Monsanto Company 11-SY-217U 1 of 467 

Petition for the Determination of Nonregulated Status for MON 87712 Soybean 

The undersigned submits this petition under 7 CFR § 340.6 to request that the 

Administrator make a determination that the article should not be regulated under 

7 CFR Part 340 

July 19, 2011 

OECD Unique Identifier: MON-87712-4 

Monsanto Petition Number: 11-SY-217U 

Submitted by:

Taiwo O. Koyejo, Ph.D. 

Monsanto Company 

800 North Lindbergh Blvd. 

St. Louis, MO 63167 

Phone: (314) 694-4021 

Fax: (314) 694-3080 

E-mail: taiwo.o.koyejo@monsanto.com 

Prepared by:

E. Bell, Ph.D., T.C. Best, B.S., A.H. Culler, Ph.D., E.C. Dickinson, B.S., J.C. Goley, 

M.S., T.O. Koyejo, Ph.D., K.E. Niemeyer, M.S., J.R. Phillips, Ph.D., G.J. Rogan, M.S., 

A. Skipwith, M.S., H. Su, M.S. 

Contributors and/or Principal Investigators:

B.M. Baltazar, Ph.D., L.D., Barberis, B.S., P.L. Bommireddy, Ph.D., C.R. Brown, M.A., 

D.B. Carson, Ph.D., F.G. Dohleman, Ph.D., M. Elrod, M.S., A. Evans, M.P.H., A.C. 

Fitzgerald, M.S., C.W. Garnaat, M.S., T. Geng, Ph.D., S.-W. Hoi, M.S., K.M. Huizinga, 

Ph.D., N. Ivleva, Ph.D., T.A. Kaempfe, B.S., M.S. Koch, Ph.D., K.R. Lawry, M.S., K.J. 

Mathis, S.A. Nolte, Ph.D., S. Paul, M.S., S.L. Phillips, M.S., C. Jiang, Ph.D., A. 

Silvanovich, Ph.D., D. Stojsin, Ph.D., S.J. Tauchman, M.S., Q. Tian, Ph.D., H. Tu, M.S., 

J.L. Vicini, Ph.D., R. Wang, M.S. 

 
Monsanto Company 11-SY-217U 2 of 467 

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 upon 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. 

©2011 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 11-SY-217U 4 of 467 

EXECUTIVE SUMMARY 

The Animal and Plant Health Inspection Service (APHIS) of the United States 

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 in the United States (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 is granted, thereby allowing unrestricted 

introduction of the article. 

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

nonregulated status for the new biotechnology-derived soybean product, MON 87712, 

any progeny derived from crosses between MON 87712 and conventional soybean, and 

any progeny derived from crosses of MON 87712 with biotechnology-derived soybean 

that have previously been granted nonregulated status under 7 CFR Part 340. 

Product Description

Soybean is one of the largest U.S. crops based on the acreage planted and quantity 

harvested each year. Global demand for soybean is expected to increase and as a major 

global supplier of soybean, the U.S. must increase soybean production to help meet 

growing demand. Increased soybean productivity in the U.S. has been accomplished by 

both increasing the area under cultivation and through yield increases per unit area. 

Based on recent trends in farm production and land area, most OECD (Organization for 

Economic Cooperation and Development) countries, including the U.S. and Canada, are 

predicted to face the challenge of expanding agricultural output by raising productivity on 

a stable or reduced land area. Thus, much of the projected expansion in soybean 

production in the future is expected to come from increased yield rather than increased 

area under production. 

Improvement in soybean yield remains one of the major objectives for plant breeders. 

Gains in major crop yields over the years can, in-part, be attributed to genetic 

improvement through traditional breeding. Breeders have crossed plants with different 

genetic backgrounds and selected traits resulting in higher yields, compositional 

improvements, and desirable production traits. Biotechnological approaches complement 

traditional soybean breeding efforts by targeting some of these same major characteristics 

as traditional breeding. 

Crop yield results from a sequential growth and development process – first the plant 

grows vegetatively and produces photosynthetic tissue, followed by flowering and the 

production of seeds, and finally seed filling and maturation. Actual yield performance is a 

complex parameter that is dependent on a number of genetic and environmental factors, 

that influence a crop’s opportunity to realize its full yield potential. Improvements in crop 

yield have been a primary focus of conventional breeding. The genetic changes that 

resulted in crop domestication and yield improvement in conventional varieties have been 
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shown by modern molecular biology analysis to have been typically achieved through the 

selection and safe use of plant genes encoding transcriptional regulator proteins. 

Agricultural biotechnology provides the opportunity to further enhance crop yields 

through the introduction of new genetic elements that use or modify existing pathways in 

the plant. 

Monsanto Company has developed the biotechnology-derived soybean line MON 87712, 

which will be used in traditional breeding programs to produce commercial soybean 

varieties with increased yield opportunity. The yield increase in MON 87712 is achieved 

using the BBX32 gene from the plant Arabidopsis thaliana that produces a protein that 

interacts with one or more endogenous transcription factors to regulate the plant’s 

day/night processes and results in increased availability of assimilates1

 in MON 87712 

compared to an appropriate comparator without this gene. Increased assimilate 

availability in MON 87712 is supported by the measurement of factors indicative of an 

extended period of photosynthetic activity in MON 87712 and evidence of changes in 

diurnal metabolism during the reproductive phase of the soybean plant, as well as by the 

significantly higher yield of MON 87712 when compared to control, as observed in 

multisite field studies in the U.S. 

Data and Information Presented Confirms the Lack of Plant Pest Potential of 

MON 87712 Compared to Conventional Soybean

The data and information presented in this petition demonstrate that MON 87712 is 

unlikely to pose an increase in plant pest potential, including weediness or adverse 

environmental impacts, compared to conventional soybean. The conclusion on the food, 

feed, and environmental safety of MON 87712 was based on multiple, well-established 

lines of evidence: 

1. Soybean is a highly domesticated crop that does not possess any of the attributes 

commonly associated with weeds and has a history of safe consumption. 

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

intact copy of the T-DNA insert in a single locus within the soybean genome. 

3. Data demonstrated that the BBX32 protein in MON 87712 is unlikely to be a 

toxin or allergen based on extensive information collected. 

4. A compositional assessment of harvested seed and forage confirmed that 

MON 87712 is compositionally equivalent to conventional soybean. 

5. An extensive evaluation of phenotypic and agronomic characteristics and 

environmental interactions of MON 87712 demonstrated no increased plant pest 

potential or adverse environmental impact compared to conventional soybean. 

 

1

 An Assimilate is a product of plant metabolism (Salisbury and Ross, 1992) from processes such 

as carbon and nitrogen fixation.
Monsanto Company 11-SY-217U 6 of 467 

6. An assessment of the potential impact on non-target organisms (NTOs) and 

endangered species indicated that MON 87712 is unlikely to have adverse effects 

on these organisms compared to conventional soybean. 

7. Evaluation of MON 87712 using typical cultivation and management practices for 

soybean concluded that deregulation of MON 87712 is expected to have minimal 

impact on soybean agronomic practices or land use, while providing the 

opportunity to increase the yield of soybean and help meet growing global 

demand for soybean. 

Soybean is a Highly Domesticated Crop Lacking Weedy Characteristics 

There is a longstanding history of safe use and consumption of conventional soybean and 

processed soybean products. Soybean is grown as a commercial crop in over 35 

countries. Domestication occurred as early as 1000 B.C. and soybean is now the most 

widely grown oilseed crop in the world, with approximately 258.4 million metric tons of 

harvested seed produced in 2010, which represented 58% of world oilseed production 

that year. 

During the period from 2004 to 2010, U.S. growers planted between 75.2 and 76.6 

million acres of soybean per year. The commercial soybean species in the U.S. (Glycine

max L. Merr.) does not exhibit weedy characteristics, does not invade established 

ecosystems, and does not outcross to weedy or wild relatives. 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 (7 CFR Part 360). Soybean does not possess 

attributes commonly associated with weeds, such as the ability to disperse, invade, or 

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 it is known that soybean 

seed can germinate quickly under adequate temperature and moisture conditions, and can 

potentially grow as a volunteer plant. However, volunteer soybean plants are generally 

killed by frost during the autumn or winter of the year it germinated. Furthermore, if 

volunteer plants survive, they do not compete well with the succeeding crop, and are 

controlled readily via mechanical or other chemical means. Finally, since wild 

populations of Glycine species are not known to exist in the U.S., there is no opportunity 

for soybean, including MON 87712, to outcross to wild or weedy relatives. 

Conventional Soybean A3525 is an Appropriate Comparator to MON 87712

Soybean variety A3525 is the parental line of MON 87712, which was derived from a 

single plant transformant of the A3525 variety. A3525 was used as the conventional 

soybean comparator to support the safety assessment of MON 87712. MON 87712 and 

the conventional control A3525 have similar genetic backgrounds with the exception of 

the BBX32 expression cassette, thus, the effect of the BBX32 expression cassette and the 

expressed BBX32 protein can be assessed in an unbiased manner using a comparative 

safety assessment. 
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Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

in MON 87712

MON 87712 was developed through Agrobacterium-mediated transformation of 

conventional soybean A3525 meristem tissue with plasmid vector PV-GMAP5779. 

PV-GMAP5779 contains two T-DNAs, each delineated by Left and Right Border regions 

to facilitate transformation. The first T-DNA, designated as T-DNA I, contains the 

BBX32 coding sequence, derived from Arabidopsis thaliana, under regulation of the e35S

promoter and the E6 3′ untranslated region. The second T-DNA, designated as 

T-DNA II, contains the cp4 epsps coding sequence under the regulation of the 

FMV/EF-1α promoter, EF-1α leader, EF-1α intron, CTP2 targeting sequence, and the E9

3′ untranslated region. During transformation, both T-DNAs were inserted into the 

soybean genome where T-DNA II, containing the cp4 epsps expression cassette, 

functioned as a marker gene for the selection of transformed plantlets. Subsequently, 

conventional self-pollinated breeding methods and segregation, along with a combination 

of analytical techniques, were used to isolate those plants that contained the BBX32

expression cassette (T-DNA I) but did not contain the cp4 epsps expression cassette 

(T-DNA II), resulting in the production of marker-free MON 87712. 

Molecular characterization of MON 87712 by Southern blot analyses confirmed that one 

copy of the BBX32 expression cassette (T-DNA I) was integrated into the soybean 

genome at a single locus. No T-DNA II or backbone DNA sequences from plasmid 

vector PV-GMAP5779 were detected in MON 87712. The complete DNA sequence of 

the insert and adjacent genomic DNA sequence in MON 87712 confirmed the integrity of 

the inserted BBX32 expression cassette within the inserted sequences and identified the 5′

and 3′ insert-to-genomic DNA junctions. Additionally, Southern blot analysis of 

MON 87712 demonstrated that the inserted DNA has been maintained through five 

generations of breeding, thereby, confirming the stability of the insert over multiple 

generations. 

Data Confirms BBX32 Protein Safety

MON 87712 contains the BBX32 expression cassette that encodes the BBX32 protein. 

BBX32 is a transcriptional accessory protein that interacts with one or more endogenous 

transcription factors to regulate the plant’s day/night processes and results in increased 

availability of assimilates in the plant. BBX32 is a protein that has homologs in food 

plants with a history of safe use. 

A multistep approach was used to characterize and assess the safety of BBX32 protein in 

MON 87712. This detailed characterization and assessment confirmed that the BBX32 

protein in MON 87712 is safe for human and animal consumption. The assessment 

involved: 1) characterization of the physicochemical and functional properties of BBX32; 

2) quantification of BBX32 levels in plant tissues; 3) comparison of the amino acid 

sequence of BBX32 in MON 87712 to known allergens, gliadins, glutenins, toxins, and 

other biologically-active proteins known to have adverse effects on mammals; 4) 

evaluation of the digestibility of BBX32 protein in simulated gastric and intestinal fluids; 

5) documentation of the presence of related proteins in several plant species currently 
Monsanto Company 11-SY-217U 8 of 467 

consumed; and 6) investigation of the potential mammalian toxicity through an oral 

gavage assay. 

Western blot analysis demonstrated that BBX32 was detected in leaf and root of 

MON 87712, and was below the limit of detection in harvested seed and forage. 

Bioinformatics analysis determined that BBX32 does not share amino acid sequence 

similarities with known allergens, gliadins, glutenins, or protein toxins. In addition, 

BBX32 was rapidly digested in in vitro assays using simulated gastric and intestinal 

fluids and did not show any adverse effects when administered to mice via oral gavage. 

The acute gavage study results, combined with undetectable levels in harvested seed and 

forage result in margins of exposure (MOE) exceeding one million for the most exposed 

population in the U.S. The protein safety assessment supports the conclusion that BBX32 

poses no meaningful risk to the environment, human, or animal health. 

MON 87712 is Compositionally Equivalent to Conventional Soybean 

Detailed compositional analyses were conducted in accordance with soybean-specific 

OECD guidelines to assess levels of key nutrients and anti-nutrients in MON 87712 

compared to levels present in the parental conventional soybean control A3525 as well as 

conventional commercial reference varieties. These compositional comparisons were 

made by analyzing the harvested seed and forage harvested from plants grown at each of 

eight field sites in the U.S. during the 2009 field season. The conventional commercial 

reference varieties used to establish a range of natural variability for the key nutrients and 

anti-nutrients in conventional commercial soybean varieties have a history of safe 

consumption. Nutrients assessed in this study included proximates, fiber, amino acids, 

fatty acids, and vitamin E in harvested seed, and proximates and fiber in forage. The 

anti-nutrients assessed in harvested seed included raffinose, stachyose, lectin, phytic acid, 

trypsin inhibitors, and isoflavones. 

The combined-site analysis was conducted to identify statistically significant differences 

(α=0.05) between MON 87712 and the conventional control A3525. The results from the 

combined-site data were reviewed using considerations relevant to food and feed safety 

and nutritional quality. These considerations included assessments of: 1) the relative 

magnitude of the differences in the mean values of nutrient and anti-nutrient components 

of MON 87712 and the conventional control, 2) whether the MON 87712 component 

mean value was within the range of natural variability of that component as represented 

by the 99% tolerance interval of the conventional commercial reference varieties grown 

concurrently in the same field trial, 3) the reproducibility of the statistically significant 

combined-site component differences at individual sites, and 4) the differences within the 

context of natural variability of commercial soybean composition published in the 

scientific literature and in the International Life Sciences Institute (ILSI) Crop 

Composition Database. 

Assessment of the analytical results concluded that the differences observed in the 

combined-site analysis were not meaningful to food and feed safety or the nutritional 

quality of MON 87712 soybean. In addition, the levels of assessed components in 

MON 87712 were compositionally equivalent to the conventional control and within the 
Monsanto Company 11-SY-217U 9 of 467 

range of variability of the conventional commercial reference varieties that were grown 

concurrently in the same field trial. It is concluded that harvested soybean seed and 

soybean forage produced from MON 87712 are compositionally equivalent to harvested 

seed and forage of the conventional soybean and that the introduction of BBX32 in 

MON 87712 does not have a meaningful impact on the composition and therefore on the 

food and feed safety, nutritional quality, and familiarity (as discussed below) of 

MON 87712 compared to conventional soybean. 

