed fractions. 

This deregulation is being sought in an environment that has rapidly adopted 

biotechnology-derived soybean varieties (James, 2007). Thirteen different 

biotechnology-derived soybean events have been granted nonregulated status by USDA 

since 1994 (www.aphis.usda.gov). In particular, biotechnology-derived herbicidetolerant soybean varieties were grown on approximately 69 million of the 75 million 

acres of soybean grown in the U.S. in 2008 (USDA-NASS, 2008). Thus, soybean 

breeders, seed manufactures, and soybean producers have developed practices and 

systems that allow for the concurrent breeding and manufacture of biotechnology-derived 

and specialty soybean and are capable of breeding and manufacturing seed, and 

producing harvested seed to meet the needs of various markets. 

Traditional plant breeding has been used to alter the fatty acid profiles of oil producing 

plants such as soybean. For example, soybean varieties with lower levels (1-3%) of the 

polyunsaturated linolenic fatty acid have recently entered the market (Fehr, 2007) and 

programs to develop high oleic soybean through conventional breeding have resulted in 

soybean varieties with an oleic content of >70% (Alt et al., 2005). More recently 

soybeans with alterations in the fatty acid profile from genetic modifications have either 

been submitted to or deregulated by USDA-APHIS (Table P-1). 
Monsanto Company 09-SY-201U Page 445 of 471

Table P-1. Deregulated or Submitted Biotechnology-derived Soybean Products with 

Altered Oil Profiles 

Phenotype ID Code(s) Institution Date Deregulated 

Altered Oil Profile MON 87769 Monsanto Submitted 

High Oleic Acid DP-3Ø5423-1 Pioneer Submitted 

Altered Oil Profile G94-1, G94-19, G-168 DuPont May, 1997 

Source: http://www.aphis.usda.gov/brs/not_reg.html

The MON 87705 soybean oil improved fatty acid profile provides important new 

formulation options for food companies interested in the development of lower saturated 

fat food products to support heart health. Conventional soybean oil typically contains 60-

65% polyunsaturated fatty acids. Because MON 87705 soybean oil has a reduced level of 

polyunsaturated fatty acids, it has higher oxidative stability without the need for 

hydrogenation, and has lower levels of saturated fats compard to commodity soybean. 

The low saturated fat/high oleic oil produced from MON 87705 soybeans is suitable for 

use in products such as vegetable oils, salad dressings, margarine, etc., or in other 

applications for which commodity soybeans are used. Additionally, the use of soybean 

oil for biodiesel and other industrial applications also is enhanced by virtue of an 

improved fatty acid profile found in MON 87705 soybean oil. 

P.D. Potential Environmental Impacts 

The soybean production environment and MON 87705 are described in detail throughout 

this petition. The discussion of impacts below is focused on specific areas of the 

production environment and related activities including land use patterns, farming 

practices, commercial activities such as seed production and marketing of soybean and 

impacts to the quality of agricultural products. 

P.D.1. Impacts on Land Use 

Soybean is grown as a commercial crop on over 75 million acres in at least 27 states in the 

U.S. (USDA-NASS, 2009a). Soybean acreage in the past five years has been relatively 

stable varying from 64.7 million to 75.7 million acres with a 10-year average of 73.3 

million acres (Table IX-1). Fluctuations in soybean acreage are due to environmental, 

agronomic and economic factors1

, as well as government programs such as the crop 

reserve program (CRP) or ethanol mandates imposed by the U.S. government. Soybean 

fields are typically highly managed agricultural areas that can be expected to be dedicated 

to crop production for many years and cultivation of MON 87705 is not expected to 

differ from typical soybean cultivation practices. The improved fatty acid profile trait 

provides growers with an option to produce a value added soybean and potentially greater 

profitability from their farming operation (see Section IX). Additionally, canola and 

 

1

 2008 NASS crop acreage report [http://www.nass.usda.gov/Newsroom/2008/06_30_2008.asp] accessed 

6/2009 
Monsanto Company 09-SY-201U Page 446 of 471

sunflower production acreage has fluctuated due to complex market demands. During 

the period of 1998 through 2008, canola acreage has ranged from 865,000 to 1,555,000 

acres. Likewise, during the same period, sunflower acreage fluctuated between 246,869 

and 669,338 acres2

. MON 87705 will likely be used in common rotations on land 

previously used for agricultural purposes. 

There is no indication that the introduction and widespread adoption of biotechnologyderived crops in general has resulted in a significant change to the total U.S. acreage 

devoted to agricultural production. The cumulative land area in the U.S. planted to 

principal crops, which include corn, sorghum, oats, barley, winter wheat, rye, durum, 

spring wheat, rice, soybean, peanuts, sunflower, cotton, dry edible beans, potatoes, 

canola, proso millet, and sugar beets, has remained relatively constant over the past 25 

years. From 1983 to 1995, the average yearly acreage of principal crops was 328 million. 

This average is statistically unchanged at 326 million acres since the introduction of 

biotechnology-derived crops in 19963

. 

Granting deregulated status to MON 87705 is not expected to significantly alter 

commercial soybean cultivation in terms of agricultural inputs and production 

management (see Section IX). Currently, biotechnology-derived herbicide tolerant 

soybean is planted on 92% of the soybean acreage (USDA-NASS, 2008) and the 

Roundup Ready soybean system has become the standard weed control program in U.S. 

soybean production. Therefore the presence of the herbicide tolerance trait in 

MON 87705 does not offer a new or different incentive to growers that would be 

expected to significantly alter commercial soybean cultivation. 

A possible cumulative impact from the introduction of herbicide-tolerant soybean 

varieties is the increase in the practice of no-till or conservation tillage by U.S. growers. 

In 1995, before the introduction of glyphosate-tolerant soybeans, approximately 27% of 

the U.S. soybean acres used no-till production. By 2004, no-till acres increased to 36% 

of the total soybean acres (Sankula, 2006). A few states provide statistics on the adoption 

of no-till acres in their state. In 2006 in Illinois, no-till farming was used on 51% of the 

soybean acres. The University of Illinois Extension Service attributed that figure to the 

fact that 90% of the state’s acres were planted to glyphosate-tolerant varieties, along with 

other factors, such as high fuel prices, improved equipment, higher yields using no-till 

practices, and better grower awareness of the advantages to soil and water quality from 

no-till farming4

. For an overview of the cumulative impacts (including tillage) on land 

use from deregulation of glyphosate-tolerant crops see Appendix N. 

Additionally, because MON 87705, with the exception of the improved fatty acid profile, 

is phenotypically and agronomically equivalent to commercial soybean varieties, there is 

no expectation that introduction of MON 87705 will significantly alter the range of 

commercial soybean cultivation. Although it is not known how many acres will be 

planted to MON 87705, it is expected that this product and/or subsequent combinations 

 

2

 Comparisons of canola and sunflower acreage established from databases at 

http://usda.mannlib.cornell.edu/usda/current/htrcp/ 

3

 Calculated from database at http://usda.mannlib.cornell.edu/usda/current/htrcp by comparing the total 

acres from 1983 to 1995 and from 1996 to 2007. 

4

 University of Illinois Extension Servive News Release: No-till is now the "Conventional" Tillage System 

for Illinois Farmers; http://web.extension.uiuc.edu/state/newsdetail.cfm?NewsID=4991
Monsanto Company 09-SY-201U Page 447 of 471

of this product with other biotechnology-derived or conventional soybean varieties 

potentially could be grown on the majority of U.S. soybean acres. 

Because of the similar oil profiles, it is possible that the introduction of MON 87705 

and/or subsequent combinations of MON 87705 with other biotechnology-derived and 

conventional soybean varieties could impact demand for oil from other oilseed crops such 

as canola and sunflower. The decision to plant a crop is based on many factors including 

demand, input costs, environmental conditions and geography. Soybean acreage does not 

tend to overlap land typically used for canola and sunflower production in the U.S. 

(Figure P-1). Additionally, canola and sunflower production acreage has fluctuated due 

to complex market demands. During the period of 1998 through 2008, canola acreage 

has ranged from 865,000 to 1,555,000 acres. Likewise, during the same period, 

sunflower acreage fluctuated between 246,869 and 669,338 acres5

. Because of these 

geographic differences and complex market dynamics, it is unlikely that the introduction 

of MON 87705 will significantly impact the planting of other oil producing crops and 

should not result in significant changes to land use patterns. If, however, the demand for 

canola and sunflower oil were to decline, growers have the option to grow non-oil 

producing crops such as corn and wheat on acreage currently dedicated to canola and 

sunflower. 

Growers make planting decisions for all crops based on market demand, commodity 

prices, and the agricultural needs and practices specific to their acres. Because of this, it 

is not anticipated that the introduction of MON 87705 will result in a significant impact 

to canola and sunflower growers or to agricultural land use in those geographic areas 

where canola and sunflower are currently grown. 

 

5

 Comparisons of canola and sunflower acreage established from databases at 

http://usda.mannlib.cornell.edu/usda/current/htrcp/ 
Monsanto Company 09-SY-201U Page 448 of 471

Figure P-1. Harvested U.S. Acres of Canola, Sunflower and Soybean 

Source: 

http://www.agcensus.usda.gov/Publications/2007/Online_Highlights/Ag_Atlas_Maps/Cr

ops_and_Plants/index.asp 
Monsanto Company 09-SY-201U Page 449 of 471

P.D.2. Potential for Non-crop and Non-agricultural Impacts 

Soybean (Glycine max) does not grow in the wild in the U.S. (Hymowitz and Singh, 

1987; CFIA, 1996; OECD, 2000). Soybean does not grow and persist in unmanaged 

habitats and would not be expected to invade and/or persist in the natural environment, 

including streams, lakes, oceans or other aquatic environments. With the exception of 

suppression of the FATB and FAD2 RNAs resulting in an improved fatty acid profile, 

MON 87705 is similar to other commercial soybean varieties currently grown in the U.S. 

in that both MON 87705 and 92% of commercially grown soybean have the cp4 epsps

gene that confers tolerance to the herbicide glyphosate (USDA-NASS, 2008). Therefore, 

MON 87705 is expected to have no significant impact to non-agricultural lands and 

aquatic systems beyond those related to commercially grown soybean. There are no 

changes in agricultural inputs needed to produce a crop from MON 87705 relative to 

commodity and specialty soybean and specifically no altered pest or disease 

susceptibility. Hence, pesticide applications on MON 87705 that may result in drift 

impacting non-crop or non-agricultural lands would be comparable to those used on 

conventional or currently available soybean varieties. 

Because MON 87705 produces the CP4 EPSPS protein conferring tolerance to the 

herbicide glyphosate it is anticipated that glyphosate will be used to control weeds in 

fields planted with MON 87705. Glyphosate has been approved for use by the U.S. 

Environmental Protection Agency and food and feed tolerances have been established in 

the U.S. for its residues, since 1979. Glyphosate has a complete and comprehensive 

regulatory data base (toxicity, environmental fate, and ecological toxicity) that has been 

evaluated by EPA and it has been determined that “glyphosate will not pose unreasonable 

risks or adverse effects to humans or the environment” (EPA, 2002). More information 

on the impacts of glyphosate to animal and plant communities and threatened and 

endangered species may be found in Section P.D.7 of this appendix and Appendix O. 

Likewise a comprehensive discussion concerning the development of glyphosate resistant 

weeds may be found in Appendix L. 

P.D.3. Potential Impacts to Agricultural Practices 

MON 87705 has been shown to be no different from conventional soybean in its 

agronomic and ecological characteristics (see Sections VII, VIII, IX and X), and has the 

same levels of resistance to insects and diseases as commercial soybean. A summary of 

agronomic practices for soybean production is presented in Section IX of this petition. 

Because glyphosate-tolerant soybean is already grown on 92% of U.S. soybean acres 

(USDA-NASS, 2008), no significant impact is expected from the introduction of 

MON 87705 on current cultivation practices, including seeding, tillage, fertilizer and 

pesticide applications and crop rotation practices for soybean (Section IX). An analysis 

of expected impact to agricultural practices for commodity seed and certified seed 

production as well as potential impacts to specialty soybean production are discussed 

below. 
Monsanto Company 09-SY-201U Page 450 of 471

Potential Impact to Soybean Commodity and Specialty Soybean Production

Monsanto will seek regulatory approval for MON 87705 and its combinations, where 

required, with other biotechnology-derived soybean varieties in all key soybean import 

countries with a functioning regulatory system to to support the flow of international 

trade. MON 87705 would provide food manufacturers and consumers with a soybean oil 

having an improved fatty acid profile and greater thermal and oxidative stability for 

multiple uses. It would also provide growers with a higher value crop resulting in greater 

profit potential for U.S. soybean producers. Soybean growers and processors are 

accustomed to accommodating a diversity of specialty soybean varieties in their normal 

handling of commodity soybean. Currently specialty soybean varieties are produced on 

approximately 12% of the U.S. soybean acreage (see Section IX.B.2). Soybean growers 

have demonstrated an ability to provide customers with their soybean product of choice. 

As discussed in Section IX, MON 87705 will be produced using IDP practices consistent 

with those used for other specialty soybean varieties. MON 87705 will be segregated 

from commodity soybean by producers as part of their contract and to maintain the value 

of the crop. Because soybean is primarily a self-pollinated crop with minimal gene 

movement and due to the identity preservation production system, no significant impact 

to commodity or other specialty soybean production is anticipated, should APHIS grant 

nonregulated status to MON 87705. 

No cumulative impacts to commodity or other specialty soybean production are expected 

from deregulation. Soybean producers have demonstrated their ongoing ability to 

produce multiple specialty soybean varieties in a diverse marketplace. 

Potential Impact to Certified Seed Production 

Certified seed production is a carefully managed process (Section IX.B.4). MON 87705 

is not expected to impact certified seed production practices or production of other 

certified commodity or specialty soybean seed for reasons described in this section. 

If MON 87705 is deregulated, seed production could occur within production systems 

already developed by seed producers for certified seed varieties (see Section X). 

MON 87705 has been thoroughly characterized and (with the exception of the modified 

fatty acid profile) is not agronomically or phenotypically different from currently 

commercial soybean. The implementation of management practices to avoid pollen from 

a biotechnology-derived crop in organic, specialty or conventional soybean seed or 

commodity seed production operations is directly impacted by the nature of soybean 

pollination (Section IX.B.4). Soybean is a highly self-pollinated species that exhibits 

very low levels of outcrossing. Numerous studies on soybean cross-pollination have 

been conducted, and the published results, with and without supplemental pollinators, are 

summarized in Table X-1. Under natural conditions, cross-pollination among adjacent 

plants in a row or among plants in adjacent rows ranged from 0 to 6.3%. In experiments 

where supplemental pollinators (usually bees) were added to the experimental area, crosspollination ranged from 0.5 to 7.74% in adjacent plants or adjacent rows. However, 

cross-pollination does not occur at these levels over long distances. Cross-pollination 

rates decrease to less than 1.5% beyond one meter from the pollen source, and rapidly 
Monsanto Company 09-SY-201U Page 451 of 471

decrease with greater distances from the source. The following cross-pollination rates at 

extended distances have been reported: 0.02% at 8.2 m of separation (Caviness, 1966), 

0.05% at 5.4 m (Ray et al., 2003), and 0% at 6.5 m (Abud et al., 2003). Hence, certified 

soybean seed producers can and have effectively implemented practices (e.g., isolation 

distances during the growing season, equipment cleaning during harvest, and post-harvest 

separation of harvested seed) that allow them to maintain commercially acceptable levels 

of varietal purity. 

No cumulative effects are anticipated to certified seed production from deregulation of 

MON 87705. The use of management practices that prevent trait movement and 

comingling of soybean varieties has resulted in production of soybean varieties with 

improved genetics over time. As a result of the implementation of these management 

practices, growers today can choose from numerous varieties of soybean including those 

used to produce commodity and specialty soybean varieties. 

Potential Impacts to Organic Soybean Production

Organic soybean markets typically enjoy a market premium offsetting the additional 

production and record-keeping costs associated with this production method (see Section 

IX.B.2). Organic farming operations as described by the National Organic Program, 

which is administered by USDA’s Agricultural Marketing Service, requires organic 

production operations to have distinct, defined boundaries and buffer zones to prevent 

unintended contact with prohibited substances or products of excluded methods from 

adjoining land that is not under an organic production management plan. Organic 

production operations must also develop and maintain an organic production system plan 

approved by an accredited certifying agent. This plan enables the production operation to 

achieve and document compliance with the National Organic Standards, including the 

prohibition of the use of excluded methods. Excluded methods include a variety of 

methods used to genetically engineer organisms or influence their growth and 

development by means that are not possible under natural conditions or processes. The 

use of biotechnology such as that used to produce MON 87705 is an excluded method 

under the National Organic Program [7 C.F.R. § 205.2]. 

Organic certification involves oversight by an accredited certifying agent of the materials 

and practices used to produce or handle an organic agricultural product. This oversight 

includes an annual review of the certified operation’s organic system plan and on-site 

inspections of the certified operation and its records. Although the National Organic 

Standards prohibit the use of excluded methods, they do not require testing of inputs or 

products for the presence of excluded methods. The presence of a detectable residue of a 

product of excluded methods alone does not necessarily constitute a violation of the 

National Organic Standards. The unintentional presence of the products of excluded 

methods will not affect the status of an organic product or operation when the operation 

has not used excluded methods and has taken reasonable steps to avoid contact with the 

products of excluded methods as detailed in an approved organic system plan. Organic 
Monsanto Company 09-SY-201U Page 452 of 471

certification certifies that organic production and handling processes have been followed, 

not that the product itself is “free” from any particular substance. 

Production systems designed prior to the introduction of MON 87705 or even prior to the 

introduction of biotechnology-derived soybean have allowed for production of soybean to 

meet varied customer demands. In addition to the market segments that produce organic 

or conventional soybean, distinct identity-preserved specialty soybean with such traits as 

clear hilum or high protein have also been grown and successfully marketed for specific 

food uses in domestic and export markets for many years (Cui et al., 2004). The choice 

to grow biotechnology-derived, organic or conventional soybean depends on many 

factors and the dynamics of the marketplace. The dynamics of the marketplace, choice 

between various varieties of soybean, and the existing production practices will not be 

impacted by the introduction of MON 87705. 

Organic soybean producers utilize production practices designed to specifically avoid the 

presence of both soybean products using conventional herbicide or other pesticide 

treatments, as well as biotechnology-derived crops. These well established practices to 

avoid “excluded methods” will continue with the introduction of MON 87705 varieties. 

They include isolation zones, use of buffer rows surrounding the organic crop, adjusted 

planting dates and varietal selection (www.attra.ncat.org). The implementation of 

management practices to avoid pollen from a biotechnology-derived crop in organic or 

conventional soybean production operations is facilitated by the nature of soybean 

pollination. As noted previously in this petition, soybean is a highly self-pollinated 

species and exhibits very low level of outcrossing. Hence, organic or conventional 

soybean producers can and have effectively implemented practices (e.g., isolation during 

the growing season, equipment cleaning during harvest, and post-harvest separation of 

harvested seed) that allow them to avoid the presence of biotechnology-derived soybean 

and maintain organic or conventional production status. 

Currently, biotechnology-derived herbicide tolerant soybean is planted on 92% of the 

soybean acreage (USDA-NASS, 2008) and the Roundup Ready soybean system – that is, 

planting Roundup Ready soybean and applying glyphosate in crop – has become the most 

widely used weed control program in U.S. soybean production. Despite the high 

adoption rates of Roundup Ready soybean by growers, organic and conventional soybean 

production remains an option for farmers who choose to produce using these production 

practices or varieties of soybean. The decision to grow organic, conventional, or 

biotechnology-derived soybean varieties is typically an economic one based on market 

dynamics. Organic soybean producers and those growing conventional soybean for 

sensitive non-biotechnology markets typically enjoy a market premium offsetting the 

additional production and record-keeping costs. While the widespread adoption of 

Roundup Ready soybean has reduced the number of conventional soybean varieties that 

are available, conventional and organically produced soybean seed is currently available 

from numerous seed suppliers (Table P-1). Additional information on organic seed 

sources is provided though the U.S. Agricultural Marketing Service (AMS) at: 
Monsanto Company 09-SY-201U Page 453 of 471

www.ams.usda.gov. Thus, growers have a choice in the soybean variety they plant, and 

this is not expected to change with the introduction of MON 87705. 

Buyers recognize that where there are biotechnology-derived crop varieties on the 

market, as with soybean, a guarantee that a commodity crop is 100% “free” of 

biotechnology-derived material is not feasible based on the limitations of testing and 

sampling methodology (Born, 2005). While the presence of a biotechnology-derived 

product like MON 87705 is unlikely in instances where producers utilize production 

practices designed to avoid biotech presence, in some instances buyer allowances 

between 0.1 to 5% biotechnology-derived commodity soybean in organic soybean are 

specified. This also is consistent with the USDA National Organic Program allowing for 

detectable residues of excluded methods (including biotechnology-derived crop products) 

as long as the producer has taken steps to avoid those methods 

(www.ams.usda.gov/nop/Q&A.html). Similarly, international regulatory organizations 

have recognized that testing and sampling methodologies limit the ability to confirm that 

commodity or specialty soybean is 100% free of biotechnology-derived material. Thus, 

they have set allowable tolerances for this material in conventional products to support 

food labeling and traceability laws. These tolerances allow from 0.9% (European Union) 

up to 5% (Japan) of the food to be biotechnology-derived in products considered 

“conventional.” Levels above the threshold may trigger special labeling. Thus, de 

minimis levels of approved biotechnology-derived soybean mav be allowable in certified 

organic or conventional soybean. 

Table P-2. Organic and Conventional Soybean Seed Sources 

Organic Soybean Seed Sources*: Conventional Soybean Seed Sources 

Albert Lea Seed House AgVenture Seeds (modified oil) 

Blue River Hybrids Campbell Seed (modified oil) 

Golden Grains Becks Hybrids (food grade) 

Great Harvest Organics Monsanto (Asgrow) 

Greis Seed Farm Schillinger Seed 

Lancaster Ag Products Pioneer 

Lawler Farm Center Soy Genetics 

Prairie Gold Seeds Stewart Seed (modified oil) 

Superior Organic Grains, Ltd Stine Seed 

Walter Seed and Honey Co Syngenta - multiple brands 

 Terral Seed 

Various State Crop Improvement 

Organizations

* From: www.organicgrains.ncsu.edu
Monsanto Company 09-SY-201U Page 454 of 471

P.D.4. Potential Impacts to Raw or Processed Agricultural Commodities 

Within this petition, extensive data have been presented relating to plant growth 

parameters, disease susceptibility, insect susceptibility, and forage and seed composition 

of MON 87705 compared to conventional soybean varieties. These data indicate that 

there are no biologically relevant differences between MON 87705 and conventional 

soybean, except for the deliberate change in the fatty acid profile of the oil and its 

glyphosate tolerance. Biotechnology-derived soybean varieties like MON 87705 undergo 

a voluntary food and feed consultation process with the FDA prior to release on the 

market. Monsanto will complete this consultation process prior to a commercial 

introduction of MON 87705. 

From a grain quality standpoint (e.g., foreign material, damaged grain, etc; see 

www.gipsa.usda.gov for soybean grain standards), the soybean produced from 

MON 87705 varieties is expected to be of comparable quality to soybean commercially 

produced in the U.S. for commodity markets based on the data presented herein. The low 

saturated fat, high oleic acid oil produced from MON 87705 soybean is suitable for use in 

any application for which commodity soybean oil is currently used, including products 

such as vegetable oils, salad dressings, margarine, etc. 

As described elsewhere in this petition, MON 87705 and the low saturated fat, high oleic 

acid soybean oil produced from MON 87705 will be produced and processed utilizing an 

identity preservation (IDP) system to capture the food quality value of the oil. Because 

other low saturated fat, high oleic acid oils such as canola and sunflower are already 

present in the commodity stream and are routinely handled by processors using standard 

industry processing methods as described in Appendix K, the introduction of 

MON 87705 will require no new inventions or processes. Processors routinely perform 

analyses for oil quality and fatty acid composition that enable them to capture the value 

of specialty soybean oils, to determine the appropriate customers and uses for various 

soybean and other oils, and to create the appropriate and requested blends of such oils for 

particular uses. 

Soybean is typically processed into two major fractions, the oil and meal, and several 

minor fractions including lecithin and protein isolate. The MON 87705 oil fraction will 

have the intended low saturated fat, high oleic acid profile, and will be produced and 

utilized within an IDP system. With the exception of low level residual oil, the other 

processed fractions derived from MON 87705 are compositionally equivalent to 

processed fractions from commodity soybean, and will be utilized in a manner similar to 

commodity soybean processed fractions (see Section VII.C). The meal, and other non-oil 

processed fractions are intended to be distributed into the commodity stream and will be 

marketed and sold in the commodity market. Because of their compositional 

equivalence, it is not anticipated that deregulation of MON 87705 will have a significant 

impact on these commodity soybean processed fractions. 

During the development of MON 87705, Monsanto has engaged in dialogue with 

relevant grain handlers, processors and food companies regarding the uses and 
Monsanto Company 09-SY-201U Page 455 of 471

applications of low saturated fat, high oleic soybean oil. This dialogue has contributed 

to the understanding of the processing and use of MON 87705 soybean and the resulting 

oil and other fractions, and to the market and trade assessments set forth in this petition. 

Before implementing an IDP system for MON 87705-derived varieties, Monsanto will 

have additional dialogue with relevant stakeholders in the value chain to discuss and 

understand the potential impacts on commodity oils and processed soybean products 

from the IDP production, handling and use of MON 87705 soybean and its low saturated 

fat, high oleic soybean oil. Based on that dialogue and any resulting refinements to the 

market and trade assessments, Monsanto will take the following steps, where applicable 

and appropriate: 

1. refine plans for production, handling and processing of MON 87705-derived 

soybean varieties and the low saturated fat, high oleic soybean oil (the IDP 

system) to address valid concerns raised by the stakeholders 

2. assess potential impacts on processed soybean products due to the presence of 

low saturated fat, high oleic soybean oil in commodity oil, and collaborate 

with stakeholders to support the evaluations needed to assess when potential 

impacts might occur, what processes may be necessary to mitigate any 

potential negative impacts, and what potential positive impacts may occur 

based on the improved oil profile 

3. engage in industry outreach and education regarding the MON 87705 soybean 

and low saturated fat, high oleic soybean oil IDP system, including product 

handling and use 

4. make available prior to commercialization a detection method or testing 

regime that enables identity verification of MON 87705 for its intended use 

Based on our market and trade assessments, because MON 87705 soybean is appropriate 

for all the same uses as commodity soybean, the only likely potential impact on raw or 

processed commodities in the event MON 87705-derived varieties reach the commodity 

channel would be a reduction in saturated and polyunsaturated fats in commodity oil. 

This reduction in saturated and polyunsaturated fats would improve the functionality of 

the oil and not impact the suitability of the soybean oil for anticipated end uses. The only 

impact would be an improvement in the nutritional profile of the oil relative to 

commodity soybean oil. Based on this analysis, there would be no significant impacts on 

raw or processed soybean commodities due to USDA-APHIS granting nonregulated 

status to MON 87705. 

Based on this assessment, Monsanto does not anticipate any significant cumulative 

impacts on either raw or processed soybean commodities resulting from the deregulation 

of MON 87705. Food ingredient and food manufacturers routinely process, manage, 

handle and blend a wide variety of specialty oils derived from oilseed crops (including 

other modified oils produced by conventionally bred varieties or previously deregulated 

events) with no adverse or cumulative effects on their manufacturing processes or on the 

quality of their products. Similarly, food manufactures will integrate low saturated fat, 
Monsanto Company 09-SY-201U Page 456 of 471

high oleic soybean oil into their existing handling and manufacturing process, and are 

expected to have the equivalent experience with low saturated fat, high oleic soybean oil, 

resulting in no adverse or cumulative effects due to USDA-APHIS granting deregulated 

status to MON 87705. 