MON 87712 Does Not Change Soybean Plant Pest Potential or Environmental 

Interactions

Plant pest potential of a biotechnology-derived crop is assessed on the basis of familiarity 

that the USDA recognizes as an important underlying concept in risk assessment. The 

concept of familiarity is based on the fact that the biotechnology-derived plant is 

developed from a conventional plant hybrid or variety whose biological properties and 

plant pest potential are well known. Familiarity considers the biology of the plant, 

knowledge of the introduced trait, the receiving environment, and the interactions among 

these factors. This provides a basis for comparative risk assessment between a 

biotechnology-derived plant and the conventional control. Thus, the phenotypic, 

agronomic, and environmental interaction assessment of MON 87712 included the 

parental conventional control A3525 as a comparator. This evaluation used a weight of 

evidence approach and considered statistical differences between MON 87712 and the 

conventional control A3525 with respect to reproducibility, magnitude, and directionality 

of any detected differences. Conventional commercial reference varieties were used to 

establish a range representative of natural variability in soybean for each characteristic 

assessed, and provided a context from which to further evaluate any statistical 

differences. Characteristics assessed included: seed dormancy and germination, pollen 

morphology, and symbiont interactions in the greenhouse, plant phenotypic observations, 

environmental interaction evaluations, and volunteer potential and persistence outside of 

cultivation evaluations conducted in the field. The phenotypic, agronomic, and 

environmental interaction assessment demonstrated that MON 87712 does not differ from 

the conventional control A3525 in any biologically meaningful way indicative of 

increased pest potential or adverse environmental impact. Thus, MON 87712 is unlikely 

to have increased weediness or plant pest potential compared to conventional soybean. 

Seed dormancy and germination characterization demonstrated that the seed of 

MON 87712 had germination characteristics not fundamentally different from seed of 

the conventional control A3525. In particular, the lack of hard seed, a well-recognized 

characteristic of weediness affecting seed germination, supports a conclusion of no 

increased weediness of MON 87712 when compared to the conventional control A3525. 

For pollen characteristics and symbiont interactions, no statistically significant 

differences were observed between MON 87712 and the conventional control A3525 for 

any of the parameters measured, including pollen viability and diameter, nodule number 

and dry weight, shoot total nitrogen, and shoot and root dry weight. These results support 

the conclusion that MON 87712 is not likely to exhibit increased plant pest potential 

compared to conventional soybean. 
Monsanto Company 11-SY-217U 10 of 467 

The field evaluation of phenotypic, agronomic, and environmental interaction 

characteristics of MON 87712 also support the conclusion that MON 87712 is not likely 

to have an increased plant pest potential compared to conventional soybean. The 

evaluations were conducted at 19 replicated field sites in soybean production regions 

across the U.S. These assessments included plant growth and development 

characteristics, as well as observations for plant responses to abiotic stressors, plantdisease, and plant-arthropod interactions. The observed phenotypic characteristics were 

not fundamentally different between MON 87712 and the conventional control A3525. 

In the combined-site analysis, no statistically significant differences were detected 

between MON 87712 and the conventional control A3525 for seedling vigor, days to 

50% flowering, days to 50% end of flowering, plant height, lodging, pod shattering, grain 

moisture, or 100 seed weight. Statistically significant differences were observed between 

MON 87712 and the conventional control A3525 for early stand count, days to 50% 

senescence, days to physiological maturity, final stand count, and yield. Although 

significantly different from the conventional control A3525, the mean values of 

MON 87712 for early stand count, and final stand count were within the range of 

commercial reference varieties for each characteristic and thus would not be adverse in 

terms of pest potential. Differences in days to 50% senescence, days to physiological 

maturity, and yield were consistent with the mode of action of the introduced trait in 

MON 87712. The increase in yield is agronomically desirable and would not contribute 

to increased weediness potential of MON 87712 without changes in a combination of 

other characteristics associated with weediness. 

Evaluations of volunteer potential and persistence outside of cultivation from field-grown 

plants provide information useful in assessing potential weediness characteristics of 

MON 87712 compared to the conventional control A3525. Evaluation of volunteer 

potential demonstrated no statistically significant differences between MON 87712 and 

the conventional control A3525. Evaluation of persistence outside of cultivation 

demonstrated a few statistically significant differences between MON 87712 and the 

conventional control A3525, however these differences were not seen across all sites and 

were small in magnitude. Taken together, these comparative assessments indicate that 

MON 87712 is not likely to have increased weediness or plant pest potential compared to 

conventional soybean. 

In summary, the phenotypic, agronomic, and environmental interaction data were 

generated and evaluated to characterize MON 87712, and to assess whether the 

introduction of the trait in MON 87712 alters the plant pest potential compared to 

conventional soybean. The evaluation, using a weight of evidence approach, considered 

the reproducibility, magnitude, and direction of detected differences between 

MON 87712 and the conventional control, and comparison to the range of the 

commercial reference varieties. The results indicated that MON 87712 does not possess 

weediness characteristics, increased susceptibility or tolerance to specific abiotic stress, 

diseases, or arthropods, or characteristics that would confer a plant pest risk or a 

significant adverse environmental impact compared to conventional soybean. 

 
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MON 87712 Will Not Adversely Affect NTOs or Threatened or Endangered Species

Evaluation of the impacts of a biotechnology-derived crop on Non-Target Organisms 

(NTO) and threatened and endangered species is a component of the plant pest risk 

assessment. Since MON 87712 does not possess pesticidal activity, all organisms that 

interact with MON 87712 are considered to be NTOs. The environmental assessment 

demonstrated that the presence of BBX32 protein in MON 87712 did not meaningfully 

alter plant-arthropod interactions, including beneficial arthropods, or disease 

susceptibility compared to the conventional control A3525. 

The biochemical information and experimental data for evaluation of MON 87712 

included molecular characterization, BBX32 protein safety assessments, information 

from the environmental interaction assessment, demonstration of compositional 

equivalence to conventional soybean, and demonstration of agronomic and phenotypic 

equivalence to conventional soybean (with the exception of presence of BBX32 protein 

in MON 87712). Taken together, these data support the conclusion that MON 87712 has 

no reasonable mechanism to harm NTOs, nor does it pose an additional risk to threatened 

and endangered species compared to the cultivation of conventional soybean. 

Outcrossing and gene introgression from MON 87712 to sexually-compatible species in 

the U.S. is unlikely since no known wild Glycine species related to cultivated soybean are 

known to be present in North America. Furthermore, should cross-pollination occur, 

MON 87712 and its progeny are not expected to exhibit a significant environmental 

impact because, as described above, evaluations have shown that the presence of BBX32 

is not likely to enhance weediness or plant-pest potential. Therefore, the likelihood and 

environmental consequence of pollen transfer from MON 87712 to other Glycine species 

is considered negligible. 

Deregulation of MON 87712 Will Not Significantly Impact Soybean Agronomic 

Practices or Land Use

Introduction of MON 87712 is expected to have minimal impact on current cultivation 

and management practices for soybean. MON 87712 has been shown to be no different 

from conventional soybean regarding pest potential and compositional characteristics, 

and has the same levels of tolerance or susceptibility to insects and diseases as 

commercial soybean. MON 87712 did not require any additional inputs to produce a 

crop. The intended increase in yield is not expected to significantly impact any of the 

agricultural practices farmers use to produce a soybean crop. Farmers understand the 

value of increased yield for their farm’s productivity and profitability, and are 

accustomed to the incremental yield improvements for varieties obtained through 

traditional breeding. Growers are also accustomed to experiencing field-to-field and yearto-year yield variation based on environmental conditions and the genetics of the varieties 

they select for planting. Therefore, growers are capable of adjusting harvesting and 

storage equipment to handle increased yields. MON 87712 will provide another option to 

farmers to pursue better yielding varieties for their farm. MON 87712 offers the potential 

to improve productivity in the U.S. soybean production system, thereby helping to meet 

the growing global demand for soybean. 
Monsanto Company 11-SY-217U 12 of 467 

Conclusion

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

MON 87712 is not likely to be a plant pest. Therefore, Monsanto Company requests a 

determination from APHIS that MON 87712 and any progeny derived from crosses 

between MON 87712 and conventional soybean or deregulated biotechnology-derived 

soybean be granted nonregulated status under 7 CFR Part 340. 
Monsanto Company 11-SY-217U 13 of 467 

TABLE OF CONTENTS 

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

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

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

TABLE OF CONTENTS ................................................................................................13

LIST OF TABLES ...........................................................................................................18

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

ABBREVIATION AND DEFINITIONS ......................................................................25

I. RATIONALE FOR THE DEVELOPMENT OF MON 87712 ...............................27

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

7 CFR § 340.6 ...............................................................................................................27

I.B. Rationale for the Development of MON 87712 ....................................................27

I.C. Submissions to Other Regulatory Agencies ..........................................................29

I.C.1. Submission to FDA .....................................................................................29

I.C.2. Submissions to Foreign Government Agencies ..........................................29

II. THE BIOLOGY OF SOYBEAN ..............................................................................30

II.A. Soybean as a Crop ................................................................................................30

II.B. History of Soybean ...............................................................................................31

II.C. Taxonomy and Phylogenetics of Soybean ...........................................................31

II.D. The Genetics of Soybean .................................................................................34

II.E. Pollination of Cultivated Soybean ...................................................................34

II.F. Cultivated Soybean as a Volunteer ..................................................................35

II.G. Characteristics of the Recipient Plant ..................................................................35

II.H. Soybean as a Test System in Product Safety Assessment ...................................35

III. DESCRIPTION OF THE GENETIC MODIFICATION ....................................36

III.A. The Plasmid Vector PV-GMAP5779 .................................................................36

III.B. Description of the Transformation System .........................................................36

III.C. The BBX32 Coding Sequence and the BBX32 Protein (T-DNA I) ....................37

III.D. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (TDNA II) .........................................................................................................................37

III.E. Regulatory Sequences .........................................................................................40

III.F. T-DNA Borders ...................................................................................................40

III.G. Genetic Elements Outside of the T-DNA Borders .............................................40

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION .....................46
Monsanto Company 11-SY-217U 14 of 467 

IV.A. Southern Blot Analysis to Determine Insert and Copy Number of 

T-DNA I in MON 87712 ...............................................................................................52

IV.A.1. Probe 1 .....................................................................................................52

IV.A.2. Probe 2 .....................................................................................................53

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

T-DNA II Sequences in MON 87712 ............................................................................56

IV.B.1. Probe 3 and Probe 5 .................................................................................56

IV.B.2. Probe 4 .....................................................................................................56

IV.C. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GMAP57779 Backbone Sequences in MON 87712 ...............................................60

IV.C.1. Plasmid Vector Backbone Probe 6 ...........................................................60

IV.C.2. Plasmid Vector Backbone Probe 7 and Probe 9 ......................................60

IV.C.3. Plasmid Vector Backbone Probe 8 ...........................................................61

IV.D. Organization and Sequence of the Insert and Adjacent DNA in 

MON 87712 ...................................................................................................................65

IV.E. PCR and DNA Sequence Analyses to Examine the MON 87712 

Insertion Site ..................................................................................................................67

IV.F. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 87712 ..........................................................................................69

IV.F.1. Probe 1 ......................................................................................................69

IV.G. Inheritance of the Genetic Insert in MON 87712 ...............................................72

IV.H. Genetic Modification Characterization Conclusion ...........................................75

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE 

BBX32 PROTEIN IN MON 87712 .................................................................................76

V.A. Identity and Function of the BBX32 Protein from MON 87712 .........................76

V.B. Characterization and Equivalence of BBX32 Protein from MON 87712 ...........76

V.C. Expression Levels of BBX32 Protein in MON 87712 ........................................78

V.D. Assessment of Potential Allergenicity of the BBX32 Protein .............................81

V.E. Safety Assessment Summary of BBX32 Protein in MON 87712 .......................81

V.E.1. The Donor Organism is Safe .....................................................................81

V.E.2. BBX32 Protein Belongs to a Common Class of Plant Proteins ................82

V.E.3. BBX32 Protein in MON 87712 is Not Homologous to a Known 

Allergen or Toxin ...................................................................................................82

V.E.4. BBX32 Protein in MON 87712 is Labile in in vitro Digestion 

Assays ....................................................................................................................82

V.E.5. BBX32 Protein in MON 87712 is Not Acutely Toxic ..............................82

V.F. BBX32 Protein Characterization and Safety Conclusion ....................................83
Monsanto Company 11-SY-217U 15 of 467 

VI. COMPOSITIONAL ASSESSMENT OF MON 87712 .........................................85

VI.A. Compositional Equivalence of MON 87712 Seed and Forage to 

Conventional Soybean ...................................................................................................85

VI.A.1. Nutrient Levels in Harvested Soybean Seed ............................................87

VI.A.2. Anti-Nutrient Levels in Soybean Seed ....................................................89

VI.A.3. Nutrient Levels in Soybean Forage .........................................................89

VI.B. Compositional Assessment of MON 87712 Conclusion ..................................115

VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT ...............................................................................117

VII.A. Characteristics Measured for Assessment ......................................................117

VII.B. Interpretation of Phenotypic and Environmental Interaction Data .................121

VII.C. Comparative Assessments of the Phenotypic, Agronomic, and 

Environmental Interaction Characteristics of MON 87712 ........................................124

VII.C.1. Seed Dormancy and Germination Characteristics ................................124

VII.C.2. Field Phenotypic, Agronomic Characteristics, and Environmental 

Interactions ...........................................................................................................127

VII.C.3. Pollen Characteristics ...........................................................................133

VII.C.4. Symbiont Interactions ...........................................................................135

VII.C.5. Volunteer Potential Assessment ...........................................................138

VIII. U.S. AGRONOMIC PRACTICES.....................................................................142

VIII.A. Introduction ...................................................................................................142

VIII.B Soybean Yield .................................................................................................142

VIII.C. Overview of U.S. Soybean Production ..........................................................144

VIII.C.1. Soybean Production .............................................................................144

VIII.C.2. Variety Development ..........................................................................150

VIII.C.3. Soybean Seed Production ....................................................................150

VIII.D. Production Management Considerations .......................................................153

VIII.D.1. Pre-Season ............................................................................................153

VIII.D.2. Planting and Early Season ...................................................................155

VIII.D.3. Mid to Late Season ..............................................................................157

VIII.D.4. Harvest Season ....................................................................................158

VIII.E. Management of Insects ..................................................................................159

VIII.F. Management of Diseases and Other Pests ......................................................159

VIII.G. Weed Management ........................................................................................161

VIII.H. Crop Rotation Practices in Soybean ..............................................................162
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VIII.I. Soybean Volunteer Management ....................................................................163

VIII.J. Stewardship of MON 87712 ...........................................................................165

VIII.K. Impact of the Introduction of MON 87712 on Agricultural Practices ..........165

IX. ENVIRONMENTAL CONSEQUENCES ...........................................................167

IX.A. Introduction ......................................................................................................167

IX.B. Plant Pest Assessment of MON 87712 Insert and Expressed Protein .............168

IX.B.1. Characteristics of the Genetic Insert and Expressed Protein .................168

IX.B.2. Compositional Characteristics ...............................................................169

IX.B 3. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics ......................................................................................................170

IX.C. Weediness Potential of MON 87712 ................................................................173

IX.D. Potential for Pollen Mediated Gene Flow ........................................................173

IX.D.1. Hybridization with Cultivated Soybean .................................................174

IX.D.2. Hybridization with Wild Annual Species within Subgenus Soja ..........174

IX.D.3. Hybridization with the Wild Perennial Species of Subgenus 

Glycine .................................................................................................................175

IX.D.4. Transfer of Genetic Information to Species with which Soybean 

Cannot Interbreed (Horizontal Gene Flow) .........................................................175

IX.E. Potential Impact on Soybean Agronomic Practices ..........................................176

IX.F. Summary of Plant Pest Assessments ................................................................176

X. ADVERSE CONSEQUENCES OF INTRODUCTION .......................................179

REFERENCES ...............................................................................................................180