P.D.5. Potential Impacts on Commercial Use of Soybean 

The decision to deregulate MON 87705 would allow for breeding of this product with 

conventional and previously deregulated biotechnology-derived soybean varieties of 

diverse genetic backgrounds. These varieties will include commercial varieties with low 

linolenic acid levels which can further enhance the oxidative stability of the soybean oil. 

In addition, MON 87705 will be combined using traditional breeding methods with other 

biotechnology-derived traits, including glyphosate tolerance (MON 89788), to deliver the 

best agronomic platform to farmers. It is expected that breeders and certified seed 

producers would use MON 87705 to develop varieties and to supply seed for planned 

commercial markets in the U.S. Monsanto anticipates that commercial use of 

MON 87705 will include export of soybean and soybean products, and has described 

import approval submission plans elsewhere in the petition. Monsanto’s stewardship 

program for this product is described in Section IX.I. 

There are no statistically significant differences observed in yield between MON 87705 

and its conventional control (Table VIII-4). However, MON 87705 represents an 

additional trait to those commonly selected for by soybean breeders. Such traits, whether 

conventionally bred or biotechnology-derived, have the potential to impact yield gains 

(Fehr, 2007). Monsanto and its breeding company partners have aggressive breeding 

programs in place which utilize increased population size and yield testing, and advanced 

molecular selection tools. This program will ensure that MON 87705 yields are 

continually increasing comparable to conventional commodity soybean, thus providing a 

competitive commercial soybean alternative to U.S. growers. 

P.D.6. Potential Impacts to Human Health and Safety 

Most human interactions with soybean occur through agricultural production, industrial 

operations, or through consumption. Therefore, this health and safety discussion will 

focus on food and feed safety as well as safety of workers exposed to MON 87705. The 

low saturated fat, high oleic soybean oil derived from MON 87705 soybean oil is suitable 

for use in products such as vegetable oils, salad dressings, margarine, etc., or in any 

application for which commodity soybean oil is used. 

Under the Federal Food, Drug, and Cosmetic Act (FFDCA), it is the responsibility of 

food and feed manufacturers to ensure that the products they market are safe and properly 

labeled. Food and feed derived from MON 87705 must be in compliance with all 

applicable legal and regulatory requirements. Biotechnology-derived crops for food and 

feed use undergo a voluntary consultation process with the FDA prior to release onto the 

market. Although a voluntary process, Monsanto routinely completes a consultation with 
Monsanto Company 09-SY-201U Page 457 of 471

the FDA prior to placing a new biotechnology derived crop product on the market. A list 

of completed consultations on biotechnology-derived crop products is available at the 

FDA website. 

Human Health 

MON 87705 is genetically modified to suppress endogenous FATB and FAD2 RNAs in 

the developing soybean seed, thereby improving the fatty acid profile to contain lower 

saturated fat, higher oleic and lower linoleic fatty acid levels. Therefore, the improved 

soybean oil from MON 87705 has enhanced heat and oxidative stability, and, due to the 

lower levels of saturated fats, a healthier fatty acid profile relative to conventional 

soybean. 

MON 87705 contains only those fatty acids that are presently found in soybean oil. 

MON 87705 has a fatty acid profile that is comparable to commercial high oleic 

vegetable oils (e.g., high oleic canola, high oleic safflower, high oleic sunflower), 

traditional oils such as olive oil that has a long-history of consumption in the diet, and 

canola oil that obtained U.S. FDA GRAS status. 

Extensive compositional analyses of forage and seed were conducted on samples from 

replicated, multi-site field trials to compare the composition of MON 87705 to a 

conventional soybean control and to commercially available soybean varieties (see 

Section VII for details). The compositional analyses of MON 87705 confirmed the 

intended changes to four major soybean oil fatty acids. All other components analyzed in 

MON 87705 seed and forage were either not statistically significantly different compared 

to a conventional control, or, if significantly different, were within the 99% conventional 

soybean tolerance interval with the exception of eicosenoic acid. This value (0.34% FA) 

fell outside the tolerance intervals. However, eicosenoic acid has a history of safe 

consumption at higher levels in other commonly consumed vegetable oils such as canola 

oil (4.3% FA), peanut oil (1.7% FA), high oleic safflower oil (0.5% FA) and high oleic 

sunflower oil (0.5% FA) (Codex 2005). 

The Dietary Guidelines for Americans (USHHS, 2005) recommend that saturated fat 

intake should be kept below 10% of total daily caloric intake and below 7% for 

individuals with elevated low density lipoprotein (LDL) blood cholesterol. The Dietary 

Guidelines also recommend that most dietary fat should come from PUFA and MUFA 

sources, such as vegetable oils. When MUFAs, such as oleic acid, are substituted for 

saturated fat in the diet, LDL cholesterol decreases. Likewise, diets high in PUFAs, such 

as the omega-6 fatty acid linoleic acid, are associated with a favorable blood lipid profile, 

and diets that contain 5-10% of energy as omega-6 PUFAs may confer benefits on 

coronary artery disease mortality. 

MON 87705 soybean oil contains approximately 6% saturated fatty acids, approximately 

76% 18:1 oleic acid and approximately 10% 18:2 linoleic acids levels. The fatty acid 

profile of MON 87705 is similar to a variety of other widely consumed vegetable oils 
Monsanto Company 09-SY-201U Page 458 of 471

such as canola and olive oils (Table P-3) for which the U.S. FDA has approved a 

qualified health claim related to coronary heart disease.56 Fatty acid profiles similarly 

higher in oleic acid than conventional soybean oil are also found in other vegetable oils 

such as high oleic safflower and sunflower oils and in nuts such as filberts, almonds, 

pistachios and pecans (Gunstone, 1994). These edible fats and oils are available for 

consumption in a wide variety of products including shortenings, salad and cooking oils, 

margarine and mayonnaise. 

Table P-3. Comparison of Fatty Acid Profiles from Plant Sources 

Oil %* Saturated 

Fat

%* Oleic 

Acid (18:1)

%* Linoleic 

Acid (18:2)

%* Linolenic 

Acid (18:3)

%* PUFAs

Canola 6 57 26 10 32

MON 87705 

Soybean

6 76 10 7 17

Conventional 

Soybean

15 23 53 8 60

Olive 13 78 7 1 8

Palm 50 38 11 1 12

Coconut 92 6 2 0 2

Source: Padley et al., 1994. 

*% of total fatty acids 

A dietary assessment was conducted in which MON 87705 replaced conventional liquid 

soybean oil in salad dressings, margarine and spreads, mayonnaise and home use of 

liquid soybean oil. The assessment indicates that intake of the saturated fat (16:0 

palmitic acid) would decrease from 14.4 g/day to 13.8 g/day representing a decrease of 

3.8% on a mean per capita basis. A summary of this assessment can be found in 

Appendix M. A decrease in dietary saturated fat intake is consistent with current dietary 

recommendations (Lichtenstein et al., 2006; USHHS, 2005; WHO/FAO, 2003). There 

was an overall increase in the total daily intake of oleic acid from 27.3 g/day to 30.3 

g/day representing an increase of 10.8% on a mean per capita basis. The oleic acid 

increase post-replacement is expected, since, similar to olive oil or canola oil, the oleic 

acid fraction in MON 87705 soybean oil is higher than the commodity soybean oil it 

replaced. Oleic acid is not an essential fatty acid and no recommended intake level has 

been set by the Institute of Medicine of the National Academies (IOM, 2002); however, 

as stated above, it is recognized that there is an inverse relationship between the intake of 

MUFAs like oleic acid and the total cholesterol (TC):HDL cholesterol (HDL-C) 

concentration ratio. 

 

56 http://www.cfsan.fda.gov/~dms/qhccanol.html, http://www.cfsan.fda.gov/~dms/qhcolive.html 
Monsanto Company 09-SY-201U Page 459 of 471

In summary, the nutritional impact from the use of MON 87705 soybean oil in targeted 

foods under the intended conditions of use, is estimated to result in changes in fatty acid 

consumption that are in line with current dietary guidelines for fatty acid intake. 

Therefore, MON 87705 is regarded to be as safe and nutritious as conventional soybean 

for food use. 

MON 87705 produces a lower saturated fat, high oleic soybean oil that has improved 

oxidative stability and, due to the lower levels of saturated fats, a healthier fatty acid 

profile relative to comodity soybean oil. As discussed previously, Monsanto will provide 

the U.S. FDA with information on identity, function, and characterization of the genes, 

including expression of the gene products. Additionally, information on the safety of the 

improved fatty acid profile in MON 87705 soybean oil, including a dietary risk 

assessment, will be provided to the U.S. FDA for evaluation including a dietary risk 

assessment. On the basis of the assessment of laboratory data and scientific literature, it 

is reasonable to conclude that MON 87705 is safe for food and feed use. 

MON 87705 also expresses the CP4 EPSPS protein throughout the plant conferring 

tolerance to glyphosate, which is the active ingredient in the Roundup family of 

agricultural herbicides. It is structurally homologous to EPSPS proteins that are part of 

the amino acid synthesis pathway of all plants (Devine et al., 1993). The safety of any 

protein(s) newly introduced into a biotechnology-derived crop needs to be assessed (Delaney 

et al., 2008; ILSI, 2004). The safety of CP4 EPSPS protein present in biotechnologyderived crops has been extensively evaluated (Harrison et al., 1996). The U.S. EPA has 

also reviewed the safety of the CP4 EPSPS protein and has established a tolerance 

exemption for the protein and the genetic material necessary for its production in or on all 

raw agricultural commodities (40 CFR § 174.523). This exemption was based on a safety 

assessment that included rapid digestion in simulated gastric fluids, lack of homology to 

known toxins and allergens, and lack of toxicity in an acute oral mouse gavage study. A 

history of safe use is supported by the lack of any documented reports of adverse effects 

since the introduction of other Roundup Ready crops in 1996. 

Roundup Ready soybean is planted on 92% of U.S. soybean acreage (USDA-NASS, 

2008). Consequently glyphosate is the most widely used herbicide in soybean. The 

toxicology of glyphosate has been extensively reviewed. A summary of food and feed 

tolerances, regulatory approvals with associated dietary exposure assessments and recent 

chronic and short-term aggregate risk assessments for glyphosate may be found in 

Appendix O. 

Worker Safety 

In the agricultural production of soybean, growers may be exposed to pesticides during 

application of chemicals to crops. Planting Roundup Ready soybean and applying 

glyphosate in crop has become the standard weed control program in U.S. soybean 

production. MON 87705 will share that Roundup Ready weed management system, so 

any adoption of MON 87705 by growers currently planting Roundup Ready soybean 

would not significantly change the commercial soybean agronomic practices, or use of 
Monsanto Company 09-SY-201U Page 460 of 471

pesticides such as herbicides, associated with soybean production (see Section IX). 

Worker safety issues related to the use of pesticides during agricultural production of 

MON 87705 will remain the same. A comprehensive human safety evaluation and risk 

assessment concluded that glyphosate has low toxicity to mammals, is not a carcinogen, 

does not adversely affect reproduction and development, and does not bioaccumulate in 

mammals (Williams et al., 2000). This petition demonstrates that, other than the 

improved fatty acid profile of the soybean oil and tolerance to the herbicide glyphosate, 

MON 87705 is agronomically and phenotypically equivalent to conventional soybean. 

Thus the cumulative impacts due to agricultural management and processing practices of 

MON 87705 would be no different than those due to management practices of 

commercial soybean varieties. Similarly, there are no changes to industrial processing of 

MON 87705 relative to the processing of commercial soybean, including specialty 

soybean. 

P.D.7. Potential Impacts to Plant, Animal and Microbial Communities Including 

Threatened or Endangered Species and Biodiversity 

The following section addresses potential impacts due to deregulation of MON 87705 to 

plant and animal communities, including soil organisms. An overview of the potential 

impact of glyphosate in the environment may be found in Appendix O. 

 In assessing the potential impact to plant and animal communities, the potential for gene 

movement and introgression from MON 87705 was evaluated because movement and 

establishment of the gene and trait to related species could have indirect impacts to plant 

and animal communities that extend beyond the original recipient organism. Monsanto 

considered two primary issues: 1) the potential for gene flow and introgression, and 2) 

the potential impact of introgression. The genus Glycine has approximately nine species, 

with G. max being placed in the subgenus Soja along with one other species, G. soja. G. 

max is sexually compatible with only G. soja and no other Glycine species. G. max is the 

only Glycine species located in the United States. Therefore, the probability of gene flow 

and introgression of MON 87705 into other species in the U.S. is essentially zero 

(Stewart et al., 2003); thus, the potential impact of introgression of MON 87707 to 

sexually compatible relatives on plant and animal communities is nonexistent if USDAAPHIS were to grant the petition for nonregulated status. Additional discussion of the 

potential environmental impact due to gene movement may be found in Sections X.D. 

Animals 

Soybean production systems in agriculture are host to many animal species. Mammals 

and birds may seasonally consume grain, and invertebrates can feed on the plant during 

the entire growing season. Animals that feed primarily on soybean are seed-feeding 

insects and rodents found in agricultural fields. Rodents, such as mice or squirrels, may 

seasonally feed exclusively on soybean seeds. Thus, these animals may have a diet 

containing significant amounts of soybean seeds. Deer may also browse in soybean 

fields on the forage and on seed left after harvest. 
Monsanto Company 09-SY-201U Page 461 of 471

Agronomic practices used to produce MON 87705 will be the same as those used to 

produce other glyphosate-tolerant soybean, including Roundup Ready soybean, that were 

planted on 92% of U.S. soybean acreage in 2008 (USDA-NASS, 2008) so any switch by 

growers from their current glyphosate-tolerant soybean to MON 87705 would not result 

in significant impacts on wildlife compared to current soybean production. 

Potential impacts to animals would be primarily based on the effects of the introduced 

protein CP4 EPSPS and the suppression of endogenous FATB and FAD2 RNAs resulting 

in the improved fatty acid profile of the MON 87705 soybean oil. As discussed 

previously, there is no meaningful risk to animal or human health from dietary exposure to 

CP4 EPSPS or the improved fatty acid profile from MON 87705 soybean oil. There are no 

toxic properties associated with either the CP4 EPSPS protein or with the improved fatty 

acid profile produced by MON 87705. Furthermore, the composition of the seed, forage 

and meal produced by MON 87705 is unchanged from conventional soybean meal. This 

information indicates that there would be no negative effects to mammals that forage on 

MON 87705. Similarly, it is expected that there would be no impact to birds or other 

animals that may consume soybean forage or soybean seed. During field trials no 

changes in insect feeding damage were observed (see Section VIII) indicating similar 

insect susceptibility for MON 87705 compared to conventional soybean. As 

MON 87705 exhibits no toxic effects on animals or pollinators of other plants in or 

around fields cultivated with MON 87705, they will not be affected. 

Ninety-two percent of soybean acreage in the U.S. is herbicide-tolerant, with most of 

these acres planted with Roundup Ready soybean (USDA-NASS, 2008). Consequently, 

glyphosate is the most widely used herbicide in soybean production. A comprehensive 

human safety evaluation and risk assessment concluded that glyphosate has low toxicity 

to mammals, is not a carcinogen, does not adversely affect reproduction and 

development, and does not bioaccumulate in mammals (Williams et al., 2000; Appendix 

O). An ecotoxicological risk assessment concluded that the use of glyphosate does not 

pose an unreasonable risk of adverse effects to non-target species, such as birds and fish, 

when used according to label directions, nor does it pose an unreasonable risk of adverse 

effects to insects outside of the application area. On the basis of this analysis, 

deregulation of MON 87705 will not result in significant impacts on animals, including 

insects that live near or in soybean fields containing MON 87705. 

Plants 

Soybean production systems in agriculture are host to many plant species. Likewise, the 

environment surrounding a soybean field varies in plant composition depending on the 

region. In certain areas, soybean fields may be bordered by other soybean, corn or other 

crops; fields may also be surrounded by wooded and/or pasture/grassland areas, as well 

as aquatic environments. Therefore, the types of vegetation, including weeds, around a 

soybean field depend on the area where the soybean is planted. A variety of weeds dwell 

in and around soybean fields; those species will also vary depending on the region where 

the soybean is planted. 
Monsanto Company 09-SY-201U Page 462 of 471

If MON 87705 is granted nonregulated status, agricultural practices that are used for 

biotechnology-derived herbicide-tolerant commercial soybean grown on the majority of 

U.S. acreage would be used for plant management during the cultivation of MON 87705. 

MON 87705 does not exhibit characteristics associated with weedy growth and will not 

compete with plants found outside of agricultural production. Weeds within fields of 

MON 87705 will be managed using mechanical, cultural, and chemical control measures, 

as weeds are now managed in commercial soybean systems. Monsanto’s glyphosate 

label provides information regarding appropriate conditions for application of Roundup 

herbicide agricultural herbicides that are designed to minimize damage to adjacent 

vegetation. Therefore, the presence of the herbicide- tolerance trait in MON 87705 and 

the improved fatty acid profile trait is not expected to have a significant impact on 

surrounding plant communities. 

Threatened and Endangered Species 

No significant impact to threatened and endangered species is expected from the 

introduction of MON 87705. Monsanto has considered the potential impact of 

MON 87705 on federally listed Threatened or Endangered Species (TES) and species 

proposed for listing, as provided under Section 7 of the Endangered Species Act. In this 

analysis, Monsanto considered the biology of MON 87705, as well as typical agricultural 

practices associated with cultivation of soybean. As previously noted, consumption of 

the CP4 EPSPS protein has no toxicity in laboratory testing with mice (see Section 

X.B.1.). MON 87705 does not express any additional proteins or natural toxicants that 

are known to directly or indirectly affect a listed TES or species proposed for listing by 

the U.S. Fish and Wildlife Service. Additionally, MON 87705 contains only those fatty 

acids that are presently found in soybean oil. MON 87705 has a fatty acid profile that is 

comparable to commercial high oleic vegetable oils (e.g., high oleic canola, high oleic 

safflower, high oleic sunflower), that are currently grown inthe U.S. 

MON 87705 is not sexually compatible with a federally listed TES or a species proposed 

for listing. The only TES animal listed that occupies habitat that is likely to include 

soybean fields and that might feed on soybean is the federally Endangered Delmarva 

Peninsula Fox Squirrel, (Sciurus niger cinereus), found in areas of the mid-Atlantic 

Eastern seaboard2

. It is known to utilize certain agricultural lands readily, and its diet 

includes acorns, nuts/seeds of hickory, beech, walnut, and loblolly pine; buds and flowers 

of trees, fungi, insects, fruit, and an occasional bird egg. Given all these factors and the 

lack of noted adverse effects of the CP4 EPSPS protein and MON 87705 on mice and 

other non-target organisms, respectively, it is concluded that MON 87705 will not have 

an effect on the Delmarva Peninsula Fox Squirrel. 

No impact to any threatened or endangered plant species is expected from the cultivation 

of MON 87705. Like other G. max, MON 87705 will likely be a poor competitor with 

native vegetation, has no sexually-compatible relatives in the U.S. and will not survive 

 

2

 http://ecos.fws.gov/tess_public/SpeciesReport.do; [Accessed May 14, 2009]. 
Monsanto Company 09-SY-201U Page 463 of 471

outside of cultivation. Thus, there is no opportunity for MON 87705 to interbreed with 

any plant species or displace natural vegetation in the U.S. 

In a TES risk assessment previously provided to USDA-APHIS in support of Roundup 

Ready alfalfa petition 04-110-01p Monsanto identified some plant but no animal species 

that may be at risk from the use of glyphosate-based herbicides in the Roundup Ready 

crop system this assessment is summarized in Appendix O. To mitigate these potential 

risks, Monsanto developed Pre-Serve (www.pre-serve.org), a web-based program 

designed to protect TES plant species from potential impacts resulting from the 

agricultural use of herbicides that contain glyphosate. Pre-Serve instructs growers to 

observe specific precautions when spraying glyphosate herbicides on Roundup Ready 

crops near TES plant species that may be at risk. 

Soil Microorganism 

No adverse effects on soil microorganisms are associated with MON 87705 nor do the 

characteristics of the CP4 EPSPS protein or the improved fatty acid profile pose any 

concern to soil microorganisms. Monsanto presented data in this petition (see Section 

VIII) demonstrating the lack of impact to symbiotic microbes associated with soybean 

plants. The B. japonicum-soybean symbiosis of MON 87705 was not changed as a result 

of the introduction of the CP4 EPSPS protein and the improved fatty acid profile 

compared to a conventional soybean control. MON 87705 contains only those fatty acids 

that are presently found in soybean oil, and has a fatty acid profile that is comparable to 

other commercial high oleic vegetable oils. The fatty acids found in MON 87705 have a 

long history of safe use in human and animal consumption, are naturally present in the 

environment, and will be broken down and utilized by soil microorganisms in a manner 

similar to fatty acids found in other food crops. 

On the basis of these observations and in conjunction with related phenotypic 

measurements for MON 87705, no impact on soil microorganisms or soil arthropods is 

expected from deregulation of MON 87705.

Biodiversity 

Analysis of available information indicates that MON 87705 exhibits no traits that would 

cause increased weediness, that its unconfined cultivation would not lead to increased 

weediness of other sexually compatible relatives (of which there are none in the United 

States), and it is unlikely to have effects on non-target organisms common to agricultural 

ecosystems or threatened or endangered species recognized by the U.S. Fish and Wildlife 

Service. The use of herbicides in agricultural fields is likely to indirectly impact 

biodiversity by decreasing weed species present in the field. Agricultural fields are 

purposefully managed to be weed-free resulting in greater economic benefit to the 

grower. Ninety-six percent of soybean acreage in the U.S. was treated with glyphosate in 

2006 (USDA-ERS, 2007). Therefore introduction of MON 87705 is unlikely to affect 

the animal or plant communities found in commercial soybean production systems due to 

both the lack of toxicity of either the CP4 EPSPS protein or the improved fatty acid 

profile. Because MON 87705 will likely replace soybean varieties that already have 
Monsanto Company 09-SY-201U Page 464 of 471

glyphosate tolerance, no significant impacts from its introduction are anticipated. For an 

overview of cumulative impacts from deregulation of glyphosate-tolerant crops see 

Appendix N. Based on this analysis, it is concluded that deregulation of soybean 

varieities containing MON 87705 would have no significant impact on biodiversity. 

No impacts to plant and animal communities or threatened and endangered species have 

been identified for MON 87705. No cumulative impacts to plant and animal 

communities or threatened and endangered species have been identified. 

P.D.8. Other Cumulative Effects 

Conventional Breeding with Other Biotechnology-derived or Conventional Soybean 

Varieties 

As previously mentioned, several biotechnology-derived soybean crop varieties have 

been deregulated or are under consideration for deregulation, and a list of the events 

codes deregulated by USDA is presented in Table P-5 below. MON 87705 may be bred 

with these deregulated biotechnology-derived soybean crop products as well as with 

conventional soybean, creating new improved varieties. APHIS has determined that none 

of the biotechnology-derived individual soybean products it has deregulated display 

increased plant pest characteristics and that any progeny derived from crosses of these 

soybean crop products with other conventional or biotechnology-derived soybean are 

unlikely to exhibit new plant pest properties. 

An assessment of the stability of the genetic insert in MON 87705 was conducted, and 

data have been presented in this petition demonstrating that MON 87705 is stable in 

progeny. Having established that the genetic material is stable and that MON 87705 is 

inherited in a Mendelian fashion, and based on experience with MON 87705 in 

Monsanto’s plant breeding program, it can be concluded that the phenotype of 

MON 87705 is likewise stable. Conventional breeding has an established history of safe 

use, and use of MON 87705 in breeding programs is expected to behave in a manner 

similar to other conventional traits and biotechnology-derived traits. For example, in an 

assessment by McCann et al. (2005), it was shown that during three years of breeding 

into multiple varieties, the composition of glyphosate-tolerant soybean remained 

equivalent to that of conventional soybean. Given that there have been no plant pest 

characteristics associated with MON 87705, or with any of the previously deregulated 

events listed below, no significant impacts are expected to other soybean varieties 

through the use of MON 87705 in breeding programs and in combination with any of the 

previously deregulated soybean crop products. 

All biotechnology-derived soybean products on the market today have satisfactorily 

completed the FDA consultation process established to review the safety of foods and 

feeds derived from biotechnology-derived crops for human and animal consumption (see 

Table P-5). As mentioned above, breeding with modified oil soybean products on this 

list, while not currently envisioned, would predictably alter the fatty acid composition of 

MON 87705 (i.e., modify the oil profile to a combination of the individual trait 
Monsanto Company 09-SY-201U Page 465 of 471

characteristics). No impacts to public health (e.g., food or feed safety) are expected due to 

combination of these events through conventional breeding because the deregulated 

events have a history of safe use and on the basis of knowledge of the type of 

modifications made to each of the deregulated events, the biochemical pathways are not 

likely to unexpectedly interact or result in the production of novel constituents. 

The decision to deregulate MON 87705 would also allow for breeding of this product 

with conventional soybean varieties of diverse genetic backgrounds. These varieties will 

include commercial varieties with low linolenic acid levels which can further enhance the 

oxidative stability of the soybean oil. In addition, MON 87705 will be combined using 

traditional breeding methods with other biotechnology-derived traits, including 

glyphosate tolerant MON 89788. This combined trait product will incorporate the 

improved fatty acid profile of MON 87705 with the superior germplasm and 4-7% yield 

advantage of MON 89788 to deliver the best agronomic platform to farmers. No impacts 

to public health (e.g., food or feed safety) or environmental safety are expected due to the 

breeding of MON 87705 with these other soybean varieties because these varieties have 

an established history of safe use. 

Furthermore, the process of conventional breeding to combine biotechnology-derived 

traits or biotechnology-derived and conventional varieties to produce combined trait 

products would likely identify and remove off-types during development of new 

varieties. Breeders use standard testing and assessment procedures to further examine 

and confirm the equivalence of the combined trait products, compared to the single event 

products, in terms of phenotypes, agronomic characteristics, and the efficacy of the traits. 

Table P-5. Deregulated or Submitted Biotechnology-derived Soybean Products 

Phenotype ID Code(s) Institution Date Deregulated 

High Oleic Acid DP-3Ø5423-1 Pioneer Submitted 

Glyphosate Tolerant MON 89788 Monsanto February, 2007 

Phosphinothricin 

Tolerant 

GU262 AgrEvo October, 1998 

Phosphinothricin 

Tolerant 

A5547-127 AgrEvo May, 1998 

Altered Oil Profile G94-1, G94-19, G-168 DuPont May, 1997 

Phosphinothricin 

Tolerant 

W62, W98, A2704-12, 

A2704-21, A5547-35 

AgrEvo August, 1996 

Glyphosate tolerant 40-3-2 Monsanto May, 1994 

Source: http://www.aphis.usda.gov/brs/not_reg.html

Economic Impacts of Biotechnology-derived Products 

In a recent study, economists Brookes and Barfoot (2006) quantified the cumulative 

economic and environmental impacts of biotechnology-derived crops grown during the 

past eleven years (1996-2006). The authors report that biotechnology-derived crops have 
Monsanto Company 09-SY-201U Page 466 of 471

resulted in substantial global economic and environmental benefits. In the U.S. the 

impact of herbicide-tolerant soybean has primarily been to reduce the cost of production. 