APPENDICES ................................................................................................................196

Appendix A: USDA Notifications ................................................................................197

Appendix B. Mode of Action of BBX32 Protein in MON 87712 ..............................200

Appendix C: Materials, Methods, and Results for Molecular Analyses of 

MON 87712 .....................................................................................................................230

Appendix D: Materials, Methods and Results for Characterization of BBX32 

Protein Produced in MON 87712 .................................................................................237

Appendix E: Materials and Methods Used for the Analysis of the Levels of 

BBX32 Protein in MON 87712 ......................................................................................244

Appendix F: Materials, Methods, and Individual-Site Results for 

Compositional Analysis of MON 87712 Soybean Seed and Forage ..........................248

Appendix G: Materials, Methods, and Individual-Site Results for Seed 

Dormancy and Germination Assessment of MON 87712 ..........................................339
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Appendix H: Materials, Methods, and Individual-Site Results from 

Phenotypic, Agronomic, and Environmental Interaction Assessment of 

MON 87712 under Field Conditions ............................................................................346

Appendix I: Materials and Methods for Pollen Morphology and Viability 

Assessment ......................................................................................................................386

Appendix J: Materials and Methods for Symbiont Assessment ...............................389

Appendix K: Materials and Methods for Volunteer Potential Assessment .............393

Appendix L: Materials and Methods for Persistence Outside of Cultivation 

Assessment ......................................................................................................................399

Appendix M: Petitioner’s Environmental Report .....................................................411
Monsanto Company 11-SY-217U 18 of 467 

LIST OF TABLES 

Table II-1. World Soybean Production in 2010 .........................................................30

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

Number, Genome Symbol, and Distribution .................................................................33

Table III-1. Summary of Genetic Elements in the PV-GMAP5779 ...........................42

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

Hybridizing Probes and Restriction Enzymes Used in MON 87712 Analysis ...........50

Table IV-2. Summary of Genetic Elements in MON 87712 ........................................51

Table IV-3. Segregation of the Expression Cassette During the Development 

of MON 87712 ..................................................................................................................74

Table V-1. Summary of BBX32 Protein Levels in Leaf, Root, Forage, and 

Seed from MON 87712 Grown in 2009 U.S. Field Trials .............................................80

Table VI-1. Summary of Differences (p<0.05) for the Comparison of 

Soybean Component Levels for MON 87712 vs. Conventional Control ....................91

Table VI-2. Summary of Combined-site Soybean Seed Nutrients for 

MON 87712 vs. Conventional Control .........................................................................103

Table VI-3. Summary of Combined-site Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................109

Table VI-4. Summary of Combined-site Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................111

Table VI-5. Literature and ILSI Database Ranges for Components in 

Soybean Forage and Seed ..............................................................................................113

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials, Laboratory or Greenhouse 

Tests .................................................................................................................................119

Table VII-2. Germination Characteristics of MON 87712 and the 

Conventional Control A3525 ........................................................................................126

Table VII-3. Field Phenotypic Evaluation Sites for MON 87712 during 2009........129

Table VII-4. Combined-Site Comparison of MON 87712 to Conventional 

Control A3525 During 2009 for Phenotypic and Agronomic Characteristics .........130

Table VII-5. Pollen Characteristics of MON 87712 Compared to the 

Conventional Control A3525 ........................................................................................134

Table VII-6. Symbiont Interaction Assessment of MON 87712 and 

Conventional Control A3525 ........................................................................................137

Table VIII-1. Soybean Production in the U.S, 2001 – 20101 .....................................147

Table VIII-2. U.S. Soybean Production by Region and State in 2010 .....................148

Table VIII-3. U.S. Soybean Production Costs and Returns in 20101 .......................149
Monsanto Company 11-SY-217U 19 of 467 

Table VIII-4. Ratings for Postemergence Control of Volunteer Soybean in 

Labeled Rotational Crops1

 ............................................................................................164

Table IX-1. Summary of Published Literature on Soybean Cross Pollination .......178

Table A-1. USDA Notifications and PermitsApproved for MON 87712 and 

Status of Trials Conducted under These Notifications ...............................................198

Table B-1. Soybean reproductive stages as defined by Fehr and Caviness 

(1981) ...............................................................................................................................203

Table B-2. Combined-Site Least Square Means, P-values, and Percent 

Differences of Yield and Yield Components of MON 87712 Compared to the 

Negative Segregant Control [MON 87712 (-)] from U.S. 2009 Field Trials (19 

sites) 210

Table B-3. Combined-Site Least Square Means, P-values, and Percent 

Differences of Yield and Yield Components of MON 87712 Compared to the 

Conventional Control, A3525 from U.S. 2009 Field Trials (19 sites) ........................211

Table B-4: Summary of significant differences between MON 87712 soybean 

and control (MON 87712(-) and A3525) leaf tissue at the R1 developmental 

stage .................................................................................................................................220

Table B-5: Summary of significant differences between MON 87712 soybean 

and control (MON 87712(-) and A3525) leaf tissue collected at the R6 

developmental stage .......................................................................................................221

Table C-1. Hybridization Conditions of Utilized Probes ..........................................233

Table D-1. Purity of the MON 87712-Produced BBX32 Protein .............................242

Table F-1. Commercial Reference Soybean Varieties ...............................................248

Table F-2. Re-expression Formulas for Statistical Analysis of Composition 

Data .................................................................................................................................254

Table F-3. Component with Observations Below the Assay Limit of 

Quantification Not Excluded from Statistical Analysis ..............................................255

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................257

Table F-5. Summary of Site ARNE Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................263

Table F-6. Summary of Site ARNE Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................265

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................267

Table F-8. Summary of Site ILCY Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................273

Table F-9. Summary of Site ILCY Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................275
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Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................277

Table F-11. Summary of Site ILHI Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................283

Table F-12. Summary of Site ILHI Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................285

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................287

Table F-14. Summary of Site ILWY Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................293

Table F-15. Summary of Site ILWY Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................295

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................297

Table F-17. Summary of Site INRC Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................303

Table F-18. Summary of Site INRC Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................305

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................307

Table F-20. Summary of Site INSH Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................313

Table F-21. Summary of Site INSH Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................315

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 

vs. Conventional Control ...............................................................................................317

Table F-23. Summary of Site KSLA Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................323

Table F-24. Summary of Site ILCY Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................325

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for 

MON 87712 vs. Conventional Control .........................................................................327

Table F-26. Summary of Site NEYO Soybean Seed Anti-nutrients for 

MON 87712 vs. Conventional Control .........................................................................333

Table F-27. Summary of Site NEYO Soybean Forage Nutrients for 

MON 87712 vs. Conventional Control .........................................................................335

Table G-1. Starting Seed of MON 87712, Conventional Control and 

Commercial Reference Varieties Used in Dormancy Assessment .............................342
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Table G-2. Dormancy and Germination Characteristics of MON 87712 and 

the Conventional Control A3525 Seed Produced at each of the Three Field 

Sites..................................................................................................................................343

Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental 

Interaction Assessment ..................................................................................................348

Table H-2. Field and Planting Information ................................................................349

Table H-3. Data Missing or Excluded from Analysis ................................................354

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to 

Conventional Control A3525 ........................................................................................364

Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control 

A3525, and the Commercial Reference Varieties .......................................................370

Table H-6. Abiotic Stress Response Evaluations of MON 87712 and 

Conventional Control A3525 Using an Observational Severity Scale ......................374

Table H-7. Disease Damage Evaluations of MON 87712 and Conventional 

Control A3525 Using an Observational Severity Scale ..............................................375

Table H-8. Arthropod-Related Damage Evaluations of MON 87712 and 

Conventional Control A3525 Using an Observational Severity Scale ......................376

Table H-9. Abundance of Pest Arthropods in Samples Collected from 

MON 87712, Conventional Control A3525, and the Commercial Reference 

Varieties ..........................................................................................................................378

Table H-10. Abundance of Beneficial Arthropods in Samples Collected from 

MON 87712, Conventional Control A3525, and the Commercial Reference 

Varieties ..........................................................................................................................382

Table J-1. Starting Seed of MON 87712, Conventional Control, and 

Commercial Reference Varieties Used in the Symbiont Assessment ........................389

Table K-1. Starting Seed for Volunteer Potential Assessment .................................394

Table K-2. Seeding Information, Soil Description, and Cropping History 

Information for Volunteer Potential Assessment ........................................................396

Table L-1. Starting Seed for Persistence Outside of Cultivation Assessment .........399

Table L-2. Seeding Information, Soil Description, and Cropping History 

Information for Persistence Outside of Cultivation Assessment ...............................402

Table L-3. Study Area Planting Information and Soil Description for 2010 

Extended Season at MOCB Site ...................................................................................402

Table L-4. Data Characteristics Evaluated at Each Site for Persistence 

Outside of Cultivation Assessment ...............................................................................403

Table L-5. Growth Stage Monitoring of MON 87712, the Conventional 

Control A3525, and the Commercial Reference Varieties in 2009 ............................406

Table L-6. Stand Counts of MON 87712, the Conventional Control A3525, 

and the Commercial Reference Varieties in 2009 .......................................................407
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Table L-7. Plant Vigor Monitoring of MON 87712, the Conventional Control 

A3525, and the Commercial Reference Varieties in 2009 ..........................................408

Table L-8. Phenotypic Comparison of MON 87712 to the Control within 

Each Site in 2009 ............................................................................................................409

Table M-1. 2010 Soybean Productivity by Region .....................................................421

Table M-2. Deregulated or Submitted Biotechnology-derived Soybean 

Products ..........................................................................................................................458
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LIST OF FIGURES 

Figure III-1. Circular Map of PV-GMAP5779 Showing Probe 1 through 

Probe 9 ..............................................................................................................................38

Figure III-2. Schematic of the Development of MON 87712 ......................................39

Figure III-3. Deduced Amino Acid Sequence of the BBX32 Protein in MON 

87712..................................................................................................................................45

Figure IV-1. Schematic Representation of the Insert and Flanking DNA 

Sequences in MON 87712 ................................................................................................49

Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number 

of T-DNA I in MON 87712: Probe 1 .............................................................................54

Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number 

of T-DNA I in MON 87712: Probe 2 .............................................................................55

Figure IV-4. Southern Blot Analysis to Detect the Presence or Absence of 

T-DNA II Sequences in MON 87712: Probe 3 and Probe 5 ........................................58

Figure IV-5. Southern Blot Analysis to Detect the Presence or Absence of 

T-DNA II Sequences in MON 87712: Probe 4..............................................................59

Figure IV-6. Southern Blot Analysis to Determine the Presence or Absence 

of PV-GMAP5779 Backbone Sequences in MON 87712: Probe 6 .............................62

Figure IV-7. Southern Blot Analysis to Determine the Presence or Absence 

of PV-GMAP5779 Backbone Sequences in MON 87712: Probe 7 and Probe 

 9.........................................................................................................................................63

Figure IV-8. Southern Blot Analysis to Determine the Presence or Absence 

of PV-GMAP5779 Backbone Sequences in MON 87712: Probe 8 .............................64

Figure IV-9. Overlapping PCR Analysis Across the Insert in MON 87712 ..............66

Figure IV-10. PCR Amplification of the MON 87712 Insertion Site .........................68

Figure IV-11. Breeding History of MON 87712 ...........................................................70

Figure IV-12. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 87712: Probe 1 .............................................................................71

Figure IV-13. Breeding Path for Generating Segregation Data for 

MON 87712 .......................................................................................................................73

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

Interpretation Methods .................................................................................................122

Figure VIII-1. U.S. soybean yield rose at an annual average rate of 0.35 bu/A 

between 1924 – 2010.......................................................................................................143

Figure VIII-2. U.S. Net Value of Soybean Production, Dollars Per Acre ................147

Figure B-1. U.S. soybean yield rose at an annual average rate of 0.35 bu/A 

between 1924 – 2010.......................................................................................................201

Figure B-2. Determination of yield in conventional soybean. ....................................204
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Figure B-3. Diurnal cycling of processes such as nitrogen and carbon 

metabolism in plants is achieved through light signaling and the circadian 

clock .................................................................................................................................206

Figure B-4. Leaf Area Index and Light Interception ................................................214

Figure B-5. Leaf Photosynthetic Rate Data ................................................................215

Figure B-6. Gene Expression Patterns for two Examples of Diurnally 

Regulated Genes in MON 87712, MON 87712(-) and A3525 ....................................218

Figure B-7: BBX32 expression in MON 87712 leads to increased yield ..................224

Figure D-1. Western Blot Analysis of MON 87712- and E. coli -produced 

BBX32 Proteins ..............................................................................................................241

Figure D-2. BBX32 Coding Sequence Comparison ...................................................243

Figure M-1. Annual U.S. soybean yield 1924 – 2010. .................................................424

Figure M-2. U.S. Net Value of Soybean Production, Dollars Per Acre ...................426

Figure M-3. Soybean Yield Losses in the U.S. Averaged Over the Period 

1996 to 2009 Attributable to Pests and Diseases .........................................................437

Figure M-4: Average historical U.S. fertilizer application to corn crop and 

corn and soybean yields over time. ...............................................................................455
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ABBREVIATION AND DEFINITIONS2

 

~ 

AA 

AACC 

ac 

ADF 

AAFCO 

AEBSF 

AOAC 

AOCS 

AOSA 

APHIS 

bp 

bu 

CEX 

CFR 

CO 

CP4 EPSPS 

CSFII 

CTAB 

CV 

DAP 

dCTP 

DDI 

DEEM 

dNTP 

DWCF 

dwt 

ECL 

EDTA 

FA 

FAARP 

FDA 

ft 

fwt 

g, μg, mg 

HCl 

HP 

H.U. 

IGEPAL 

Approximately 

amino acid 

American Association of Cereal Chemists 

Acres 

acid detergent fiber 

Association of Americal Feed Control Officers 

4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride 

Association of Official Agricultural Chemists 

American Oils Chemists Society 

Association of Official Seed Analysts 

Animal and Plant Health Inspection Service 

Base pair 

Bushels 

Cation exchange 

Code of Federal Regulations 

CONSTANS protein 

5-Enolpyruvylshikimate-3-phosphate synthase protein from 

Agrobacterium sp. strain CP4 

Continuing Survey of Food Intakes by Individuals 

Cetyltrimethylammonium bromide 

coefficient variation 

days after pollination 

Deoxycytidine triphosphate 

Daily Dietary Intake 

Dietary exposure evaluation model 

Deoxynucleotide triphosphate 

dry weight conversion factor 

dry weight 

Enhance chemiluminescence 

ethylenediamine tetraacetic acid 

fatty acid 

Food Allergy Research and Resource Program 

Food and Drug Administration 

feet 

fresh weight 

gram, microgram, milligram 

hydrogen chloride 

High performance 

hemagglutinating unit 

octylphenoxypolyethoxyethanol 

 

2

 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7. 

Bedford/St. Martin's, Boston, MA. 
Monsanto Company 11-SY-217U 26 of 467 

IgG 

In 

kb 

kDa 

LB 

LOD 

LOQ 

MALDI-TOF 

MS 

MES 

mm, nm 

MOE 

MWCO 

NA 

ND 

NDF 

NFDM 

NOAEL 

NTO 

OECD 

OSL 

OSR 

PRESS 

PBS 

PCR 

PEG 

PVDF 

PVP 

RT 

SDS-PAGE 

SGF 

SIF 

SOP 

SPS 

TBST 

TCEP 

T/C/R 

TIU 

Tm 

TSSP 

U.S. 

USDA 

UTR 

Immunoglobulin G 

Inch 

kilobase 

kiloDalton 

Laemmli Buffer 

Limit of detection 

limit of quantitation 

matrix-assisted laser desorption/ionization time-of-flight 

mass spectrometry 

2-(N-morpholino)ethanesulfonic acid 

millimeter, nanometer 

Margin of exposure 

Molecular weight cut-off 

Not applicable 

Not determined 

neutral detergent fiber 

Non-fat dry milk 

No observable adverse effect level 

Non-target organism 

Organization for Economic Cooperation and Development 

Over-season leaf 

Over-season root 

predicted residual sum of squares 

Phosphate Buffered Saline 

Polymerase Chain Reaction 

Polyethylene glycol 

polyvinylidene fluoride 

Polyvinylpyrrolidone 

Room temperature 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis 

Simulated gastric fluid 

Simulated intestinal fluid 

Standard operating proceedure 

Sucrose Phosphate Synthase 

Tris-buffered saline containing Tween-20 

Tris(2-carboxyethyl)phosphine 

test/control/reference 

Trypsin Inhibitor Unit 

melting temperature 

tissue-specific site pool 

United States 

United States Department of Agriculture 

Untranslated Region 
Monsanto Company 11-SY-217U 27 of 467 

I. RATIONALE FOR THE DEVELOPMENT OF MON 87712 

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

7 CFR § 340.6 

The Animal and Plant Health Inspection Service (APHIS) of the United States (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 § 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 for the new biotechnology-derived soybean product, MON 87712, 

any progeny derived from crosses between MON 87712 and conventional soybean, and 

any progeny derived from crosses of MON 87712 with any biotechnology-derived 

soybean that have previously been granted nonregulated status under 7 CFR Part 340. 

I.B. Rationale for the Development of MON 87712 

Monsanto Company has developed the biotechnology-derived soybean line MON 87712, 

which will be used in traditional breeding programs to produce commercial varieties with 

increased yield opportunity. The yield increase in MON 87712 is achieved using the 

BBX32 gene from the plant Arabidopsis thaliana that produces a protein that interacts 

with one or more endogenous transcription factors to regulate the plant’s day/night 

processes and results in increased availability of assimilates (products of plant 

metabolism) in MON 87712 compared to an appropriate comparator without this gene. 

Increased plant nutrient availability in MON 87712 is supported by the measurement of 

factors indicative of an extended period of photosynthetic activity in MON 87712 and 

evidence of changes in diurnal metabolism during the reproductive phase of the soybean 

plant, as well as by the significantly higher yield of MON 87712 when compared to 

control, as observed in multisite field studies in the U.S. Appendix B provides 

supplemental information on the function of the BBX32 protein in MON 87712. The 

data demonstrate how the plant BBX32 protein affects existing diurnal processes in 

soybean to increase yield in MON 87712. Higher yielding soybeans offer the opportunity 

for benefits to growers and the soybean food and feed chain, and help meet global 

demand for soybean. 

Soybean is one of the largest U.S. crops based on the acreage planted and quantity 

harvested each year. In 2010, soybean was planted on 76.6 million acres in the U.S., 

where the harvested soybean seed had an average yield of 43.5 bushels per acre and total 

production was 3.33 billion bushels, resulting in a net value greater than $38.91 billion 

(USDA-NASS, 2011d; 2011a). Global demand for soybean is expected to increase over 

the next five years (USDA-ERS, 2011), and as a major global supplier of soybean, the 
Monsanto Company 11-SY-217U 28 of 467 

U.S. must increase soybean production to help meet growing demand. Although the U.S. 

soybean supply has trended upward since 1924 (USDA-NASS, 2011b), it has not 

outpaced soybean use. Stock levels for soybean were projected to slip to 140 million 

bushels at the end of 2010, which would mark the third lowest level in 10 years (USDAERS, 2011). Such low inventories indicate that future soybean productivity must not only 

be large enough to refill present stockpiles to levels that provide increased food and feed 

security, but must also be adequate enough to accommodate end-user demand until the 

next crop is harvested the following year. 

Increased soybean productivity in the U.S. has been accomplished by both increasing the 

area under cultivation and through yield increases per unit area. From 1924 to 2010, 

soybean acreage increased almost 50-fold and yield rose at an average annual rate of 

approximately 0.35 bu/A (0.8%) in the U.S. (USDA-NASS, 2011b). Annual 

improvement in U.S. soybean yield is attributed to rapid producer adoption of agricultural 

improvements such as genetic and agronomic innovations that provide producers with 

means for reducing “on-farm” yield constraints (Specht et al., 1999). Agricultural 

production depends on continuing infusions of genetic resources for yield stability and 

growth (Day-Rubenstein and Heisey, 2006). 

Based on recent trends in farm production and land area, most OECD countries, 

including the U.S. and Canada, are predicted to face the challenge of expanding 

agricultural output by raising productivity on a stable or reduced land area (OECD-FAO, 

2008). Therefore, much of the projected expansion in soybean production in the future is 

expected to come from increased yield rather than increased area under production 

(OECD-FAO, 2008). 

Improvement in soybean yield remains one of the major objectives for plant breeders. 

Gains in major crop yields over the years can be attributed to genetic improvement 

through traditional breeding. Breeders have crossed plants with different genetic 

backgrounds and selected traits resulting in higher yields, compositional improvements, 

and desirable production traits. Biotechnological approaches complement traditional 

soybean breeding efforts by targeting some of the major characteristics as traditional 

breeding. 

Crop yield results from a sequential growth and development process – first the plant 

grows vegetatively and produces photosynthetic tissue, followed by flowering and the 

production of seeds, and finally seed filling and maturation. Yield is a complex trait that 

is dependent on a number of genetic and environmental factors, that influence a crop’s 

opportunity to realize its full yield potential. Improvements in crop yield have been a 

primary focus of conventional breeding. The genetic changes that resulted in crop 

domestication and yield improvement in conventional varieties have been shown by 

modern molecular biology analysis to have been typically achieved through the selection 

and safe use of plant genes encoding transcriptional regulator proteins. Agricultural 

biotechnology provides the opportunity to further enhance crop yields through the 

introduction of new genetic elements that use or modify existing pathways in the plant. 
Monsanto Company 11-SY-217U 29 of 467 

In summary, MON 87712 is of benefit to growers, the food and feed chain, and the 

society as a whole, as it provides increased yield opportunity, will help global efforts to 

provide an adequate supply of soybeans, and help sustain a robust domestic and global 

livestock market for soybean and soybean products. 

I.C. Submissions to Other Regulatory Agencies 

Under the Coordinated Framework for Regulation of Biotechnology, the responsibility 

for regulatory oversight of biotechnology-derived crops falls primarily on three U.S. 

agencies: U.S. Food and Drug Administration (FDA), the United States Department of 

Agriculture (USDA), and in the case of plant incorporated protectants, the Environmental 

Protection Agency (EPA). Deregulation of MON 87712 by USDA constitutes only one 

component of the overall regulatory oversight and review of this product. As a practical 

matter, MON 87712 cannot be released and marketed until FDA and USDA have 

completed their reviews and assessments under their respective jurisdictions. 

I.C.1. Submission to FDA

MON 87712 falls within the scope of the 1992 FDA policy statement concerning 

regulation of products derived from new plant varieties, including those developed 

through biotechnology (U.S. FDA, 1992). In compliance with this policy, Monsanto will 

initiate a consultation with the FDA on the food and feed safety and compositional 

assessment of MON 87712. 

I.C.2. Submissions to Foreign Government Agencies 

To support commercial introduction of MON 87712 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. These will include submissions to a number of foreign 

government regulatory authorities, including: the Ministry of Agriculture, People’s 

Republic of China; Japan’s Ministry of Agriculture, Forestry, and Fisheries, Ministry of 

Environment, and the Ministry of Health, Labor, and Welfare; the Canadian Food 

Inspection Agency and Health Canada; the Intersectoral Commission for Biosafety of 

Genetically Modified Organisms, Mexico; the European Food Safety Authority, as well 

as to 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 11-SY-217U 30 of 467 

II. THE BIOLOGY OF SOYBEAN 

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), 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 widely grown oilseed in the world, with approximately 258.4 million 

metric tons (MMT) of harvested seed produced in 2010, which represented 58% of world 

oilseed production that year (ASA, 2011). Soybean is grown as a commercial crop in 

over 35 countries. The major producers of soybean are the U.S., Brazil, Argentina, 

China, India, Paraguay and Canada, which accounted for approximately 95% of the 

global soybean production in 2010 (ASA, 2011); also see Table II 1. Approximately 35% 

of the 2010 world soybean production was produced in the U.S. (ASA, 2011). For the 

fifth consecutive year, U.S. whole soybean exports hit record levels in 2010, with exports 

exceeding 43.3 MMT (ASA, 2011). Approximately 45 MMT of soybeans were crushed 

in the U.S. in 2010 and used to supply the feed industry for livestock use or the food 

industry for edible vegetable oil and soybean protein isolates (ASA, 2011). U.S. stock 

levels were 3.8 MMT at the end of the year (ASA, 2011). 

Table II-1. World Soybean Production in 2010 

Country Production (million metric tons)

U.S. 90.6 

Brazil 70.0 

Argentina 49.5 

China 15.2 

India 9.6 

Paraguay 7.5 

Canada 4.3 

Other 11.6 

Source: Soy Stats, World Soybean Production (ASA, 2011). 

Soybean is now the second most planted field crop in the U.S. after corn. According to 

data from USDA-NASS (2011d), soybean was planted on approximately 76.6 million 

acres in the U.S. in 2010, producing 3.33 billion bushels of soybean with a value of $38.9 

billion (USDA-NASS, 2011d). 

Soybean is used in various food products, including tofu, soy 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 

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 29% of all the vegetable oils consumed globally, and was the second 
Monsanto Company 11-SY-217U 31 of 467 

largest source of vegetable oil worldwide, slightly behind palm oil at approximately 33% 

share (ASA, 2011). 

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. Industrial edible and 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 the American Soybean Association (ASA, 2011). 

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 could only be traced back to the Chou 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., 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 

disintegration of Chinese dynasties (Hymowitz and Newell, 1981). 

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, the 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 harvested seeds as a staple food by other cultures (Hymowitz and Newell, 

1981). 

Starting in the late 16th century and throughout the 17th century, soybean was used by 

the Europeans, and in the 17th century, soy sauce was a common item of trade from the 

east to the west (Hymowitz and Newell, 1981). 

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 seeds 

were deposited at the New York State Agricultural Society, the Massachusetts 

Horticultural Society, and the Commissioner of Patents. The two societies and the 

Commissioner of Patents sent soybean seeds 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. (Singh and Hymowitz, 1999). 

II.C. Taxonomy and Phylogenetics of Soybean 

Cultivated soybean, Glycine max (L.) Merr., is a diploidized tetraploid (2n=40), which 

belongs to the family Fabaceae, the subfamily Papilionoideae, the tribe Phaseoleae, the 

genus Glycine Willd., and the subgenus Soja (Moench) F.J. Herm. 
Monsanto Company 11-SY-217U 32 of 467 

Family: Fabaceae 

Subfamily: Papilionoideae 

Tribe: Phaseoleae 

Genus: Glycine Willd. 

Subgenus: Soja (Moench) F.J. Herm. 

Species: Glycine max (L.) Merr. 

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). 
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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 

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 (Hymowitz, 2004). 

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 ovoid to 

subspherical seeds are produced in the pods (Hymowitz, 2004). 

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. Glycine 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 et al., 1979; Skorupska et 

al., 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 
Monsanto Company 11-SY-217U 35 of 467 

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 

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 the soybean seed 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 conventional soybean variety A3525, used as the recipient for the BBX32 expression 

cassette insertion that produced MON 87712, was developed by Asgrow Seed Company. 

A3525 is a mid−maturity group III soybean variety. 

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

Soybean variety A3525 is the parental line of MON 87712 and was used as the 

conventional soybean comparator (hereafter referred to as the conventional control) in the 

safety assessment of MON 87712. MON 87712 and the conventional control A3525 

have similar genetic backgrounds with the exception of the BBX32 expression cassette, 

thus, the effect of the BBX32 expression cassette could therefore be assessed in an 

unbiased manner in the comparative safety assessment. In addition, reference materials 

were used to establish ranges of natural variability or responses representative of 

commercial soybean varieties. Conventional commercial reference varieties refer to 

commercial soybean varieties that were derived only through conventional methods. 

Commerial reference varieties refer to commercial soybean varieties that were derived 

through conventional methods and/or Roundup Ready soybean varieties. The commercial 

reference varieties used at each location were selected based on their availability and 

agronomic fit for the respective geographic region. 
Monsanto Company 11-SY-217U 36 of 467 

III. DESCRIPTION OF THE GENETIC MODIFICATION 

MON 87712 was developed through Agrobacterium tumefaciens-mediated 

transformation of conventional soybean A3525 meristem tissue utilizing transformation 

plasmid vector PV-GMAP5779. This section describes the plasmid vector, the donor 

genes, and the regulatory elements used in the development of MON 87712 and the 

deduced amino acid sequence of the BBX32 protein produced in MON 87712. In this 

section, transfer DNA (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. 

III.A. The Plasmid Vector PV-GMAP5779 

PV-GMAP5779 was used for the transformation of conventional soybean to produce 

MON 87712 and is shown in Figure III-1; PV-GMAP5779 is approximately 11.4 kb and 

contains two T-DNAs, each delineated by Left and Right Border regions to facilitate 

transformation. The first T-DNA, designated as T-DNA I, contains the BBX32 coding 

sequence under regulation of the e35S promoter and the E6 3′ untranslated region. The 

second T-DNA, designated as T-DNA II, contains the cp4 epsps coding sequence under 

the regulation of the FMV/EF-1α promoter, EF-1α leader, EF-1α intron, CTP2 targeting 

sequence, and the E9 3′ untranslated region. During transformation, both T-DNAs were 

inserted into the soybean genome (Section III.B) where T-DNA II, containing the 

cp4 epsps expression cassette, functioned as a marker gene for the selection of 

transformed plantlets. Subsequently, conventional self-pollinated breeding methods and 

segregation, along with a combination of analytical techniques, were used to isolate those 

plants that contained the BBX32 expression cassette (T-DNA I) but did not contain the 

cp4 epsps expression cassette (T-DNA II). 

The backbone region of PV-GMAP5779, which is located outside both of the T-DNAs 

contains two origins of replication (ori-V, ori-pBR322) for maintenance of the plasmid 

vector in bacteria, a bacterial selectable marker gene (aadA), and a coding sequence for 

repressor of primer (rop) protein for the maintenance of the plasmid vector copy number 

in E. coli. A description of the genetic elements and their prefixes (e.g., P-, L-, I-, TS-, 

OR-, B-, CS-, and T-) in PV-GMAP5779 is provided in Table III-1. 

III.B. Description of the Transformation System 

MON 87712 was developed through Agrobacterium-mediated transformation of soybean, 

based on the method described by Martinell et al. (2002), which allows for the generation 

of transformed plants without the utilization of callus. Briefly, meristem tissues were 

excised from the embryos of germinated conventional seed. After co-culturing with the 

Agrobacterium carrying the vector, the meristems were placed on selection medium 

containing glyphosate, carbenicillin, cefotaxime, and ticarcillin/clavulanate acid mixture, 

to inhibit the growth of untransformed plant cells and excess Agrobacterium. The 

meristems were then placed in media conducive to shoot and root development. Rooted 

plants with normal phenotypic characteristics were selected and transferred to soil for 

growth and further assessment. 
Monsanto Company 11-SY-217U 37 of 467 

The R0 plants generated through this transformation were self-pollinated to produce R1

plants, and the unlinked insertions of T-DNA I and T-DNA II were segregated. A 

non-lethal dose of glyphosate was applied to R1 plants and those plants with minor 

herbicide injury were selected for further analyses, whereas plants showing no injury, 

indicating that they contained the cp4 epsps coding sequence from T-DNA II, were 

eliminated from further development. Subsequently, plants that were homozygous for 

T-DNA I were identified by a quantitative non-polymerase chain reaction (non-PCR) 

based analysis. MON 87712 was selected as the lead event based on superior phenotypic 

characteristics and its molecular profile. The major development steps of MON 87712 

are depicted in Figure III-2. The result of this process was the production of marker-free 

MON 87712 soybean. 