In the early years of adoption the savings were between $25-34/ha. In more recent years 

the cost savings have risen to $60-78/ha based on comparison of herbicide regimes that 

would be required to deliver a comparable level of weed control. It is estimated that the 

cumulative U.S. farm income benefit from 1996-2006 was approximately $8.73 billion 

USD. Other soybean production countries (Argentina, Brazil, Canada, South Africa) have 

also experienced cost savings from the adoption of biotechnology-derived herbicidetolerant soybean varieties. In addition to cost savings, countries like Romania and 

Mexico have also experienced yield gains (31% and 9%, respectively) from 

improvements in weed control. 

It is not known how many U.S. acres MON 87705 will be grown on when 

commercialized. However, MON 87705 may provide benefits to U.S. soybean producers 

similar to herbicide-tolerant biotechnology-derived soybean commercialized on broad 

acres across the U.S., and provide growers with another high-value specialty soybean 

product option. 

P.E. Highly Uncertain, Unique or Unknown Risks 

MON 87705 has been thoroughly characterized and data submitted in the petition 

demonstrate that it poses no increased plant pest risk compared to conventional soybean. 

USDA-APHIS has previously deregulated thirteen biotechnology-derived soybean crop 

products. MON 87705 offers a low saturated fat, high oleic soybean oil alternative that 

has significantly improved heat and oxidative stability. Introduction of previous 

biotechnology-derived crops have resulted in no unexpected effects on the quality of the 

human environment as defined under NEPA and have provided benefits to growers, 

consumers and the environment. In this respect, a decision to deregulate a new 

biotechnology-derived soybean product is not precedent setting nor are the effects to the 

quality of the human environment highly uncertain or unpredictable. 

P.F. Summary 

MON 87705 has been thoroughly characterized and the extensive body of information 

presented in Sections I through X of this petition demonstrates that MON 87705 does not 

present a plant pest risk, has no significant impact on threatened or endangered species or 

biodiversity, and will not impact the commercial interests of soybean producers or those 

involved in the marketing and sale of soybean and soybean products. The introduction 

and adoption of specialty soybean crop products have benefited farm income in the U.S. 

The amount of land devoted to farming (specifically to corn or soybean) has not changed 

with the introduction of biotechnology-derived crops. Similarly, no significant change in 

the use of agricultural land or amount of land devoted to farming would be expected to 

occur with the commercial introduction of MON 87705. MON 87705 will utilize 

common cultivation practices typically employed for production of commodity soybean 

and many types of specialty soybean, and management practices typically used for 
Monsanto Company 09-SY-201U Page 467 of 471

specialty soybean (i.e., IDP production practices). Hence, agricultural practices would 

not be impacted if MON 87705 were deregulated. 

The opportunity for growers to produce soybean with a low saturated fat, high oleic acid 

fatty acid profile that has significantly improved heat and oxidative stability relative to 

conventional soybean is a positive benefit from an economic and human health 

perspective. Additionally, MON 87705 soybean and its fractions have, other than the 

intended changes to fatty acid composition, been demonstrated to be equivalent to 

conventional soybean. Therefore, MON 87705 soybean and its fractions are as safe and 

as wholesome for food and feed purposes as conventional soybean. For these reasons, 

the proposed action to grant nonregulated status to MON 87705 does not represent a 

significant impact to the environment. 
Monsanto Company 09-SY-201U Page 468 of 471

Appendix P References 

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oleate content in three soybean populations. Crop Science 45:2005-2007. 

Born, H. 2005. Marketing organic grains. http://www.attra.ncat.org/attrapub/PDF/marketingorganicgrains.pdf [Accessed May 19, 2009]. 

Brookes, G and P. Barfoot. 2006. Global impact of biotech crops: socio economic and 

environmental effects 1996-2006. PG Economics Ltd., UK. 

CFIA. 1996. The biology of Glycine max (L.) merr. (soybean). Biology Document BIO1996-10, O. Canadian Food Inspection Agency, Ontario 

Codex. 2005. Codex Standard for named vegetable oils. Codex Alimentarus. 

Cui, Z., A.T. James, S. Miyazaki, R.F. Wilson, and T.E. Carter Jr. 2004. The breeding of 

specialty soybeans for traditional and new soyfoods. Pp 264-322 in Proceedings of the 

American Oil Chemists' Society. 

Delaney, B., J.D. Astwood, H. Cunny, R.E. Conn, C. Herouet-Guicheney, S. Macintosh, 

L.S. Meyer, L. Privalle, Y. Gao, J. Mattsson, and M. Levine. 2008. Evaluation of protein 

safety in the context of agricultural biotechnology. Food and Chemical Toxicology. 46 

Suppl 2:S71-97. 

Devine. M.D., S.O. Duke and C. Fedtke. 1993. Inhibition of Amino Acid Biosynthesis. 

Pages 251-294 in Physiology of Herbicide Action. PTR Prentice-Hall, Inc., Englewood 

Cliffs, New Jersey. 

Dutton, H.J. 1963. Kinetics of linolenate hydrogenation. Journal of the American Oil 

Chemists’ Society 40:35-39. 

Dutton, H.J., C.R. Lancaster, C.D. Evans, and J.C. Cowan. 1951. The flavor problem of 

soybean oil. VIII. Linolenic Acid. Journal of the American Oil Chemists’ Society 

28:115-118. 

EPA. 2002. Biopesticides registration action document (Bacillus thuringiensis Cry2Ab2 

protein and its genetic material necessary for its production in cotton). U.S. 

Environmental Protection Agency Chemical PC Code 006487. 

FDA. 2006. Food Labeling: Trans Fatty Acids in Nutrition Labeling, Nutrient Content 

Claims, and Health Claims. 
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Fehr, W.R. 2007. Breeding for modified fatty acid composition in Soybean. Crop Science 

47(S3):S72–S87 

Graef, G., B.J. LaVallee, P. Tenopir, M. Tat, B. Schweiger, A.J. Kinney, J.H.V. Gerpen, 

and T.E. Clemente. 2009. A high-oleic-acid and low-palmitic-acid soybean: Agronomic 

performance and evaluation as a feedstock for biodiesel. Plant Biotechnology Journal

7:411-421. 

Gunstone, F.D. 1994. Fatty acid structure. Pages 1-20 in The Lipid Handbook. 2nd ed. 

F.D. Gunstone, J.L. Harwood and F.B. Padley (eds.). Chapman & Hall, Cambridge, Great 

Btitain. 

Harrison, L.A., M.R. Bailey, M.W. Naylor, J.E. Ream, B.G. Hammond, D.L. Nida, B.L. 

Burnette, T.E. Nickson, T.A. Mitsky, M.L. Taylor, R.L. Fuchs, and S.R. Padgette. 1996. 

The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-

phophate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is 

not toxic to acutely gavaged mice. Journal of Nutrition 126: 728-740. 

Hu, F.B., M.J. Stampfer, J.E. Manson, E. Rimm, G.A. Colditz, B.A. Rosner, C.H. 

Hennekens, and W.C. Willett. 1997. Dietary fat intake and the risk of coronary heart 

disease in women. The New England Journal of Medicine 337(21):1491- 1492. 

Hymowitz, T., and R.J. Singh. 1987. Taxonomy and Speciation. Soybean Monograph, 

Soybeans: Improvement, Production and Uses:23-48 

ILSI. 2004. Nutritional and Safety Assessments of Foods and Feeds Nutritionally 

Improved through Biotechnology. http://www.ift.org/cms/?pid=1000362. 

IOM. 2002. Dietary fats: Total fats and fatty acids. Pages 8-57 and 422-541 in Dietary 

Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein 

and Amino Acids. Panel on Macronutrients, . Institute of Medicine. National Academies 

Press, Washington D.C. 

James, C. 2007. Global status of commercialized biotech/GM crops: 2007. International 

Service for the Acquisition of Agri-biotech Applications. Cornell University, Ithaca, 

New York. 

Judd, J.T., B.A. Clevidence, R.A. Muesing, J.Wittes, M.E. Sunkin, and J.J. Podczasy. 

1994. Dietary trans fatty acids: Effects on plasma lipids and lipoproteins of healthy men 

and women. American Journal of Clinical Nutrition 59:861-868. 

Knothe, G. 2005. Dependence of biodiesel fuel properties on the structure of fatty acid 

alkyl esters. Fuel Processing Technology 86:1059-1070 
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Lichtenstein, A.H., L.J. Appel, M. Brands, M. Carnethon, S. Daniels, H.A. Franch, B. 

Franklin, P. Kris-Etherton, W.S. Harris, B. Howard, N. Karanja, M. Lefevre, L. Rudel, F. 

Sacks, L. Van Horn, M. Winston, and J. Wylie-Rosett. 2006. Diet and lifestyle 

recommendations revision 2006: a scientific statement from the American Heart 

Association Nutrition Committee. Circulation 114:82-96. 

McCann, M.C., K. Liu, W.A. Trujillo and R.C. Dobert. 2005. Glyphosate-tolerant 

soybeans remain compositionally equivalent to conventional soybeans (Glycine max. L.) 

during three years of field testing. Journal of Agriculture and Food Chemistry 53:5331-

5335. 

Mensink, R.P. and M.B. Katan. 990. Effect of dietary trans fatty acids on high-density 

and low-density lipoprotein cholesterol levels in healthy subjects. New England Journal 

of Medicine 232(7):439-445 

OECD. 2000. Consensus document on the biology of Glycine max (L.) merr. (soybean). 

OECD ENV/JM/MONO(2000)9. Organization for Economic Co-operation and 

Development, Paris. 

Okkerse, C., A. de Jonge, J.W.E. Coenen, and A. Rozendall. 1967. Selective 

hydrogenation of soybean oil in the presence of copper catalysts. Journal of the American 

Oil Chemists’ Society 44:152-156. 

Padley, F.B., F.D. Gunstone and J.L. Harwood. 1994. Occurrence and characteristics of 

oils and fats. Pages 47-224 in The Lipid Handbook. 2nd ed. F.D. Gunstone, J.L. Harwood 

and F.B. Padley (eds.). Chapman & Hall, Cambridge, Great Btitain. 

Ray, J.D., T.C. Kilen, A.C. Abel, and R.L. Paris. 2003. Soybean natural cross-pollination 

rates under field conditions. Environmental Biosafety Research 2:133-138. 

Sankula, S. 2006. Quantification of the impacts on US agriculture of biotechnologyderived crops planted in 2005. National Center for Food and Agriculture Policy, 

Washington, D.C. 

Soyatech. 2008. Statistics. Soyatech, Manitoba, Canada. 

Stewart, C.N., M.D. Halthill, and S.I. Warwick. 2003. Transgene introgression from 

genetically modified crops to their wild relatives. Nature Reviews 4:E1-E17. 

USDA-ERS. 2007. Adoption of genetically engineered crops in the U.S. U.S. Department 

of Agriculture, Washington, D.C 

USDA-NASS. 2009a. Crop Production 2008 Summary. U.S. Department of Agriculture, 

Washington, D.C 
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USDA-NASS. 2008. Acreage. U.S. Department of Agriculture, National Agricultural 

Statistics Service. Washington, D.C. 

USDA-NASS. 2006. Crop production 2005 summary. U.S. Department of Agriculture 

National Agricultural Statistics Service. 

USHHS. 2005. Dietary Guidelines for Americans. HHS Publication No: HHS-ODPHP2005-01-DGA-A. U.S. Department of Health and Human Services, Washington, D.C. 

www.health.gov/dietaryguidelines [Accessed June 7, 2009]. 

WHO. 2003. Diet, Nutrition and the Prevention of Chronic Diseases. Report of a joint 

WHO/FAO Expert consultation. WHO Technical Report Series 915:55-56. 

Williams, G.M., R. Kroes, and I.C. Munro. 2000. Safety evaluation and risk assessment 

of the herbicide Roundup and its active ingredient, Glyphosate, for humans. Regulatory 

Toxicology and Pharmacology 32:117-165. 

Zock, P.L. and M.B. Katan. 1992. Hydrogenation alternatives: effects of trans fatty acids 

and stearic acid versus linoleic acid onserum lipids and lipoproteins in humans. Journal of 

Lipid Research 33:399-410. 
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Petition for the Determination of Nonregulated Status for Improved 

Fatty Acid Profile MON 87705 Soybean 

ADDENDENDUM I 

Market Impact of MON 87705 High Oleic Soybean Oil

Submitted April 7, 2011 

OECD Unique Identifier: MON-87705-6 

Monsanto Petition Number: 09-SY-201U 

 
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Introduction 

Overall total use of oils derived from plant sources (vegetable oil) for food and industrial 

applications exceeded 14 million tons in North America in 2009. Due to its abundance, reliable 

supply and price, soybean oil is one of the most highly used of the vegetable oils (Attachment I, 

Table 3.1, pg 62). In 2009, soybean oil accounted for approximately 8.2 million tons of food and 

1.2 million ton of industrial vegetable oil uses (See Attachment I, Part 1b, pg 17-18). Soybean 

oil use in foods peaked at roughly 9.3 million tons in the 2006/2007 timeframe just before the 

U.S. Food and Drug Administration required food labels to report trans-fat content in 2006. 

(http://www.fda.gov/Food/LabelingNutrition/ConsumerInformation). The demand for soybean 

oil for frying and baking applications has declined in recent years due to the desire to lower 

unhealthy trans-fat in the diet and changes in food labeling policy. 

High oleic soybean oil produced using MON 87705 has a fatty acid profile that has reduced 

saturated fats, and higher monounsaturated fat. These properties, particularly the reduction in 

polyunsaturated fats (18:2), result in increased oil oxidative stability greatly improving oil 

performance in frying and baking applications. The decrease in saturated fat (16:0, 18:0) further 

improves the nutritional profile of the oil for use in food preparation. MON 87705 will be bred 

into Vistive® low-linolenic soybean further improving the oil profile by lowering the level of 

linolenic acid and eliminating the need to hydrogenate the oil. 