III.C. The BBX32 Coding Sequence and the BBX32 Protein (T-DNA I) 

The expression cassette present in MON 87712 contains the coding region for BBX32 

protein from Arabidopsis thaliana (Figure III-3) (Holtan et al., 2011; Khanna et al., 2009; 

Putterill et al., 1995). The presence of BBX32 modulates aspects of diurnal biology to 

increase yield by the increased capacity for growth and reproductive development (refer 

to Appendix B for more details). 

III.D. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA II) 

The cp4 epsps expression cassette (T-DNA II) that is not present in MON 87712 encodes 

a 47.6 kDa CP4 EPSPS protein consisting of a single polypeptide of 455 amino acids 

(Padgette et al., 1996b). The cp4 epsps coding sequence is the codon optimized coding 

sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS 

protein (Barry et al., 1997; Padgette et al., 1996a). CP4 EPSPS protein confers tolerance 

to glyphosate and was used as a selectable marker during the transformation selection 

process. Plants that did not contain the cp4 epsps expression cassette were isolated 

through conventional self-pollinated breeding methods and segregation, along with a 

combination of analytical techniques. 
Monsanto Company 11-SY-217U 38 of 467 

Probe 

Number 

DNA Probe 

Type 

Start Position 

(bp) 

End Position 

(bp) 

Total Length 

(~kb) 

1 T-DNA I Probe 1 1 1610 1.6 

2 T-DNA I Probe 2 1556 2549 1.0 

3 T-DNA II Probe 1 3078 4383 1.3 

4 T-DNA II Probe 2 4304 6048 1.7 

5 T-DNA II Probe 3 5954 7075 1.1 

6 Backbone Probe 1 2550 2720 0.2 

7 Backbone Probe 2 7487 9245 1.8 

8 Backbone Probe 3 9116 10620 1.5 

9 Backbone Probe 4 10543 11428 0.9 

Figure III-1. Circular Map of PV-GMAP5779 Showing Probe 1 through Probe 9 

A circular map of the plasmid vector PV-GMAP5779 used to develop MON 87712 is 

shown. PV-GMAP5779 contains two T-DNAs, designated as T-DNA I and T-DNA II. 

Genetic elements and restriction sites used in Southern analyses (with positions relative 

to the size of the plasmid vector) are shown on the exterior of the map. The probes used 

in the Southern analyses are shown on the interior of the map. 

*The Left and Right border regions of T-DNA II share 100% identity with T-DNA I, which were covered by Probe 1 

and Probe 2 and thus not included in the T-DNA II probes. 

11428 bp

CS-BBX32

T-E6

B-Right Border Region

B-Right Border Region

P-FMV/EF-1α

L-EF-1α

I-EF-1α

TS-CTP2 CS-cp4 epsps

T-E9

aadA

OR-ori-pBR322

CS-rop

OR-ori V

PV-GMAP5779

B-Left Border Region

P-e35S EcoR I 478

Nco I 1146

EcoR I 3636

Nco I 4815

1

2

3

4

5

6

7

8

9

T-DNA I

T-DNA II
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Figure III-2. Schematic of the Development of MON 87712 

Transformed meristem tissue from A3525 with PV-GMAP5779 in 

Agrobacterium tumefaciens

Selected transformants and generated rooted shoots from the 

transformed meristem tissues 

Screening of transformed plants for the presence of T-DNA I (BBX32

expression cassette) and absence of the T-DNA II 

(cp4 epsps expression cassette) 

Identified MON 87712 as lead candidate based on analysis of the 

genomic insert and evaluation of progeny generations in laboratory and 

field assessments 

Assembled Agrobacterium binary plasmid vector PV-GMAP5779 and 

transferred to Agrobacterium tumefaciens strain ABI 
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III.E. Regulatory Sequences 

The BBX32 coding sequence in T-DNA I is under the regulation of the e35S promoter, 

and the E6 3′ untranslated region. The e35S promoter is the promoter for 35S RNA of 

cauliflower mosaic virus (CaMV) (Odell et al., 1985) containing the duplicated enhancer 

region (Kay et al., 1987), which functions to direct transcription in plant cells. The E6 3′

untranslated region is the 3′ untranslated region of the E6 gene from Gossypium 

barbadense (cotton) encoding a fiber protein involved in early fiber development (John, 

1996), which functions to direct polyadenylation of the RNA transcripts. 

T-DNA II contains the cp4 epsps coding sequence under the regulation of the 

FMV/EF-1α promoter, EF-1α leader, EF-1α intron, the CTP2 targeting sequence, and the 

E9 3′ untranslated region. The FMV/EF-1α promoter is the chimeric promoter consisting 

of enhancer sequences from the 35S RNA promoter of figwort mosaic virus (FMV) 

(Richins et al., 1987) combined with the promoter of the EF-1α gene from Arabidopsis 

thaliana encoding elongation factor EF-1α (Axelos et al., 1989), which functions to direct 

transcription in plant cells. The EF-1α leader is the 5′ leader (exon 1) sequence of the 

EF-1α gene from Arabidopsis thaliana that encodes elongation factor EF-1α (Axelos et 

al., 1989), which is involved in regulating gene expression. The EF-1α intron is the 

intron with flanking splice sites of the EF-1α gene from Arabidopsis thaliana encoding 

elongation factor EF-1α (Axelos et al., 1989), which is involved in regulating gene 

expression (Curie et al., 1991). The CTP2 targeting sequence is the targeting sequence of 

the ShkG gene from Arabidopsis thaliana encoding the EPSPS transit peptide region 

(Herrmann, 1995; Klee et al., 1987), which functions to direct transport of the CP4 

EPSPS protein to the chloroplast. The E9 3′ untranslated region is the 3′ untranslated 

region from Pisum sativum (pea) rbcS gene family encoding the small subunit of ribulose 

bisphosphate carboxylase protein (Coruzzi et al., 1984), which functions to direct 

polyadenylation of the mRNA. 

III.F. T-DNA Borders 

PV-GMAP5779 contains Left and Right Border regions (Figure III-1 and Table III-1) 

that were derived from Agrobacterium tumefaciens (Barker et al., 1983; Depicker et al., 

1982; Zambryski et al., 1982). The border regions each contain a 24-25 bp nick site that 

is the site of DNA exchange during transformation. Left and Right Border regions 

separate the T-DNA from the plasmid backbone region and are involved in the efficient 

transfer into the soybean genome. Because PV-GMAP5779 is a 2T-DNA vector, it 

contains two Left Border regions and two Right Border regions, where one border region 

set flanks T-DNA I and the other border region set flanks T-DNA II. 

III.G. Genetic Elements Outside of the T-DNA Borders 

Genetic elements that exist outside of the T-DNA border regions are those that are 

essential for the maintenance or selection of PV-GMAP5779 in bacteria and are referred 

to as plasmid backbone. The origin of replication, ori V, is required for the maintenance 

of the plasmid in Agrobacterium and is derived from the broad host plasmid RK2 (Stalker 

et al., 1981). The origin of replication, ori-pBR322, is required for the maintenance of 
Monsanto Company 11-SY-217U 41 of 467 

the plasmid in E. coli and is derived from the plasmid vector pBR322 (Sutcliffe, 1979). 

Coding sequence rop encodes the repressor of primer (ROP) protein which is necessary 

for the maintenance of plasmid vector 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 87712 has been confirmed by 

Southern blot analyses (see Section IV.B). 
Monsanto Company 11-SY-217U 42 of 467 

Table III-1. Summary of Genetic Elements in the PV-GMAP5779 

Genetic Element 

Location in 

Plasmid 

Vector Function (Reference) 

T-DNA I 

B1-Left Border 

Region 

1-442 DNA region from Agrobacterium tumefaciens

containing the left border sequence used for transfer of 

the T-DNA (Barker et al., 1983) 

Intervening 

Sequence 

443-510 Sequence used in DNA cloning 

P2-e35S 511-1123 Promoter from the 35S RNA of cauliflower mosaic 

virus (CaMV) (Odell et al., 1985) containing the 

duplicated enhancer region (Kay et al., 1987) that 

directs transcription in plant cells 

Intervening 

Sequence 

1124-1147 Sequence used in DNA cloning 

CS3-BBX32 1148-1825 Coding sequence of the BBX32 gene from Arabidopsis 

thaliana encoding a zinc finger protein (B-box type) 

(Khanna et al., 2009; Putterill et al., 1995) which 

modulates aspects of diurnal biology (Holtan et al., 

2011) 

Intervening 

Sequence 

1826-1839 Sequence used in DNA cloning 

T4-E6 1840-2154 3′ UTR region of the E6 gene from Gossypium 

barbadense (cotton) encoding a fiber protein involved 

in early fiber development (John, 1996) that directs 

polyadenylation of mRNA 

Intervening 

Sequence 

2155-2192 Sequence used in DNA cloning 

B-Right Border 

Region 

2193-2549 DNA region from Agrobacterium tumefaciens

containing the right border sequence used for transfer of 

the T-DNA (Depicker et al., 1982; Zambryski et al., 

1982) 

Backbone 

Intervening 

Sequence 

2550-2720 Sequence used in DNA cloning 

T-DNA II

B-Right Border 

Region 

2721-3077 DNA region from Agrobacterium tumefaciens

containing the right border sequence used for transfer of 

the T-DNA (Depicker et al., 1982; Zambryski et al., 

1982) 

 
Monsanto Company 11-SY-217U 43 of 467 

Table III-1. Summary of Genetic Elements in the PV-GMAP5779 (continued) 

Intervening 

Sequence 

3078-3099 Sequence used in DNA cloning 

P-FMV/EF-1α 3100-4139 Chimeric promoter consisting of enhancer sequences from 

the 35S RNA promoter of figwort mosaic virus (FMV) 

(Richins et al., 1987) combined with the promoter of the 

EF-1α gene from Arabidopsis thaliana encoding 

elongation factor EF-1α (Axelos et al., 1989) that directs 

transcription in plant cells 

L5- EF-1α 4140-4185 Leader (exon 1) sequence of the EF-1α gene from 

Arabidopsis thaliana that encodes elongation factor EF-1α

(Axelos et al., 1989) that is involved in regulating gene 

expression 

I

6

- EF-1α 4186-4807 Intron with flanking splice sites of the EF-1α gene from 

Arabidopsis thaliana encoding elongation factor EF-1α

(Axelos et al., 1989) that involved in regulating gene 

expression (Curie et al., 1991) 

Intervening 

Sequence 

4808-4816 Sequence used in DNA cloning 

TS7-CTP2 4817-5044 Targeting sequence of the ShkG gene from Arabidopsis 

thaliana encoding the EPSPS transit peptide region that 

directs transport of the protein to the chloroplast 

(Herrmann, 1995; Klee et al., 1987) 

CS-cp4 epsps 5045-6412 Codon optimized coding sequence of the aroA gene from 

the Agrobacterium sp. strain CP4 encoding the CP4 

EPSPS protein that provides herbicide tolerance (Barry et 

al., 2001; Padgette et al., 1996a) 

Intervening 

Sequence 

6413-6418 Sequence used in DNA cloning 

T-E9 6419-7061 3′ UTR sequence from Pisum sativum (pea) rbcS gene 

family encoding the small subunit of ribulose bisphosphate 

carboxylase protein (Coruzzi et al., 1984) that directs 

polyadenylation of the mRNA 

Intervening 

Sequence

7062-7075 Sequence used in DNA cloning 

B-Left Border 

Region

7076-7486 DNA region from Agrobacterium tumefaciens containing 

the left border sequence used for transfer of the T-DNA 

(Barker et al., 1983) 

Backbone

Intervening 

Sequence 

7487-7581 Sequence used in DNA cloning 

 
Monsanto Company 11-SY-217U 44 of 467 

Table III-1. Summary of Genetic Elements in the PV-GMAP5779 (continued) 

aadA 7582-8470 Bacterial 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 

8471-9000 Sequence used in DNA cloning 

OR8-ori-pBR322 9001-9589 Origin of replication from pBR322 for maintenance of 

plasmid in E. coli (Sutcliffe, 1979) 

Intervening 

Sequence 

9590-10016 Sequence used in DNA cloning 

CS-rop 10017-10208 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 

10209-10945 Sequence used in DNA cloning 

OR-ori V 10946-11342 Origin of replication from the broad host range plasmid 

RK2 for maintenance of plasmid in Agrobacterium

(Stalker et al., 1981) 

Intervening 

Sequence 

11343-11428 Sequence used in DNA cloning 

1

B, Border. 

2

P, Promoter. 

3

CS, Coding Sequence. 

4

T, Transcription Termination Sequence. 

5

L, Leader. 

6

I, Intron. 

7

TS, Targeting Sequence. 

8

OR, Origin of Replication. 

 
Monsanto Company 11-SY-217U 45 of 467 

Figure III-3. Deduced Amino Acid Sequence of the BBX32 Protein in MON 87712 

The amino acid sequence of the BBX32 protein was deduced from the full-length coding 

nucleotide sequence present in PV-GMAP5779 (see Table III-1 for more detail). 
Monsanto Company 11-SY-217U 46 of 467 

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION 

A multi-faceted approach was taken to characterize the genetic modification that 

produced MON 87712. The results confirmed that MON 87712 contains a single copy of 

the BBX32 expression cassette (T-DNA I) that is stably integrated at a single locus and is 

inherited according to Mendelian principles over multiple generations (Section IV.G.). 

Additionally, the results confirmed that T-DNA II and plasmid vector backbone 

sequences are not detected in MON 87712. These conclusions were based on several 

lines of evidence: 1) Southern blot analyses to assay the entire soybean genome for the 

presence of T-DNA I sequences and the absence of T-DNA II and plamid vector 

backbone sequences derived from PV-GMAP5779, and to confirm that a single copy of 

T-DNA I was inserted at a single site and is stably inherited; 2) DNA sequence analyses 

to determine the exact sequence of the inserted DNA and the DNA sequences flanking 

the 5' and 3' ends of the insert; 3) DNA sequence comparison of the inserted DNA 

sequence to the T-DNA I sequence in PV-GMAP5779 to confirm that only the expected 

sequences were integrated; and 4) sequence comparison of the DNA sequences flanking 

5' and 3' end of the T-DNA I insert to the insertion site sequence 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 

MON 87712 contains a single copy of T-DNA I that 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 T-DNA I, as well as the presence or absence of T-DNA II and plasmid vector 

backbone sequences. The Southern blot strategy was designed to ensure that all potential 

inserted segments would be identified. The entire soybean genome was assayed with 

probes that spanned the complete plasmid vector PV-GMAP5779 to detect the presence 

of T-DNA I, as well as the absence of T-DNA II and plasmid vector backbone sequences. 

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 0.1 copies per genome equivalent. Two sets of 

restriction enzymes were specifically chosen to fully characterize T-DNA I and detect 

any potential segments from the plasmid vector PV-GMAP5779. The restriction enzyme 

sets were chosen such that each enzyme set cleaves once within the inserted T-DNA and 

at least once within the known DNA sequence flanking the 5' or 3' end of the insert. As a 

result, the enzyme sets produce overlapping segments that contain the entire insert 

sequence and adjacent 5' and 3' flanking DNA sequence. Therefore, at least one segment 

containing a portion of the insert with the adjacent 5'flanking DNA generated by one set 

of the enzyme(s) is of a predictable size and overlaps with another predictable size 

segment containing a portion of the insert with the adjacent 3' flanking DNA generated 

by another set of the enzyme(s). This two set enzyme design ensures that the entire insert 

is identified in a predictable hybridization pattern. Additionally, this two enzyme set 

design also maximizes the possibility of detecting an insertion elsewhere in the genome 

that could be overlooked if that band co-migrated with an expected band. 
Monsanto Company 11-SY-217U 47 of 467 

To determine the number of copies and the insertion sites of T-DNA I, and the presence 

or absence of T-DNA II and the plasmid vector backbone sequences, duplicated samples 

that consisted of equal amounts of digested DNA were run on the agarose gel. One set of 

samples was run for a longer 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. The molecular weight 

markers on the left of the figures were used to estimate the sizes of the bands present in 

the long run lanes of the Southern blots, and the molecular weight markers on the right of 

the figures were used to estimate the sizes of bands present in the short run lanes of the 

Southern blots. Southern blot results demonstrated that MON 87712 contains a single 

copy of T-DNA I at a single insertion site in the soybean genome, and no additional 

genetic elements, including backbone sequences, from PV-GMAP5779 were detected in 

MON 87712 (Figure IV-2 through Figure IV-8). 

The PCR and DNA sequence analyses complement the Southern blot analyses. PCR and 

DNA sequence analyses performed on MON 87712 determined the complete DNA 

sequence of the insert and adjacent DNA sequences, confirmed the organization of the 

elements within the insert, and determined the 5′ and 3′ insert-to-plant junctions 

(Figure IV-9 and Figure IV-10). In addition, DNA sequencing analyses confirmed each 

genetic element in the insert and the sequence of the insert matches the corresponding 

sequence in PV-GMAP5779. Furthermore, genomic organization at the MON 87712 

insertion site was determined by comparing the 5' and 3' flanking sequences of the insert 

to the sequence of the insertion site in conventional soybean. 

The stability of T-DNA I present in MON 87712 across multiple generations (R3, R4, R5, 

R6, and R7) was demonstrated by Southern blot fingerprint analysis. Genomic DNA from 

five generations of MON 87712 (Figure IV-11) was digested with one of the enzyme sets 

used for the insert and copy number analysis and was hybridized with a probe that detects 

restriction segments that encompass the entire T-DNA I insert (Figure IV-12). This 

fingerprint strategy consists of two border segments that assess not only the stability of 

T-DNA I, but also the stability of genomic DNA directly adjacent to T-DNA I. 

Generational stability analysis demonstrated that the expected Southern blot fingerprint 

of MON 87712 was maintained through five generations of the breeding history, thereby 

confirming the stability of T-DNA I in MON 87712. 

Segregation analysis was employed to examine the genetic behavior of the T-DNA I 

insert in MON 87712. The results from this analysis showed heritability and stability of 

the insert occurred as expected across multiple generations (Figure IV-13, Table IV-3) 

which corroborates the molecular insert stability analysis and establishes that T-DNA I is 

inherited according to Mendelian principles of inheritance. 

A circular map of PV-GMAP5779 annotated with the probes used in the Southern blot 

analysis is presented in Figure III-1. A linear map depicting restriction sites within the 

insert, as well as the DNA flanking the insert in MON 87712 is shown in Figure IV-1. 

Based on the plasmid map and the linear map of the insert, a table summarizing the 

expected DNA segments for Southern analyses is presented in Table IV-1. The genetic 

elements within the MON 87712 insert are summarized in Table IV-2. The results from 
Monsanto Company 11-SY-217U 48 of 467 

the Southern blot analyses are presented in Figure IV-2 through Figure IV-8. PCR 

amplification of the MON 87712 insert and the insertion site in conventional control for 

DNA sequence analysis are shown in Figure IV-9 and Figure IV-10, respectively. The 

generations used in the generational stability analysis are depicted in the breeding history 

shown in Figure IV-11 and the results from the generational stability analysis are 

presented in Figure IV-12. The breeding path for the segregation data is shown in 

Figure IV-13 and the results for the segregation analysis are presented in Table IV-3. 

Materials and methods used for the characterization of the insert in MON 87712 are 

found in Appendix C. 
Monsanto Company 11-SY-217U 

Figure IV-1. Schematic Representation of the Insert and Flanking DNA Sequences in MON 87712 DNA derived from the T-DNA I of PV-GMAP5779 integrated in MON 87712. Right-angled arrows indintegrated T-DNA I and the beginning of the flanking sequence. Identified on the map are the genetic elemen

well as restriction sites for enzymes with positions relative to the size of the DNA sequence (flanks and insert) analyses. The relative locations of the T-DNA I probes and the expected sizes of restriction segments are indic

diagram is not drawn to scale. Locations of T-DNA I probes are approximate. Probes are described in Figure III

Nco I 148

EcoR I 289

EcoR I 3750

Nco I 4418

Spe 

B-Left Border Region r1

P-e35S

CS-BBX32

T-E6

B-Right Border Region r1

5' Flank 3' Flank 

Probe 1

Probe 2

1 

Nco I

EcoR I and Spe I

~ 4.3 kb ~ 2.2 kb

~ 3.5 kb ~ 2.2 kb
Monsanto Company 11-SY-217U 

Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and RestrictMON 87712 Analysis 

Southern Blot Analysis T-DNA I T-DNA II Backbone Southern Blot Figure IV-2 IV-3 IV-4 IV-5 IV-6 IV-7 IV-8 Probe Used 1 2 3, 5

4

6 7, 9 8 

Probing Target 

Digestion enzyme Expected Band Sizes on each Southern Blot 

PV-GMAP5779 Nco I ~7.8 kb ~3.7 kb ~3.7 kb ~7.8 kb ~3.7 kb ~7.8 kb ~3.7 kb ~7.8 kb* ~3.7 kb ~7.8 kb ~3.7 kb* ~7.8 Probe Templates1 N/A ~~2 ~~2 ~1.3 kb ~1.1 kb ~~2 ~~2 ~1.8 kb ~0.9 kb ~~2 MON 87712 

Nco I ~4.3 kb 

~2.2 kb ~2.2 kb None None None None Non

EcoR I and Spe I

~3.5 kb ~2.2 kb ~2.2 kb None None None None Non

*these bands were detected by the probes due to sequence homology to the other digested segment of PV-GMAP1probe template spikes were used as positive hybridization controls in Southern blot analyses when multiple prob

the blot simultaneously. 

2‘~~’ indicates that probe template was not used. 3‘--’ indicates that the particular restriction enzyme or the combination of the enzymes was not used in the analys
Monsanto Company 11-SY-217U 51 of 467 

Table IV-2. Summary of Genetic Elements in MON 87712 

Genetic Element

Location in 

Sequence Function (Reference) 

T-DNA I 

Sequence 

flanking 5' end 

of insert 

1-3493 DNA adjacent to the 5' end of the inserted DNA 

B1-Left Border 

Regionr1

3494-3714 DNA region from Agrobacterium tumefaciens

containing the left border sequence used for transfer of 

the T-DNA (Barker et al., 1983) 

Intervening 

Sequence 

3715-3782 Sequence used in DNA cloning 

P2-e35S 3783-4395 Promoter from the 35S RNA of cauliflower mosaic 

virus (CaMV) (Odell et al., 1985) containing the 

duplicated enhancer region (Kay et al., 1987) that 

directs transcription in plant cells 

Intervening 

Sequence 

4396-4419 Sequence used in DNA cloning 

CS3-BBX32 4420-5097 Coding sequence of the BBX32 gene from Arabidopsis 

thaliana encoding a zinc finger protein (B-box type) 

(Khanna et al., 2009; Putterill et al., 1995) which 

modulates aspects of diurnal biology (Holtan et al., 

2011) 

Intervening 

Sequence 

5098-5111 Sequence used in DNA cloning 

T4-E6 5112-5426 3′ UTR region of the E6 gene from Gossypium 

barbadense (cotton) encoding a fiber protein involved 

in early fiber development (John, 1996) that directs 

polyadenylation of mRNA 

Intervening 

Sequence 

5427-5464 Sequence used in DNA cloning 

B-Right Border 

Regionr1 

5465-5507 DNA region from Agrobacterium tumefaciens

containing the right border sequence used for transfer of 

the T-DNA (Depicker et al., 1982; Zambryski et al., 

1982) 

Sequence 

flanking 3'end 

of insert 

5508-7472 DNA adjacent to the 3' end of the inserted DNA 

1

B, Border. 

2

P, Promoter. 

3

CS, Coding Sequence. 

4

T, Transcription Termination Sequence. 

r1Superscripts in Left and Right Borders indicate that the border sequences in MON 87712 were truncated 

compared to the border sequences in PV-GMAP5779. 
Monsanto Company 11-SY-217U 52 of 467 

IV.A. Southern Blot Analysis to Determine Insert and Copy Number of T-DNA I in 

MON 87712 

The copy number and insertion sites of T-DNA I sequences in the soybean genome were 

evaluated by digesting the R3 generation of MON 87712 and the conventional control 

genomic DNA samples with two sets of restriction enzymes, Nco I and a combination of 

EcoR I and Spe I and hybridized Southern blots with probes that span the T-DNA I 

(Figure III-1). Each restriction digest is expected to produce a specific banding pattern 

on the Southern blots (Table IV-1). Any additional copies and/or integration sites would 

be detected as additional bands. 

The restriction enzyme Nco I cleaves once within the inserted DNA and once within the 

known 5' and 3' flanking sequences (Figure IV-1). Therefore, if T-DNA I sequences 

were present as a single copy at a single integration site in MON 87712, the digestion 

with Nco I was expected to generate two border segments with expected sizes of ~4.3 kb 

and ~2.2 kb (Figure IV-1 and Table IV-1). The combination of EcoR I and Spe I cleaves 

once within the inserted DNA and once within the known 5′ and 3′ flanking sequences in 

MON 87712 (Figure IV-1). Therefore, if T-DNA I sequences were present as a single 

copy at a single integration site in MON 87712, the digestion with EcoR I and Spe I was 

expected to generate two border segments with expected sizes of ~3.5 kb and ~2.2 kb 

(Figure IV-1 and Table IV-1). 

The Southern blots were hybridized with probes spanning the entire T-DNA I sequence 

(Figure III-1, Probe 1 and Probe 2). Each Southern blot contained a negative control and 

positive controls. Conventional control genomic DNA digested with Nco I spiked with 

either digested PV-GMAP5779 DNA and/or probe template(s) served as positive 

hybridization controls. Conventional control genomic DNA digested with appropriate 

restriction enzymes was used as a negative control. The results of this analysis are shown 

in Figure IV-2 and Figure IV-3. 

IV.A.1. Probe 1 

Conventional control genomic DNA digested with Nco I (Figure IV-2, lane 1 and lane 7) 

or a combination of EcoR I and Spe I (Figure IV-2, lane 3 and lane 9) and hybridized 

with Probe 1 (Figure III-1) showed no detectable hybridization bands, as expected. 

Conventional control genomic DNA digested with Nco I and spiked with PV-GMAP5779 

DNA previously digested with Nco I produced two expected size bands at ~7.8 kb and 

~3.7 kb (Figure IV-2, lane 5 and lane 6). Detection of the spiked controls indicates that 

the probe hybridized to its target sequences. 

MON 87712 DNA digested with Nco I and hybridized with Probe 1 (Figure III-1) 

produced two expected bands at ~4.3 kb and ~2.2 kb (Figure IV-2, lane 2 and lane 8). 

The ~4.3 kb band represents the 5′ end of the inserted DNA and the adjacent DNA 

flanking the 5′ end of the insert. The ~2.2 kb band represents the 3′ end of the inserted 

DNA and the adjacent DNA flanking the 3′ end of the insert. 
Monsanto Company 11-SY-217U 53 of 467 

MON 87712 DNA digested with a combination of EcoR I and Spe I and hybridized with 

Probe 1 produced the two expected bands at ~3.5 kb and ~2.2 kb (Figure IV-2, lane 4 and 

lane 10). The ~3.5 kb band represents the 5′ end of the inserted DNA and the adjacent 

DNA flanking the 5′ end of the insert. The ~2.2 kb band represents the 3′ end of the 

inserted DNA and the adjacent DNA flanking the 3′ end of the insert. The results 

presented in Figure IV-2 indicate that the sequence covered by Probe 1 resides at a single 

detectable locus of integration in MON 87712. 

There is a faint line on the blot in lane 1 through lane 6 at ~0.7 kb, which is continuous 

between the lanes, indicating that it did not result from hybridization with any DNA. 

This conclusion is confirmed by the fact that this line at ~0.7 kb is absent in the 

corresponding lanes in the short run. 

IV.A.2. Probe 2 

Conventional control genomic DNA digested with Nco I (Figure IV-3, lane 1 and lane 7) 

or a combination of EcoR I and Spe I (Figure IV-3, lane 3 and lane 9) and hybridized 

with Probe 2 (Figure III-1) showed no detectable hybridization bands, as expected. 

Conventional control genomic DNA digested with Nco I and spiked with PV-GMAP5779 

DNA previously digested with Nco I produced one expected band at ~3.7 kb (Figure IV3, lane 5 and lane 6). Detection of the spiked controls indicates that the probe hybridized 

to its target sequences. 

MON 87712 DNA digested with Nco I and hybridized with Probe 2 (Figure III-1), 

produced one expected band at ~2.2 kb (Figure IV-3, lane 2 and lane 8). The ~2.2 kb 

band represents the 3′ end of the inserted DNA and the adjacent DNA flanking the 3′ end 

of the insert. 

MON 87712 DNA digested with a combination of EcoR I and Spe I (Figure IV-3, lane 4 

and lane 10) and hybridized with Probe 2 produced one expected band at ~2.2 kb. The 

~2.2 kb band represents the 3′ end of the inserted DNA and the adjacent DNA flanking 

the 3′ end of the insert. The results presented in Figure IV-3 indicate that the sequence 

covered by Probe 2 resides at a single detectable locus of integration in MON 87712. 
Monsanto Company 11-SY-217U 54 of 467 

Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number of 

T-DNA I in MON 87712: Probe 1 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the 

T-DNA I sequence (Figure III-1, Probe 1). Each lane contains ~10 μg of digested 

genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~0.1 genome equivalent] 

7 Conventional Control (Nco I) 

8 MON 87712 (Nco I) 

9 Conventional Control (EcoR I and Spe I) 

10 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1kb DNA Extension Ladder (Invitrogen) on 

the ethidium bromide stained gel. 

 

1 2 3 4 5 6 7 8 9 10

Long Run Short Run

40

40 20

15

10

8.1

7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5

20 

15

10 8.1 7.1 6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5
Monsanto Company 11-SY-217U 55 of 467 

Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number of 

T-DNA I in MON 87712: Probe 2 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the 

T-DNA I sequence (Figure III-1, Probe 2). Each lane contains ~10 μg of digested 

genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~0.1 genome equivalent] 

7 Conventional Control (Nco I) 

8 MON 87712 (Nco I) 

9 Conventional Control (EcoR I and Spe I) 

10 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1kb DNA Extension Ladder (Invitrogen) on the 

ethidium bromide stained gel. 