Monsanto has conducted a market and trade assessment to evaluate the potential impacts on 

commodity soybean oil and other vegetable oils due to the introduction of high oleic soybean oil. 

The assessment considered that other vegetable oils including high oleic vegetable oils are 

currently on the market and are routinely handled by oil processors and food formulators. The 

system responsible for production, harvest, processing and handling of soybean and other 

vegetable oils includes critical control points as well as economic incentives for identity 

preserved soybean oils and other vegetable oils. Typically, the processor will assess the fatty 

acid profile of the oil sold to a food manufacturer prior to sale. If the fatty acid profile were 

different from commodity soybean oil, the oil could be blended (a common industry practice) to 

reach the appropriate fatty acid content based on specific food applications. On the basis of this 

assessment it is concluded that the introduction of high oleic soybean oil will have little if any 

potential for negative market impacts. In the event that comingling did occur, the economic 

impact of comingling to a food manufacturer would be minimal and remedied through blending. 

A summary of the intended uses for high oleic soybean oil, current processes for handling 

vegetable oils and potential impacts to commodity soybean oil as well as other vegetable oils is 

provided in this document. 

 

® Vistive is a registered trademark of Monsanto Technology LLC 
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Intended Uses of High Oleic Soybean Oil 

Commodity soybean oil does not have the optimum fatty acid profile to satisfy many of the 

needs of the food industry1

. The fatty acid profile for soybean oil does not provide optimal 

oxidative stability compared to other vegetable oils unless modified via hydrogenation or 

through blending with other vegetable oils. Hydrogenation creates unhealthy trans-fat making 

soybean oil less desirable for use in some applications. In addition, due to low oxidative 

stability, commodity soybean oil is not highly stable for cooking and frying when used in these 

applications, causing several operational challenges such as more frequent exchange of vegetable 

oil stocks and/or replacement of oil and polymerization, leading to frequent costly equipment 

cleaning. For these reasons, in recent years, soybean oil has lost share to other vegetable oils in 

food applications (Wilson, 2004; Attachment I, Part 2, pg 38). 

Given recent food labeling policy changes and modifications to dietary guidelines, the food 

industry is seeking vegetable oils that not only have enhanced functionality but minimize levels 

of trans- and saturated fat; the balance of fatty acids (high oleic, low saturated fat, low linolenic) 

is often difficult to achieve with current vegetable oil options. Soybean oil competes with other 

oils such as palm, canola, sunflower, peanut and corn. These oils are generally blended to satisfy 

food companies seeking to address functional or nutrition related needs. High oleic soybean oil 

can provide a complementary option either as a straight replacement of blended oils or as a 

blending alternative to end users. While blending achieves the food company’s objective, it is 

costly due to increased handling, average oil price and required technical support (Attachment I, 

Part 4, pg 75). 

Through conventional plant breeding, MON 87705 will be bred with Vistive low-linolenic 

soybean. Vistive low-linolenic soybean was developed through conventional plant breeding to 

produce lower levels of linolenic acid allowing food companies to avoid hydrogenation of 

soybean oil helping to eliminate unhealthy trans fatty acids from the diet. The low linolenic 

soybean has a mutation in the Fad3-1c and Fad3-1b genes that result in reduced levels of 

linolenic acid. The Vistive low-linolenic soybean oil is currently commercially sold in the 

United States. The soybean oil from MON 87705 in the Vistive low-linolenic soybean 

background has the following specification: palmitic acid (< 4.0%); stearic acid (< 4.0%); oleic 

acid (55 - 85%); linoleic acid (8 – 30%); and linolenic acid (< 4.0%). The modification of the 

fatty acid profile provides an equal or better alternative to vegetable oil options available today 

and offers the opportunity to reduce the practice of blending which has become the solution for 

fry applications to avoid trans-fats. The observed fatty acid profile of MON 87705 soybean and 

MON 87705 in a Vistive low-linolenic soybean genetic background are presented in Table 1. 

 

1

http://qualisoy.com 
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Figure 1. Fatty acid synthesis in MON 87705 in a Vistive low-linolenic genetic background 

Table 1. Fatty acid composition (% weight of soybeans) of MON 87705 soybeans and 

MON 87705 in a Vistive low-linolenic soybean background 

Year C16:0 C18:0 C18:1 C18:2 C18:3 

MON 87705

20071 2.36 3.31 76.47 10.10 6.69 

MON 87705 in a Vistive low-linolenic soybean background 

20072 2.60 3.05 77.27 13.77 2.63 

20082

 2.47 2.97 75.30 15.60 2.96 

20093

 2.31 3.29 71.21 19.03 3.20 

20104

 2.20 3.43 76.83 13.73 2.47 

1

Samples were obtained from field trials conducted in Chile in the 2007/2008 growing season. See USDA Petition 

Number 09-201-01p. 2

Grown in Jerseyville, IL. 

3

Samples were obtained from8 U.S. locations (2 sites each in the states of IA, IL, IN, and OH). Value is mean of 

the 8 individual site values. 

4

Samples were obtained from3 U.S. locations (2 sites in IL and one site in OH). Value is mean of the 3 individual 

site values. 

The safety of MON 87705 soybeans in a Vistive low-linolenic soybean genetic background is 

based on the long-standing history of safe consumption of the levels and types of fatty acids 

contained in commercial vegetable oils with a fatty acid profile similar to MON 87705 soybeans 
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in Vistive low-linolenic background. Monsanto has completed a safety assessment2

 of high oleic 

soybean oil and high oleic soybean oil is generally recognized, among qualified experts, as 

having been adequately shown to be safe under the conditions of its intended use. MON 87705 

soybean in a Vistive low-linolenic soybean genetic background does not contain any new fatty 

acids that are not presently found in conventional food oils and the fatty acid profile of this oil is 

similar to many other commercial oils currently available including canola, olive oil, high oleic 

safflower oil, and high oleic sunflower oil (See Table 2). The oil from MON 87705 soybeans in 

a Vistive low linolenic soybean genetic background provides options for food formulation that is 

virtually trans fat-free, and, unlike palm oil, is low in saturated fat. 

 Table 2. Fatty acid composition (% weight) of MON 87705 in Vistive background in 

comparison to soybean oil and other high oleic vegetable oils 1

Oil Saturated 

fats trans fats Oleic acid Linoleic 

acid 

Linolenic 

acid 

Other fatty 

acids 

Soybean oil, all purpose 15.2 0.7 22.6 50.1 6.5 4.9 

PH Soybean 2 24.7 34.1 31.4 4.5 0.2 5.1 

MON 87705 in Vistive 

background 3 6.8 0.22 71.7 16.9 2.9 1.5 

HOSO (305423) 4 14.27 0 70.6 5.5 7.2 3.4 

Olive oil 13.8 0 71.3 9.8 0.8 4.3 

HOLLCO 5 3 0 84 7 2 4 

High oleic canola 6.5 0.8 70 14.3 2.6 5.8 

Canola 7.6 1.6 60.6 17.7 6.4 6.1 

High oleic Sunflower 9.7 0 82.6 3.6 0.2 3.9 

1 The fatty acid profiles of PH soybean oil (partially hydrogenated soybean oil), olive oil, canola oil, high oleic canola oil, and 

high oleic sunflower oil were obtained from the USDA nutrient database (USDA 2008, National Nutrient Database for 

Standard Reference. U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Nutrient Data 

Laboratory; Beltsville, Maryland. Available from: http://www.nal.usda.gov/fnic/foodcomp/search/). 2 PH Soybean: partially hydrogenated soybean oil. 3 MON 87705 in Vistive background fatty acid composition is based on the pooled analysis of 8 oil lots. 4 HOSO (305423): high oleic soybean oil from Pioneer/DuPont, defined in Delaney (2008). 5 HOLLCO: High oleic low linolenic canola oil, values from Möllers (2004). 

 

 

2

See FDA GRAS assessment at: 

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodIngredientsandPac

kaging/ucm061846.htm#Q1 
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Vegetable oils are used in twelve broadly grouped end use sectors: snacks, salad oils (use in 

mayonnaise, salad dressings and other food products), margarine, bottled oils (home use), par 

fry, ingredient, confectionary, baked goods, industrial fry, restaurant, institutional, and food 

service (Attachment I, Part 2, pg 37). High oleic soybean oil is targeted for ten of the twelve end 

use sectors with bottled and salad oils not considered a commercial target for high oleic soybean 

oil because commodity soybean oil currently serving these sectors does not require 

hydrogenation and products currently have acceptable shelf lives. 

Industrial uses such as bio lubricants and biodiesel are also possible end uses of high oleic 

soybean oil. High oleic soybean oil is a renewable resource and is an attractive option as the 

price of crude petroleum continues to increase. The increased levels of oleic acid in high oleic 

soybean oil enhance the stability and lubricity of the oil making it a superior bio lubricant 

compared to petroleum based oils and a good feedstock for biodiesel (Graef et. al, 2009). 

Food and industrial end use sectors are currently supplied by commodity soybean oil as well as 

domestic and imported blends of other vegetable oils (e.g. palm oil). Thus, high oleic soybean 

oil would potentially replace commodity soybean oil and oil blends currently used for these 

applications. 

Market Potential of High Oleic Soybean Oil 

In order to assess the market potential for high oleic soybean oil, Monsanto commissioned a 

study that included an in-depth analysis of the vegetable oil market in North America that was 

performed by LMC International (Attachment I). The study was conducted to assess the 

potential demand for vegetable oil with the fatty acid properties of high oleic soybean oil. 

Factors considered included current uses of vegetable oils and their physical properties, health 

attributes of the oil, availability and price. The market potential estimate assumed that high oleic 

soybean oil was used in the target food sectors described above as a direct replacement for 

hydrogenated soybean oil and other vegetable oils (such as canola, corn, palm and sunflower) 

used in food applications (Attachment I, part 5; pg 88). The demand for high oleic soybean oil is 

price sensitive relative to other vegetable oils. To estimate of the maximum high oleic soybean 

the lowest price premium ($50/metric ton) was used. At the lowest price premium, high oleic 

soybean would achieve a demand for the oil estimated at 3.5 million metric tons. On the basis of 

this analysis, approximately 16 million acres of high oleic soybean would satisfy the demand if 

high oleic soybean oil were to replace current uses of hydrogenated soybean and other high oleic 

vegetable oils in food and industrial applications (Attachment I, Part 5, pg 90)3

. Additional acres 

would likely be planted to supply high oleic soybean oil for industrial uses. Based on 

assumptions regarding product adoption and acceptance by the industry, Monsanto estimates it 

would take at least a decade for high oleic soybeans to achieve this acreage and at peak 

 

3

 One bushel of soybean = 60 lbs and contains 10.7 lb oil (Wilson, 2004); average yield of soybean = 43.7 bushels 

per acre in 2010 (USDA NASS: http://www.nass.usda.gov) 
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penetration, this level would represent approximately 23% of total planted soybean acres4

. 

Subsequently, there would continue to be adequate supply of both commodity and high oleic 

soybeans to serve the vegetable oil market needs. 

Production and Handling of Vegetable Oils 

Crop production and distribution systems as well as oil processors and food manufactures have 

demonstrated the ability to handle multiple vegetable oils derived from numerous crop species. A 

summary of the current practices including critical control points for production of soybean oil 

and specialty soybean oil (e.g. Vistive low linolenic) is provided below. There are numerous 

vegetable oils on the market today including: palm, coconut, palm kernel, cottonseed, soybean, 

groundnut, rapeseed and sunflower (Attachment I, Part 2, pg 38). Other minor use oils include 

olive, corn, high oleic canola, high oleic sunflower oil and low linoleic soybean oil (Vistive). 

The processing industry (e.g. Cargill, Bunge, ADM, etc.) and end users of such oils are highly 

adept at handling these vegetable oils in their normal course of business. As a result, processors 

have developed methods to segregate multiple vegetable oil feedstocks as they are financially 

motivated to prevent mishaps with their customers. High oleic soybean, for planting and 

subsequent processing, would be introduced to the market place as an option for processors to 

supply food companies and those who seek a U.S. based vegetable oil option. 

Once key global market approvals are obtained, oil from MON 87705 will be available as an 

option for use by food processors and will be produced in an identity preserved (IDP) fashion 

similar to the system that has been successfully implemented for production of Vistive low 

linolenic soybean and other specialty vegetable oils. IDP practices are implementd for value 

added specialty soybean to capture the enhanced value of the product and ensure that the enduser or processor receives the soybean with the desired identity, fatty acid profile and quality. 

Vistive low-linolenic soybean was made available to growers in 2005 and has been grown on 

over 3.0 million acres since its initial launch. 

A summary of the quality control systems associated with seed production, crop production, 

processing and end uses of high oleic soybean oil is presented in Figure 2. 

 

 

4

 Assumes 70 million acres of soybean are planted. 
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Figure 2. Production and use checkpoints for high oleic soybean 

Seed Production and Processing. Monsanto is a leader in crop biotechnology having 

successfully introduced numerous biotech crops to the marketplace. Monsanto has developed 

and implemented seed quality practices to assure that soybean seed meets the standards 

established for purity of a trait. These standards apply to all soybean seed sold by Monsanto and 

are based upon measures that seed producers put in place to assure the genetic purity of 

improved planting seed. This system is used to assure that farmers receive seed of known 

quality with a minimum level of off types. 

The first step in the process for production of high oleic soybean is the production, processing 

and delivery of high quality seed to the grower. The entire seed production process at the 

majority of the seed companies and tollers operate using International Organization for 

Standardization (ISO) certification or standards and; therefore, include internal and external 

audits (ISO, 2009). ISO standards ensure desirable characteristics of seeds and services, such as 

quality, safety, reliability, and efficiency. The ISO standards represent an international 

consensus on good management practices with the aim of ensuring that the organization can 

consistently deliver excellent product or services. The standards not only must meet the 

customer’s requirements and applicable seed regulatory requirements, but must aim to enhance 

customer satisfaction and achieve continual improvement of its performance in pursuit of these 

objectives (ISO, 2009). 