 

1 2 3 4 5 6 7 8 9 10

Long Run Short Run

40 20 15

10 8.1 7.1 6.1 5.1 4.1

3.1

2.0

1.6

1.0

0.5

40

20 15

10

8.1 7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5
Monsanto Company 11-SY-217U 56 of 467 

IV.B. Southern Blot Analysis to Determine the Presence or Absence of T-DNA II 

Sequences in MON 87712 

The presence or absence of T-DNA II sequences in MON 87712 was evaluated by 

digesting the R3 generation of MON 87712 and the conventional control genomic DNA 

samples with two sets of restriction enzymes: Nco I and a combination of EcoR I and 

Spe I. Each Southern blot was hybridized with overlapping probes spanning the 

T-DNA II sequence, except for the border regions (Figure III-1, Probe 3, Probe 4, and 

Probe 5), since the border sequences of T-DNA II share 100% homology to the border 

sequences of T-DNA I. Therefore, these border regions were covered by Probe 1 and 

Probe 2. If T-DNA II sequences are present in MON 87712, then probing with the 

T-DNA II sequences would result in the detection of hybridization bands. The results of 

these analyses are shown in Figure IV-4 and Figure IV-5. 

IV.B.1. Probe 3 and Probe 5 

Conventional control genomic DNA digested with Nco I (Figure IV-4, lane 1 and lane 8) 

or a combination of EcoR I and Spe I (Figure IV-4, lane 3 and lane 10) and hybridized 

with Probe 3 and Probe 5 (Figure III-1) showed no detectable hybridization bands, as 

expected. Conventional control genomic DNA digested with Nco I and spiked with 

PV-GMAP5779 DNA previously digested with Nco I produced two expected bands at 

~7.8 kb and ~3.7 kb (Figure IV-4, lane 5). Conventional control genomic DNA digested 

with Nco I and spiked with probe templates (Figure III-1, Probe 3 and Probe 5) generated 

from PV-GMAP5779 produced the expected bands at ~1.3 kb and ~1.1 kb, respectively 

(Figure IV-4, lane 6 and lane 7). Detection of the spiked controls indicates that the 

probes hybridized to their target sequences. 

MON 87712 DNA digested with Nco I (Figure IV-4, lane 2 and lane 9) or a combination 

of EcoR I and Spe I (Figure IV-4, lane 4 and lane 11) and hybridized with Probe 3 and 

Probe 5 (Figure III-1) produced no detectable bands, as expected. The results presented 

in Figure IV-4 indicate that MON 87712 contains no detectable T-DNA II elements from 

Probe 3 and Probe 5 of PV-GMAP5779. 

IV.B.2. Probe 4 

Conventional control genomic DNA digested with Nco I (Figure IV-5, lane 1 and lane 7) 

or a combination of EcoR I and Spe I (Figure IV-5, lane 3 and lane 9) and hybridized 

with Probe 4 (Figure III-1) produced endogenous hybridization signals. These signals are 

not visible in the reported figure (Figure IV-5). However, these endogenous signals are 

observed at >40 kb (Nco I digest, lane 1, lane 2, and lane 5 through lane 8) and ~1.0 kb 

(EcoR I and Spe I digest, lane 3, lane 4, lane 9, and lane 10) in a darker exposure (data 

not shown). These hybridization signals most likely result from Probe 4 hybridizing to 

homologous sequences residing in the soybean genome. 

Conventional control genomic DNA digested with Nco I and spiked with PV-GMAP5779 

DNA previously digested with Nco I produced two expected bands at ~7.8 kb and 

~3.7 kb (Figure IV-5, lane 5 and lane 6) in addition to the endogenous hybridization 
Monsanto Company 11-SY-217U 57 of 467 

bands discussed above. Detection of the spiked controls indicates that the probe 

hybridized to its target sequence. 

MON 87712 DNA digested with Nco I (Figure IV-5, lane 2 and lane 8) or a combination 

of EcoR I and Spe I (Figure IV-5, lane 4 and lane 10) and hybridized with Probe 4 

(Figure III-1) produced no detectable bands other than the endogenous hybridization 

bands discussed above. The results presented in Figure IV-5 indicate that MON 87712 

contains no detectable T-DNA II elements from Probe 4 of PV-GMAP5779 other than 

the endogenous soybean sequences. 
Monsanto Company 11-SY-217U 58 of 467 

Figure IV-4. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II 

Sequences in MON 87712: Probe 3 and Probe 5 

The blot was hybridized with two 32P-labeled probes that spanned portions of the 

T-DNA II sequence (Figure III-1, Probe 3 and Probe 5). Each lane contains ~10 μg of 

digested genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with Probe 3 and Probe 5 [~1.0 genome equivalent] 

7 Conventional Control (Nco I) spiked with Probe 3 and Probe 5 [~0.1 genome equivalent] 

8 Conventional Control (Nco I) 

9 MON 87712 (Nco I) 

10 Conventional Control (EcoR I and Spe I) 

11 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1kb DNA Extension Ladder (Invitrogen) 

on the ethidium bromide stained gel. 

 

1 2 3 4 5 6 7 8 9 10 11

Long Run Short Run

40 20

15

10

8.1

7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0

40 20 15

10

8.1

7.1 6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5
Monsanto Company 11-SY-217U 59 of 467 

Figure IV-5. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II 

Sequences in MON 87712: Probe 4 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the 

T-DNA II sequence (Figure III-1, Probe 4). Each lane contains ~10 μg of digested 

genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~0.1 genome equivalent] 

7 Conventional Control (Nco I) 

8 MON 87712 (Nco I) 

9 Conventional Control (EcoR I and Spe I) 

10 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1kb DNA Extension Ladder (Invitrogen) on 

the ethidium bromide stained gel. 

 

40 20

15

10

8.1

7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5

1 2 3 4 5 6 7 8 9 10

Long Run Short Run

40 20 15 10 8.1 7.1 6.1 5.1

4.1

3.1

2.0

1.6

1.0

0.5
Monsanto Company 11-SY-217U 60 of 467 

IV.C. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GMAP57779 Backbone Sequences in MON 87712 

The presence or absence of PV-GMAP5779 backbone sequences in the soybean genome 

was evaluated by digesting the R3 generation of MON 87712 and the conventional 

control genomic DNA samples with two sets of restriction enzymes: Nco I and a 

combination of EcoR I and Spe I. Digested genomic DNA was hybridized with 

overlapping probes spanning the main backbone sequence of PV-GMAP5779 

(Figure III-1, Probe 7, Probe 8, and Probe 9) and one individual probe covering the 

backbone sequence between T-DNA I and T-DNA II (Figure III-1, Probe 6). If backbone 

DNA sequences were present in MON 87712, then hybridizing with overlapping probes 

corresponding to the backbone sequence should result in the detection of hybridization 

bands on the Southern blot. The results of this analysis are shown in Figure IV-6 through 

Figure IV-8. 

IV.C.1. Plasmid Vector Backbone Probe 6 

Conventional control genomic DNA digested with Nco I (Figure IV-6, lane 1 and lane 7) 

or a combination of EcoR I and Spe I (Figure IV-6, lane 3 and lane 9) and hybridized 

with Probe 6 (Figure III-1) showed no detectable hybridization bands, as expected. 

Conventional control genomic DNA digested with Nco I and spiked with PV-GMAP5779 

previously digested with Nco I produced two expected bands at ~7.8 kb and ~3.7 kb, 

respectively (Figure IV-6, lane 5 and lane 6). The ~3.7 kb band was expected, because 

this segment contained Probe 6 sequence. The ~7.8 kb band was also detected because a 

small region of the intervening sequence contained in Probe 7 sequence in this ~7.8 kb 

segment is identical to a portion of Probe 6. Detection of the spiked controls indicates 

that the probe hybridized to its target sequence. 

MON 87712 genomic DNA digested with Nco I (Figure IV-6, lane 2 and lane 8) or in 

combination with EcoR I and Spe I (Figure IV-6, lane 4 and lane 10) and hybridized with 

Probe 6 (Figure III-1) produced no detectable bands, as expected. The data indicate that 

MON 87712 contains no detectable backbone elements from Probe 6 of PV-GMAP5779. 

IV.C.2. Plasmid Vector Backbone Probe 7 and Probe 9 

Conventional control genomic DNA digested with Nco I (Figure IV-7, lane 1 and lane 8) 

or a combination of EcoR I and Spe I (Figure IV-7, lane 3 and lane 10) and hybridized 

with Probe 7 and Probe 9 (Figure III-1) showed no detectable hybridization bands, as 

expected. Conventional control genomic DNA digested with Nco I and spiked with 

PV-GMAP5779 previously digested with Nco I produced two expected bands at ~7.8 kb 

and ~3.7 kb, respectively (Figure IV-7, lane 5). The ~7.8 kb band was expected, because 

this segment contained Probe 7 and Probe 9 sequence. The faint ~3.7 kb band (stronger 

signal observed in darker exposure, data not shown) was also detected because a small 

region of the intervening sequence contained in Probe 6 sequence of this ~3.7 kb segment 

is identical to a portion of Probe 7 (refer to Section IV.C.1, Probe 6). 
Monsanto Company 11-SY-217U 61 of 467 

Conventional control genomic DNA digested with Nco I and spiked with probe templates 

(Figure III-1, Probe 7 and Probe 9) generated from PV-GMAP5779 produced the 

expected bands at ~1.8 kb and ~0.9 kb, respectively (Figure IV-7, lane 6 and lane 7). 

Detection of the spiked controls indicates that the probes hybridized to their target 

sequences. 

MON 87712 genomic DNA digested with Nco I (Figure IV-7, lane 2 and lane 9) or a 

combination with EcoR I and Spe I (Figure IV-7, lane 4 and lane 11) and hybridized with 

Probe 7 and Probe 9 (Figure III-1) produced no detectable bands, as expected. The data 

indicate that MON 87712 contains no detectable backbone elements from Probe 7 and 

Probe 9 of PV-GMAP5779. 

IV.C.3. Plasmid Vector Backbone Probe 8 

Conventional control genomic DNA digested with Nco I (Figure IV-8, lane 1 and lane 7) 

or a combination of EcoR I and Spe I (Figure IV-8, lane 3 and lane 9) and hybridized 

with Probe 8 (Figure III-1) showed no detectable hybridization bands, as expected. 

Conventional control genomic DNA digested with Nco I and spiked with PV-GMAP5779 

previously digested with Nco I produced an expected band at ~7.8 kb (Figure IV-8, lane 5 

and lane 6). Detection of the spiked controls indicates that the probe hybridized to its 

target sequences. 

MON 87712 genomic DNA digested with Nco I (Figure IV-8, lane 2 and lane 8) or in 

combination with EcoR I and Spe I (Figure IV-8, lane 4 and lane 10) and hybridized with 

Probe 8 (Figure III-1) produced no detectable bands, as expected. The data indicate that 

MON 87712 contains no detectable backbone elements from Probe 8 of PV-GMAP5779. 
Monsanto Company 11-SY-217U 62 of 467 

Figure IV-6. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GMAP5779 Backbone Sequences in MON 87712: Probe 6 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the PVGMAP5779 backbone sequence (Figure III-1, Probe 6). Each lane contains ~10 μg of 

digested genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~0.1 genome equivalent] 

7 Conventional Control (Nco I) 

8 MON 87712 (Nco I) 

9 Conventional Control (EcoR I and Spe I) 

10 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from λ DNA/Hind III Fragments (Invitrogen) on the 

ethidium bromide stained gel. 

 

23.1

9.4

4.4

6.6

2.3

2.0

0.6

23.1

9.4

4.4

6.6

2.3

2.0

0.6

1 2 3 4 5 6 7 8 9 10

Long Run Short Run
Monsanto Company 11-SY-217U 63 of 467 

Figure IV-7. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GMAP5779 Backbone Sequences in MON 87712: Probe 7 and Probe 9

The blot was hybridized with two 32P-labeled probes that spanned portions of 

PV-GMAP5779 backbone sequence (Figure III-1, Probe 7 and Probe 9). Each lane 

contains ~10 μg of digested genomic DNA isolated from leaf tissue. Lane designations 

are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with Probe 7 and Probe 9 [~1.0 genome equivalent] 

7 Conventional Control (Nco I) spiked with Probe 7 and Probe 9 [~0.1 genome equivalent] 

8 Conventional Control (Nco I) 

9 MON 87712 (Nco I) 

10 Conventional Control (EcoR I and Spe I) 

11 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from λ DNA/Hind III Fragments (Invitrogen) on the 

ethidium bromide stained gel. 

 

1 2 3 4 5 6 7 8 9 10 11

9.4

23.1

6.6

4.4

2.3

2.0

0.6

9.4

23.1

6.6

4.4

2.3

2.0

0.6

Long Run Short Run
Monsanto Company 11-SY-217U 64 of 467 

Figure IV-8. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GMAP5779 Backbone Sequences in MON 87712: Probe 8 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the 

PV-GMAP5779 backbone sequence (Figure III-1, Probe 8). Each lane contains ~10 μg 

of digested genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Nco I) 

2 MON 87712 (Nco I) 

3 Conventional Control (EcoR I and Spe I) 

4 MON 87712 (EcoR I and Spe I) 

5 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

6 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~0.1 genome equivalent] 

7 Conventional Control (Nco I) 

8 MON 87712 (Nco I) 

9 Conventional Control (EcoR I and Spe I) 

10 MON 87712 (EcoR I and Spe I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from λ DNA/Hind III Fragments (Invitrogen) on the 

ethidium bromide stained gel. 

9.4

23.1

6.6

4.4

2.3

2.0

0.6

9.4

23.1

6.6

4.4

2.3

2.0

0.6

1 2 3 4 5 6 7 8 9 10

Long Run Short Run
Monsanto Company 11-SY-217U 65 of 467 

IV.D. Organization and Sequence of the Insert and Adjacent DNA in MON 87712 

The organization and sequence of the elements within the MON 87712 insert was 

confirmed by DNA sequence analyses. PCR primers were designed with the intent to 

amplify three overlapping DNA regions that span the entire length of the T-DNA I insert 

and the associated DNA flanking the 5' and 3' ends of the insert (Figure IV-9). The 

amplified DNA segments were subjected to DNA sequence analyses. The analyses 

determined that the DNA sequence of the MON 87712 insert is 2014 bp long (Table IV2) and is identical to the corresponding T-DNA I sequence of PV-GMAP5779 as 

described in Table III-1. From the sequence analyses, 3493 base pairs flanking the 5' end 

of the MON 87712 insert and 1965 base pairs flanking the 3' end of the MON 87712 

insert (Table IV-2) were also determined. 

 
Monsanto Company 11-SY-217U 66 of 467 

Figure IV-9. Overlapping PCR Analysis Across the Insert in MON 87712 

PCR was performed on both conventional control genomic DNA and MON 87712 

genomic DNA using three pairs of primers to generate overlapping PCR fragments from 

MON 87712 for sequencing analysis. To verify the PCR products, 4 μl of each of the 

PCR reactions was loaded on the gel. The expected product size for each amplicon is 

provided in the illustration of the insert in MON 87712 that appears at the bottom of the 

figure. This figure is a representative of the data generated in the study. Lane 

designations are as follows: 

Lane Description 

1 1 kb DNA Ladder 

2 Conventional Control 

3 MON 87712 

4 No template DNA control 

5 Conventional Control 

6 MON 87712 

7 PV-GMAP5779 

8 No template DNA control 

9 Conventional Control 

10 MON 87712 

11 No template DNA control 

12 1 kb DNA Ladder 

Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 

1 kb DNA Ladder (Invitrogen) on the ethidium bromide stained gel. P-e35S CS-BBX32

T-E6

B-Right Border Region r1

5' 

Flank 

3' 

Flank

Product A: ~3.8 kb Product B: ~1.9 kb Product C: ~2.6 kb

1 2 3 4 5 6 7 8 9 10 11

12 

Product A Product B Product C

3.1

0.5

1.0

1.6

2.0

12

5.1

4.1

3.1

0.5

1.0

1.6

2.0

12

5.1

4.1

B-Left Border Region r1
Monsanto Company 11-SY-217U 67 of 467 

IV.E. PCR and DNA Sequence Analyses to Examine the MON 87712 Insertion Site 

PCR and sequence analyses were performed on genomic DNA extracted from 

MON 87712 and the conventional control to examine the integrity of the DNA insertion 

site in MON 87712. The PCR was performed with a forward primer specific to the 

genomic DNA sequence flanking the 5' end of the insert paired with a reverse primer 

specific to the genomic DNA sequence flanking the 3' end of the insert (Figure IV-10). 