Commercially certified soybean seed must meet state and federal seed standards and labeling 

requirements. The Association of Official Seed Certifying Agencies (AOSCA) standards for 

certified soybean seed are as follows: 98% pure seed (minimum), 2% inert matter (maximum), 
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0.05% weed seed (maximum, not to exceed 10 per lb.), 0.60% total of other crop seeds 

(maximum), 0.5% other varieties (maximum, includes off-colored beans and off-type seeds), 

0.10% other crop seeds (maximum, not to exceed three per lb.), and 80% germination and hard 

seed (minimum) (AOSCA, 2009). Seed that meets or exceeds these standards are provided in 

appropriately labeled seed bags to growers 

Production of Soybean Grain Containing High Oleic Soybean Oil. Specialty soybean (e.g. 

Vistive low linolenic) is currently produced under contracts issued by processors or elevators. It 

is expected that high oleic soybean will be produced using this system as well. 

There are three major factors needing to be satisfied before a farmer will produce a specialty 

soybean containing a modified soybean oil, like high oleic. These factors have become evident 

in Monsanto’s experience in the crop production of Vistive low linolenic soybeans. 

1. Yield of the soybean must be comparable to commodity soybeans routinely planted 

2. The option to market, price & deliver the grain must be comparable to commodity grain 

3. The premium paid to the farmer to offset the cost to identity preserve the high oleic 

containing grain must provide incremental income opportunity above the production of 

commodity grain. The income opportunity cannot be realized by the farmer unless the grain 

is delivered to the processor within specifications or in its identity preserved state. 

Therefore, the motivation is a financial incentive for the farmer to avoid comingling with 

commodity grain, keeping all the grain identity preserved within this closed loop system of 

seed – farmer - processor. Many farmers willingly choose to plant all their acreage to this 

specialty soybean because it eliminates any risk of contamination from inadvertent errors that 

may occur during the harvest. If a farmer chooses to produce the specialty grain for the 

processor, the farmer will arrange to store the grain on farm or at their local elevator, if the 

elevator is participating in the specialty program with the processor. 

The steps involved in securing grain production are: 

1. Monsanto will sell and distribute high oleic soybean seed to the farmer after the farmer has 

signed and agreed to the conditions of Monsanto’s Technology and Stewardship Agreement. 

The agreement will mandate that the farmer sell any soybean produced into an identity 

preservation channel (See Figure 2). 

2. Processors will be responsible to contract with elevators and growers the acreage needed to 

fulfill the expected demand for high oleic soybean. 

3. After harvest, the farmer will deliver the grain produced to the location specified in his 

contract, either a participating elevator or processor. If to the elevator, the elevator will keep 

the grain segregated from commodity grain and pay the farmer his premium, provided it 

passes the analytical testing. The elevator will deliver the grain to the processor as delivery 

windows and crush schedules have been established. Upon delivery of grain by the farmer, 

samples will be analyzed from every truckload by Near Infrared Transmittance (NIT) 

developed by Monsanto. This will confirm the grain contains high oleic soybean oil as 

required by the production contract. Upon confirmation of grain that meets specification of 

high oleic soybean grain, the processor or elevator will approve the premium payment to the 

farmer. 
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In the event that high oleic soybean do not meet the minimum specifications established for 

oil quality, the soybean would be isolated from other high oleic or commodity soybean and 

blended with an appropriate quantity of high oleic soybean to meet the specifications 

required for a food application. Monsanto has been conducting field trials with high oleic 

soybean since 2005 and has considerable experience with trait performance in various 

genetic backgrounds and under various climatic conditions. Trait performance has been 

consistent over several years of breeding (See Table 10). All soybean varieties that 

Monsanto will introduce must meet specifications for agronomic performance and oil quality. 

While it is not envisioned that any of the high oleic soybean varieties will underperform, 

blending with other high oleic soybean is the most likely remedy. 

Grain Elevator. Grain elevators play an important role in specialty programs with their long 

term storage of the grain. Since processing facilities crush soybeans throughout the calendar 

year, soybeans used to supply these crush plants need to be stored year round. Farmers typically 

prefer to empty their storage prior to planting of the new crop and prior to temperature warm up 

in the spring. The warm spring and summer weather present challenges as condensation can 

build up in the bins creating moisture related issues and grain eating insects become more active. 

Farmers prefer to avoid these high management grain conditions. Commercial elevators have 

expert grain managers on staff to monitor grain quality and keep grain in condition in all weather 

situations presented throughout the year. 

Commercial grain elevators are also better equipped to ship grain to processors during times of 

severe weather or when farmers cannot get to their bins when roads are impassable. 

Processors will therefore enter into supply contracts with commercial grain elevators for these 

reasons. Contracts will be “acre” based, established to fill bin capacity that has been agreed 

upon by both parties. Grain elevators will then contract with farmers directly, providing harvest 

storage terms to the farmer with premiums to be paid that have been agreed upon with the 

processor. The elevator pays the farmer the specialty grain premium upon successful analytical 

testing by NIT performed at the elevator location. NIT testing equipment will be provided by 

Monsanto to participating elevators identical to the equipment provided to processors. 

In order for the elevator to be reimbursed for the premiums paid out to farmers that have 

delivered to them, the elevator must in turn preserve the identity of the grain as it is delivered to 

the processor. 

Every load delivered to the processor by the elevator will be analyzed using NIT technology. 

Processors will approve the premium payment to the elevator after analysis of the grain confirms 

high oleic soybean oil. 

Processing Oil and Meal. Specialty high oleic soybean grain will be introduced into the 

processing plant as it is running commodity soybean grain. Upon the transition from commodity 

soybeans in the plant, the processor will ensure that the equipment is lined out and operating 

within normal and acceptable limits and parameters using commodity soybean. High oleic 

soybeans will then be moved through the plant continuously until all specialty grain located at 
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the plant is gone. After all of the specialty high oleic soybeans have moved through the seed 

prep building, the bins and conveyors feeding the seed prep building will be filled with 

commodity soybeans to keep the plant running continuously. Commodity soybeans will then be 

processed as normal. 

Upon the transition from commodity soybeans to the high oleic soybeans, a sample of crude oil 

is required to confirm the presence of the appropriate fatty acid composition (FAC) unique to 

high oleic oil. Until the fatty acid composition of the crude oil exiting the extractor is of the 

appropriate composition, as determined by the plant manager, off spec crude oil will be sent to a 

tank that will be designated for flush oil. This will contain all oil before and after the collection 

of “on-spec” high oleic oil. High oleic crude oil that is collected with the appropriate FAC will 

be designated for further refining, bleaching, and deodorization. The processor’s lab will be 

used to aid in identifying the appropriate time to start collecting crude high oleic oil exiting the 

extractor that will undergo further refining. The lab will use gas chromatography equipment to 

analyze the crude oil to determine when on spec high oleic oil can be collected from the 

extractor. A tank approved for high oleic oil will be assigned by a processor employee. After all 

high oleic grain has been processed, the seed extraction area will be flushed with commodity 

soybeans, and regular commodity oil will be produced. During the flush, crude oil exiting the 

extractor will be sampled and analyzed to confirm the equipment has been flushed to an 

acceptable level, yielding commodity soybean oil. 

Processes are established by the processor to ensure the transfer of high oleic oil to the crude oil 

tank farm to minimize loss and maximize quality. The lab at the processor will utilize 

appropriate analytical methods to determine when the high oleic oil is of proper quality to send 

to the main crude tank(s) that will supply oil for further refining. Commodity soybean flush oil 

prior to and after the crush of the high oleic oil will be collected in tanks designated as flush, and 

will be blended with commodity soybean oil to bring the flush oil into a specification range 

necessary to be sold as commodity soybean oil. 

Food Company. Oil will be supplied to the food company by oil processors and suppliers. The 

processor or supplier will test the oil and assure that it meets specific customer requirements 

including quality factors (e.g. peroxide value, oxidative stability index, color, flavor) and oil 

composition (customized blends, specified fatty acid composition). Foodservice distributors 

typically obtain the oil from the oil processors and deliver it to each foodservice outlet 

(individual stores, caterers, cafeterias, etc). From the time the oil is packaged, until it is utilized 

at the specific customer’s facility, there is proper identification of the oil through labeling and 

manufacturing codes allowing for sufficient product traceability if needed. Individual facilities 

will utilize proprietary inventory and ordering systems that are in place to insure that the 

appropriate oil is ordered, delivered and utilized. 

High oleic soybean oil will be used by the food industry in several ways. One specific type of 

food company customer will be foodservice operators including quick service restaurants, casual 

dining and full service restaurants who will use high oleic soybean oil as a fry medium and in 
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meal preparation. Food companies will also use high oleic soybean oil as a food ingredient in a 

range of food products including as spray oil on crackers and snacks, or as a component of oil 

blends used in shortenings and other foods. Each individual food company has in place systems 

for ingredient (oil) ordering, receipt, storage, access and lot identification at specific 

manufacturing locations, as well as finished product (the food which incorporates high oleic 

soybean oil) batch identification, manufacturing facility, storage, shipment to distribution 

centers, customer order picking, customer order shipment and receipt. Appropriate procedures 

are currently in place to insure traceability from receipt of the ingredient through distribution to a 

specific retailer’s facility. Food companies routinely conduct mock recalls including use of the 

media should the need arise. Food manufacturing facilities also comply with federal and state 

requirements for good manufacturing practices and product traceability. Supply chain 

consultants can be employed to confirm appropriate systems have been established that meet 

ingredient and product traceability requirements. 

Stewardship of High Oleic Soybean 

Monsanto is committed to product stewardship and to implementing BIO’s "Excellence through 

Stewardship" program and Product Launch Stewardship Policy5

. Monsanto considered Annex 2 

“Special Use traits in Commodity Crops” 6

 to develop launch plans for high oleic soybean oil 

including: (1) identifying relevant stakeholders for the trait and crop and engaging them in 

dialogue regarding use of high oleic soybean oil and potential impacts to vegetable oil markets, 

(2) conducting a market and trade assessment, including securing regulatory approvals in key 

export countries prior to full commercial launch, (3) developing a risk mitigation plan, and (4) 

undertaking appropriate outreach, necessary to educate stakeholders and implement the 

management plan for high oleic soybean oil. These actions protect against adverse impacts to 

trade of soybean due to the introduction of a new biotechnology improved soybean. 

Stakeholder Dialogue 

Monsanto is committed to dialogue with key industry stakeholder groups and has held several 

meetings with the National Oil Processors Association (NOPA) as well as other key industry 

associations such as the North American Export Grain Association (NAEGA), National Grain 

and Feed Association (NGFA), North American Milling Association (NAMA), American Bakers 

Association (ABA), and Grocery Manufacturers Association (GMA). Soybean grower 

organizations: American Soybean Association (ASA), United States Soybean Board (USB), and 

many state soybean associations. In addition QUALISOY, a collaborative program sponsored by 

the USB that serves as an independent, third part resource for information on trait-enhanced 

soybean oils, has been kept informed on the plans for this product, along with leaders in dietary 

and nutrition fields. 

 

 

5

 http://www.monsanto.com/ourcommitments/Pages/product-stewardship.aspx 6 http://www.bio.org/letters/Product_Launch_Stewarship_12_10_09.pdf; 
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Market and Trade Assessment

Monsanto has conducted a market and trade assessment to determine the impact of the 

introduction of high oleic soybean oil. Soybean is a globally traded commodity with the U.S. 

being the top global producer (Soyatech, 2010). Biotechnology-derived crops and their use as 

food and feed are subject to regulation in many countries. In order to support continued trade in 

soybean, Monsanto is pursuing regulatory approval for MON 87705 in all key soybean import 

countries with a functioning regulatory system to support the flow of international trade. 

International regulatory authorities are evaluating the biotech component as well as the modified 

oil fraction. It is expected that uses of high oleic oil soybean will be similar on a global basis. 

There would be no impact to human heath or to the use of any of the other processed fractions 

produced from soybean due to comingling of high oleic soybean with commodity soybean. 

Monsanto has completed a GRAS assessment of the high oleic soybean oil (MON 87705 in a 

Vistive background)7 and has completed the biotechnology consultation on MON 87705 with the 

U.S. FDA8

. The GRAS assessment has been evaluated by FDA and the agency had no further 

questions. The biotechnology consultation considered the food and feed safety impacts due to 

the genetic modification process, RNAi suppression of two endogenous enzymes resulting in 

modification to fatty acid metabolism, as well as exposure to the CP4 EPSPS protein. The 

GRAS assessment considered the nutritional impact of total replacement of commodity soybean 

oil with high oleic soybean oil. High oleic soybean oil is similar to other high oleic vegetable 

oils that are commonly consumed producing no harmful effects to humans. Hence, Monsanto as 

well as a panel of qualified scientific experts have concluded that high oleic soybean oil is 

generally recognized as safe. 

Information provided to USDA showed that the impact due to the suppression of the two 

endogenous enzymes is restricted to seed. With the exception of the intended changes in fatty 

acid composition and presence of the CP4 EPSPS protein, the soybean meal and other processed 

fractions used for animal feed and human food applications are compositionally equivalent to 

commodity processed soybean fractions9

. Monsanto provided information to USDA in the 

petition demonstrating that the composition of the meal was compositionally equivalent to meal 

derived from conventional soybean and safe and wholesome for food or feed applications. 

Monsanto has assessed the impact of the introduction high oleic soybean oil on commodity and 

other vegetable oils. In response to stakeholder dialogue, Monsanto was specifically requested 

by NOPA to address the impact of comingling of commodity soybean oil with high oleic 

soybean oil to bottled oils (100% commodity soybean oil) and severe heat processing (e.g. 

impact to frying applications and sensory properties of prepared foods). This assessment has 

been shared with key stakeholders such as NOPA, NAEGA, ASA, Qualisoy, USB and GMA. 

 

7

http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodIngredientsandPa

ckaging/ucm061846.htm#Q1 

8

Biotechnology notice: BNF 121: http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=bioListing; 

GRAS notice: GRN 306 - http://www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=grasListing&page=2 9

 USDA Petition Number 09-201-01p, Section VII.C. 
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Hypothetical impacts were assessed using the following scenario: (1) high oleic soybean grain 

or oil went unnoticed and was comingled ( e.g. harvest mistake, inadvertent mixing of grain 

during transport, mistaken delivery of grain, etc...), (2) the comingled soybean were delivered to 

an oil processor and mixed with commodity grain or soybean oil, (3) the oil or grain was not 

analyzed for oil composition by anyone in the food supply chain, and (4) the comingled oil was 

used in the food or feed supply chain. This scenario is highly unlikely given existing critical 

control points embedded in the production handling and processing system and because high 

oleic soybean oil is expected to be produced in a contractual identity preserved system in order to 

preserve its premium value. Processors will originate the supply of high oleic soybeans and as 

they have extensive experience managing, segregating, testing, formulating and blending other 

sources of high oleic vegetable oils such as high oleic canola and high oleic sunflower oil. 

Bottled vegetable oil is not an intended use for high oleic soybean oil. However, bottle oil was 

used to assess potential impacts at the request of NOPA. It represents a readily measurable 

impact that could occur if high oleic soybean were comingled with 100% commodity soybean. 

Mixing high oleic soybean with commodity soybean will impact to the levels of fatty acids in 

commodity soybean. This effect is measurable and can potentially translate into downstream 

effects to food labeling, oil quality, and functionality. Even though such comingling is 

improbable, potential impacts were assessed at several different comingling levels ranging from 

4% and up to 30% using four different parameters: (1) food ingredient labeling, (2) nutritional 

facts panel labeling (3) functionality of the oil, and (4) sensory evaluation. 

Food Ingredient Labeling Codex Specification. Effects were assessed on the ability to meet 

USP Food Chemical Codex (USP FCC) soybean oil labeling specification. In Table 3 the 

impact of varying amounts of high oleic soybean oil comingled with commodity soybean oil 

on the ability to meet specifications for major fatty acids is evaluated. The USP FCC is a 

compendium of internationally recognized standards for the purity and identity of food 

ingredients and is used as a set of agreed standards between buyers and manufacturers of 

food ingredients10. Based on this analysis the most sensitive fatty acid is 18:2 linoleic acid. 

If high oleic soybean were co-mingled with commodity soybean oil at levels above 14%, the 

linoleic acid (C 18:2) content of the resulting oil following processing would fall below the 

USP FCC specification for commodity soybean oil. At levels above 15% oleic (C 18:1), 

linoleic acid would be out of the specifications for soybean oil. 

To put this into perspective, 15% comingling could occur due to a farmer mistakenly 

harvesting approximately three acres of high oleic soybean (assuming yield = 43 

bushels/acre) and comingling it with commodity soybean in a grain truck hauling the 

standard load limit with a capacity of 900 bushels. If the grain were delivered to a grain 

elevator or directly to a processor, it would be mixed with other commodity soybean grain 

thereby diluting the high oleic soybean considerably with commodity soybean. The capacity 

of grain elevators or bin storage tanks is highly variable; however, bin capacities up to and 

 

10 http://www.usp.org/fcc/ 
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exceeding 1⁄2 million bushels are quite common. Hence, it would take roughly 83 fully 

loaded trucks containing 100% high oleic soybean or multiple mistakes during harvest of 

high oleic soybean to achieve a 15% level of comingling. 

Nutrition Facts Panel Labeling. Figure 3 and 4 demonstrate the amount of high oleic 

soybean oil that would be required to be comingled with commodity soybean oil in order to 

impact the Nutrition Facts Panel for major fatty acid categories. Based on this analysis, 

monounsaturated fat is the most sensitive fatty acid category and comingling up to 5% high 

oleic soybean oil could be tolerated before the Nutrition Facts Panel label for 

monounsaturated fatty acids were impacted on retail bottled vegetable oil. Up to 15% high 

oleic soybean could be comingled with commodity soybean oil before effects to 

polyunsaturated fatty acid labeling would be impacted. 

The threshold for effects is lower than USP FCC labeling situation discussed above. In this 

case it would take a mistake during harvest of one acre of high oleic soybean comingled with 

900 bushels of commodity soybean in a 900 bushel grain truck or 28 fully loaded trucks 

containing 100% high oleic soybean would need to be delivered to a 1⁄2 million bushel grain 

elevator containing commodity soybean to trigger an effect to the nutritional facts panel 

labeling. 

The impact due to both of the comingling scenario discussed above would be minimal. As 

discussed previously, there would be no impacts to human health and the changes in fatty 

acids would benefit consumers from a nutritional perspective. Mandatory labeling is 

required for saturated fats, in this case comingling levels of up to 28% could be tolerated 

without impacting the level of saturated fats reported on the label. At 28%, the level of 

saturated fat would be lower, enhancing the nutritional profile of the product (Figure 4). 

Oil Functionality; Severe Heat Processing. High oleic soybean oil has an improved stability 

profile compared to commodity soybean oil. An assessment of the impact of comingling of 

high oleic soybean oil with commodity soybean grain and oil was conducted. Information in 

Figure 5 demonstrates that comingling of high oleic soybean oil with commodity soybean oil 

increases the OSI stability index for commodity soybean oil. This outcome is expected since 

soybean oil oxidative stability is drastically influenced by the proportion of monounsaturates 

to polyunsaturates, and high oleic soybean oils are estimated to have improved oxidative 

stability compared to conventional soybean oil (Frankel, 2005). 

Food Sensory Assessment. A sensory assessment of high oleic soybean oil was performed to 

evaluate consumer acceptability of high oleic soybean oil. Figure 6 demonstrates the sensory 

results for high oleic soybean oil in the most challenging environment high temperature food 

frying. In this experiment, commodity soybean oil and blends (5% to 15%) of commodity 

and high oleic soybean oil were used to prepare French fries. Food testers were asked to 

evaluate the quality of the fries using the Sensory Quality System described by King et al 

(2003). The outcome from the sensory evaluation showed that high oleic soybean oil 

comingled with commodity soybean oil blends were essentially the same as commodity 
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soybean oil over the six-day period during which the experiment was conducted. Thus, 

comingling of high oleic soybean oil with commodity soybean oil would have no impact to 

the sensory profile of commodity soybean oil should inadvertent comingling occur (Figure 

6). 

The comingling scenarios and impacts above are presented as the most conservative scenarios. 

Oilseed processors and users of vegetable oils are accustomed to the presence of numerous 

vegetable oils of differing fatty acid makeup that are available concurrently for blending and use 

in various food applications. The comingling levels described here where an impact could occur 

in functionality or labeling would happen only in instances where common control points were 

ignored. Such levels of comingling are highly unlikely to occur due to economic incentives to 

growers, legal contracts, stewardship SOP’s (seed quality to end user), and demonstrated 

competency in managing inadvertent comingling with other vegetable oils that have a fatty acid 

profile similar to high oleic soybean oil. In addition, fatty acid analytical methods are widely 

available and used currently by oil processors and food manufacturers. 

Industry Outreach 

Monsanto has held conversations with the soybean and food industry key stakeholders 

mentioned above regarding the oil composition, stewardship plan and performance of MON 

87705. Additionally, Monsanto has consulted with NOPA and industry members of NOPA’s 

biotechnology committee as well as the ASA and USB regarding the fatty acid composition of 

high oleic soybean oil and potential changes to commodity soybean oil due to comingling. 

NOPA and members of their biotechnology committee were provided information related to the 

fatty acid composition of high oleic soybean oil derived from MON 87705 in the Vistive lowlinolenic genetic background as well as oil properties and mixing effects information described 

previously. NOPA and biotechnology committee members agree that potential impacts related 

to the unintended mixing of commodity soybean oil and high oleic soybean oil could be 

remedied through blending, a common industry practice. Given the control points in the system 

and potential market impact of high oleic soybean oil, a risk mitigation plan (as described in 

Annex 2 of BIO’s Product Launch Stewardship Policy) for high oleic soybean is not warranted. 

NOPA and the committee members continue to provide input on various aspects of the intended 

commercialization. 

Summary 

High oleic soybean offers an opportunity for soybean growers to recapture markets for vegetable 

oil previously occupied by soybean oil and create added value for U.S. soybean producers. 

Given the abundance of vegetable oils on the market and demonstrated ability of the system to 

adapt to consumer preferences incorporating new oils into existing food manufacturing 

processes, the market impact of high oleic soybean is expected to be minimal and easily 

managed. Monsanto has conducted and is implementing a product stewardship plan that is based 
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upon consideration of BIO’s Launch Stewardship guidance and upon experience with previous 

successful product introductions completed by Monsanto. Monsanto continues to engage 

stakeholders and educate them on the benefits of high oleic soybean and proper stewardship 

practices. 
Page 18 of 127Table 4. Impact of comingling of high oleic soybean oil on Food Codex specifications of commodity soybea

1FCC – Food Chemical Codex specification for named vegetable oils 2HO – Oil profile represents high oleic MON 87705 soybean bred into a low linolenic soybean background. 

Legend. The impact of mixing high oleic soybean oil on the fatty acid profile of commodity soybean oil is depic

The impact to the fatty acid profile was assessed at various percentages of high oleic soybean oil mixed with comThe “break point” reflects the percentage of high oleic soybean oil at which the fatty acid levels in soybean oil w

the USP FCC specification range for soybean oil. 
Page 19 of 127Figure 3. Impact of comingling high oleic soybean oil with bottled commodity soybeal oil on nutritional faand polu unsaturated fatty acids. 

Legend. The impact of mixing high oleic soybean oil on nutritional facts labeling for bottled soybean oil is d

above. The impact to nutritional facts panel labeling was assessed at various percentages of high oleic soybeacommodity soybean oil. The blue arrows indicate the levels of high oleic soybean oil that would impact the n

labeling.
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Figure 4. Impact of comingling high oleic soybean oil with bottled commodity soybeal 

oil on nutritional facts labeling to saturated fatty acids. 

Legend. The impact of mixing high oleic soybean oil on nutritional facts labeling for bottled 

soybean oil is depicted in the figure above. The impact to nutritional facts panel labeling was 

assessed at various percentages of high oleic soybean oil mixed with commodity soybean oil. 

The blue arrows indicate the levels of high oleic soybean oil that would impact the nutritional 

facts labeling for mandatory fatty acid reporting (saturated fats).
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Figure 5. Oxidative stability index of high oleic soybean blended with commodity soybean 

oil. 

Legend. The impact of comingling of high oleic soybean oil was assessed on the oxidative 

stability index of the blended oil. The OSI stability index is an indicator of soybean oil stability. 

All oils and fats have a resistance to oxidation, which depends on the degree of saturation, 

antioxidant and prooxidant concentration, and prior abuse. Oxidation is slow until this resistance 

is overcome, at which point oxidation accelerates and becomes more rapid. The length of time 

prior to the acceleration of oxidation is referred to as the ‘induction period,’ and the point of 

maximum rate change is referred to as the Oxidative Stability Index or Oil Stability Index (OSI), 

and is reported in hours (Frankel, 2005).

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

0 10 20 30 40 50 60 70 80 90 100

OSI

@ 110oC

% High Oleic in Commercial Soy Oil
Page 22 of 127Figure 6. Sensory assessment of high oleic soybean oil compared to other vegetable oils. Legend. A sensory assessment of the quality of French fries prepared in blends of high oil/commodity soybean 

over a period of six days. A modification of the procedure described by King et al (2002) was used for the evaluqualities of the oil blends tested were essentially the same over the six-day period. Scale: 5 = no difference, 4 = 

3 = slight difference, 2 = different, 1 = very different. 
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References 

AOSCA 2009. Seed Certification Handbook. Association of Official Seed Certifying Agencies. 

Moline, Illinois. 

Delaney, B., Appenzeller, L. M., Munley, S. M., Hoban, D., Sykes, G. P., Malley, L. and 

Sanders, C. (2008) Subchronic feeding study of high oleic acid soybeans (Event DP-3Ø5423-1) 

in Sprague-Dawley rats, Food and Chemical Toxicology, 46, 3808-3817. 

Frankel, E.N., 2005. Pages 21, 201-205 in Lipid Oxidation. The Oily Press, Bridgewater, 

England. 

Graef, G., LaVallee, B.J., Tenopir, P., Tat, M., Schweiger, B., Kinney, A.J., Van Gerpen, J.H. 

and T.E. Clemente. 2009. A high-oleic-acid and low-palmitic-acid soybean: agronomic 

performance and evaluation as a feedstock for biodiesel. Plant Biotechnology Journal, 7:411-

421. 

ISO. 2009. Selection and Use of the ISO 9000 Family of Standards. International Organization 

for Standardization. http://www.iso.org [Accessed March, 2010]. 

King. S., Gillette, M., Titman, D., Adams, J., and M. Ridgely. 2002. The sensory quality 

system: a global quality control system. Food Quality and Preferences, 13: 385-395. 

Möllers, C. (2004) Potential and future prospects for rapeseed oil. In: Gunstone, F. D. (ed.) 

Rapeseed and Canola Oil. Production, Processing, Properties and Uses. CRC Press LLC, Boca 

Raton, FL, pp. 186-217. 

Soyatech. 2010. Statistics. Soyatech, Manitoba, Canada. 

Wilson, 2004. Seed composition. P. 621-629. In Boerma, H.R. and Specht, J.E. (eds.) 

Sopybeans: Improvement, production and uses. ASA-CSSA-SSSA, Madison, Wisconsin. 1144 

pp. 
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ATTACHMENT I 

THE MARKET POTENTIAL FOR A HIGH OLEIC SOYBEAN OIL 

CONFIDENTIAL BUSINESS INFORMATION 

 
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