The amplified PCR product from the conventional control was subjected to DNA 

sequence analysis. Sequence alignments were performed between the conventional 

control sequence and the sequences flanking the 5' and 3' end of the MON 87712 

T-DNA I insert. The alignment analyses indicated a 42 base pair deletion from the 

conventional genomic DNA occurred upon T-DNA I insertion in MON 87712. This 

deletion presumably resulted from double stranded break repair mechanisms in the plant 

during the Agrobacterium-mediated transformation process (Salomon and Puchta, 1998). 

 
Monsanto Company 11-SY-217U 68 of 467 

Figure IV-10. PCR Amplification of the MON 87712 Insertion Site 

PCR analysis was performed to evaluate the insertion site. PCR was performed on 

conventional control DNA and MON 87712 DNA using Primer A, specific to the 5' 

flanking sequence, and Primer B, specific to the 3' flanking sequence of the insert in 

MON 87712. The DNA generated from the conventional control PCR was used for 

sequencing analysis. This illustration depicts the MON 87712 insertion site in the 

conventional control (upper panel) and the MON 87712 insert (lower panel). 

Approximately 9 μl of each of the PCR reactions was loaded on the gel. This figure is 

representative of the data generated in the study. Lane designations are as follows: 

Lane Description 

1 1 kb DNA Ladder 

2 Conventional Control 

3 MON 87712 

4 No template DNA control 

5 1 kb DNA Ladder 

Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from the 1kb DNA 

Ladder (Invitrogen) on the ethidium bromide stained gel. 

3.1

0.5

1.0

1.6

2.0

12

4.1

3.1

0.5

1.0

1.6

2.0

12

4.1

Primer A Primer B B-Left Border Region r1 P-e35S CS-BBX32 T-E6 B-Right Border Region r1

5' 

Flank 

3' 

Flank

Primer A Primer B

1 4 2 3 5
Monsanto Company 11-SY-217U 69 of 467 

IV.F. Southern Blot Analysis to Examine Insert Stability in Multiple Generations of 

MON 87712 

In order to demonstrate the stability of the insert in MON 87712, Southern blot analysis 

was performed using genomic DNA obtained from five generations of MON 87712. For 

reference, the breeding history diagram of MON 87712 and the generations that were 

tested are indicated in Figure IV-11. The MON 87712 R3 generation was used for the 

molecular characterization analyses as shown in Figure IV-2 through Figure IV-8. To 

analyze insert stability, four additional generations, R4, R5, R6, and R7, were evaluated by 

Southern blot analysis and compared to the R3 generation. Genomic DNA, isolated from 

each of the selected generations of MON 87712, was digested with Nco I (Figure IV-1) 

and hybridized with Probe 1 (Figure III-1). Probe 1 was designed to detect both 

segments generated by the Nco I digest. Any instability associated with the insert would 

be detected as novel bands within the fingerprint on the Southern blot. This Southern 

blot contains the same controls as described in Section IV.A.1. 

IV.F.1. Probe 1 

Conventional control genomic DNA digested with Nco I and hybridized with Probe 1 

showed no detectable hybridization bands (Figure IV-12, lane 1), as expected. 

Conventional control genomic DNA digested with Nco I and spiked with PV-GMAP5779 

DNA previously digested with Nco I produced two expected bands at ~7.8 kb and 

~3.7 kb (Figure IV-12, lane 2 and lane 3). Detection of the spiked controls indicates that 

the probe hybridized to its target sequences. 

MON 87712 DNA extracted from generations R3, R4, R5, R6, and R7 and digested with 

Nco I and hybridized with Probe 1 (Figure III-1) produced two expected bands at ~4.3 kb 

and ~2.2 kb (Figure IV-12, lane 4 through lane 8). These bands are consistent with the 

~4.3 kb and ~2.2 kb bands detected in the MON 87712 R3 generation (Figure IV-2, lane 2 

and lane 8). The ~4.3 kb band represents the 5′ end of the inserted DNA and the adjacent 

DNA flanking the 5′ end of the insert. The ~2.2 kb band represents the 3′ end of the 

inserted DNA and the adjacent DNA flanking the 3′ end of the insert. Therefore, this 

result indicates that the single copy of T-DNA I in MON 87712 is stably maintained 

across the five generations. 
Monsanto Company 11-SY-217U 70 of 467 

Figure IV-11. Breeding History of MON 87712 

The R3 generation was used for the molecular characterization and commercial 

development of MON 87712. R0 corresponds to the transformed plant. All generations 

were self pollinated (). Generations used for insert stability analysis (R3, R4, R5, R6, 

and R7) are indicated in bold text. 

R6













R5

R0

R1

R2

R4 

R3 

R7


Monsanto Company 11-SY-217U 71 of 467 

Figure IV-12. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 87712: Probe 1 

The blot was hybridized with one 32P-labeled probe that spanned a portion of the 

T-DNA I sequence (Figure III-1, Probe 1). Each lane contains ~10 μg of digested 

genomic DNA isolated from leaf tissue. Lane designations are as follows: 

Lane Description

1 Conventional Control (Nco I) 

2 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~1.0 genome equivalent] 

3 Conventional Control (Nco I) spiked with PV-GMAP5779 (Nco I) [~0.1 genome equivalent] 

4 MON 87712 (R3) (Nco I) 

5 MON 87712 (R4) (Nco I) 

6 MON 87712 (R5) (Nco I) 

7 MON 87712 (R6) (Nco I) 

8 MON 87712 (R7) (Nco I) 

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Extension Ladder (Invitrogen) on 

the ethidium bromide stained gel. 

 

 

40 20

15

10

8.1

7.1

6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5

12 3 4 5 6 7 8
Monsanto Company 11-SY-217U 72 of 467 

IV.G. Inheritance of the Genetic Insert in MON 87712 

During development of MON 87712, segregation data were generated to assess the 

heritability and stability of the T-DNA I present in MON 87712. Chi square (χ2) analysis 

was performed over several generations to confirm the segregation and stability of 

T-DNA I in MON 87712. The Chi square analysis is based on testing the observed 

segregation ratio to the expected segregation ratio according to Mendelian principles. 

The MON 87712 breeding path for generating segregation data is described in Figure IV13. The transformed R0 plant was self-pollinated to produce R1 seed. From the R1

segregating population, which consisted of 88 total plants, an individual plant 

(subsequently designated as MON 87712) homozygous for the BBX32 gene was 

identified via Invader® analysis. Invader is a non-PCR based assay that can be used to 

accurately quantify DNA copy number in plant genomes (Gupta et al., 2008). 

The selected R1 MON 87712 plant was self-pollinated to give rise to a population of R2

plants, which were in turn self-pollinated to obtain the R3 generation. At each generation, 

the fixed homozygous plants were tested for the expected segregation pattern of 1:0 

(positive:negative) for the BBX32 gene using the Invader analysis. 

Homozygous R3 MON 87712 plants were crossed to a Monsanto proprietary soybean 

line (EXP0224AAC) that did not contain the BBX32 gene to produce F1 hemizygous 

seed. The resulting F1 plants were tested for the copy number of the BBX32 gene by 

Endpoint Taqman zygosity analysis. A hemizygous F1 plant was selected and then selfpollinated to produce F2 seed. This process of self-pollination and Endpoint Taqman 

zygosity analysis was repeated for the F2, F3, and F4 plants. Subsequently, assessment at 

each of these generations was based on zygosity, and the BBX32 gene was predicted to 

segregate at a 1:2:1 (homozygous positive:hemizygous positive:homozygous negative) 

ratio for progeny derived from a hemizygous parental plant according to Mendelian 

inheritance principles. 

A Chi square (χ

2

) analysis was used to compare the observed segregation ratios to the 

expected ratios according to Mendelian inheritance principles. The χ2 was calculated as: 

χ

2

 = ∑ [( | o - e | )2 / e] 

where o = observed frequency of the phenotype and e = expected frequency of the 

phenotype. The level of statistical significance was predetermined to be 5% (α = 0.05). 

The results of the χ

2 analysis of the segregating progeny of MON 87712 are presented in 

Table IV-3. The χ

2

 value in the F2, F3, and F4 generations indicated no statistically 

significant difference between the observed and expected 1:2:1 segregation ratio. These 

results support the conclusion that the BBX32 coding sequence in MON 87712 resides at 

a single locus within the soybean genome and is inherited according to Mendelian 

inheritance principles. These results are also consistent with the molecular 

characterization data that indicate MON 87712 contains a single, intact copy of the 

BBX32 expression cassette that was inserted into the soybean genome at a single locus. 
Monsanto Company 11-SY-217U 

Figure IV-13. Breeding Path for Generating Segregation Data for MON 87712 Self pollinated. 

* Chi-square analysis conducted on segregation data from the F2, F3, and F4 generations. 

Note: An individual hemizygous plant from each of the F1, F2, and F3 populations was self-pollinated to produce the population of the 

Transformed and regenerated R 0 plant



R1

Plant #2 (MON 87712) selected 

R2



(homozygous positive)

R3 (homozygous positive)

Breeding path continued

 Cross with a Monsanto 

proprietary variety that did not contain the BBX32 gene

F1 (hemizygous )



F2 (expected segregation of 1:2:1)*

homozygous positive : hemizygous : homozygous negative



F3 (expected segregation of 1:2:1)*

homozygous positive : hemizygous : homozygous negative



F4 (expected segregation of 1:2:1)*

homozygous positive : hemizygous : homozygous neg


Monsanto Company 11-SY-217U 

Table IV-3. Segregation of the Expression Cassette During the Development of MON 87712 

1:2:1 Segregation 

Generation

Total 

Plants1

Observed # 

Plants 

Homozygous 

Positive

Observed # 

Plants 

Hemizygous

Observed # 

Plants 

Homozygous 

Negative

Expected # 

Plants 

Homozygous 

Positive

Expected # 

Plants 

Hemizygous

Expected # 

Plants 

Homozygou

Negative

F2 218 59 98 61 54.50 109.00 54.50 F3 184 48 94 42 46.00 92.00 46.00 F4 182 44 87 51 45.50 91.00 45.50 

1Plants were evaluated for the presence of the BBX32 gene by Endpoint Taqman zygosity analysis. “Total plants” refer

plants in which zygosity could be determined using the assay. 
Monsanto Company 11-SY-217U 75 of 467 

IV.H. Genetic Modification Characterization Conclusion 

Molecular characterization of MON 87712 by Southern blot analyses confirmed that one 

copy of the BBX32 expression cassette was integrated into the soybean genome at a 

single locus. No T-DNA II or backbone DNA sequences from plasmid vector 

PV-GMAP5779 were detected in MON 87712. 

PCR and DNA sequence analyses performed on MON 87712 and conventional control 

determined the following: the complete DNA sequence of the insert and adjacent DNA 

sequences in MON 87712; the organization of the genetic elements within the insert; the 

expected sequence of each element in the inserted DNA; and the 5′ and 3′ insert to 

genomic DNA junctions. The PCR and DNA sequence analysis identified a 42 base pair 

deletion that occurred at the insertion site in MON 87712. 

Southern blot analysis of MON 87712 demonstrated that the inserted DNA has been 

maintained through five generations of breeding, 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 T-DNA I in MON 87712 at a 

single chromosomal locus. 
Monsanto Company 11-SY-217U 76 of 467 

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE BBX32 

PROTEIN IN MON 87712 

Characterization of the introduced protein in a biotechnology-derived crop product is 

important to establishing its food, feed, and environmental safety. As described in 

Section IV, MON 87712 contains a BBX32 expression cassette that upon translation 

results in the BBX32 protein. This section summarizes: 1) the functionality of BBX32; 

2) the characterization of MON 87712 BBX32; 3) the levels of MON 87712 BBX32 in 

plant tissues; 4) assessment of the potential allergenicity of MON 87712 BBX32 and 5) 

the food, feed, and environmental safety assessment of MON 87712 BBX32. The data 

support a conclusion that MON 87712 is safe for the environment and human or animal 

consumption based on several lines of evidence, all of which are summarized below. 

V.A. Identity and Function of the BBX32 Protein from MON 87712 

MON 87712 has been demonstrated to provide increased yield (See Appendix B), due to 

the insertion of the Arabidopsis thaliana BBX32 gene. The BBX32 protein is a member 

of the of the B-box zinc finger family from Arabidopsis thaliana. This family represents 

a subgroup of zinc finger proteins that contain one or more B-box domains with 

specialized tertiary structures that are stabilized by binding zinc ions. The B-box domain 

is predicted to be involved in protein-protein interactions (Khanna et al., 2009). BBX32 

contains a single annotated protein domain, the B-Box B1 domain (Khanna et al., 2009). 

The B-box zinc finger family is found in many plant species; for example, the soybean Bbox family contains 61 genes (Preuss et al., submitted for publication). Homologs of 

BBX32 are found in many agronomically-important species, suggesting that the function 

of BBX32 in its source plant, Arabidopsis thaliana, is conserved in other plant species. 

BBX32 acts as a plant transcriptional accessory protein. As described in Appendix B, 

BBX32 in MON 87712 plays a role in the transcriptional regulation of the plant’s 

day/night processes, and results in increased availability of assimilates in the plant. Plant 

nutrient assimilation and utilization are critical processes to drive yield improvement. 

V.B. Characterization and Equivalence of BBX32 Protein from MON 87712 

The safety assessment of crops derived through biotechnology includes characterization 

of the functional and physicochemical properties of, and confirmation of the safety of, the 

introduced protein. The expression level of BBX32 protein in MON 87712 is low, and 

insufficient for use in the subsequent safety evaluations. Therefore, recombinant BBX32 

protein was produced in Escherichia coli, using an expression vector with a BBX32

coding sequence that matched that of the BBX32 coding sequence in MON 87712. In 

order to establish the suitability of the E. coli-produced BBX32 for use in safety 

evaluations, the E. coli-produced and the MON 87712-produced BBX32 preparations 

were characterized and assessed for equivalence. 
Monsanto Company 11-SY-217U 77 of 467 

As reported in section V.C, the level of expression of BBX32 in MON 87712 is very low. 

Due to its low level of expression, the MON 87712-produced BBX32 protein was not 

isolated to a high level of purity, but instead was enriched in leaf extract to a purity of 

approximately 0.0001%. This low purity limited the physicochemical characterization of 

MON 87712-produced BBX32 and the overall equivalence analysis to characteristics that 

could be evaluated by western blot. E. coli-produced BBX32 was characterized using a 

panel of analytic techniques including western blot analysis, N-terminal sequence 

analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 

(MALDI-TOF MS) to confirm identity, sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE) to establish the apparent molecular weight of BBX32, and 

analysis of binding activity to a specific partner protein to demonstrate protein-protein 

binding functionality. 

MON 87712-produced BBX32 protein was characterized by western blot analysis to 

establish identity via immunoreactivity with an anti-BBX32 antibody. Equivalence 

between MON 87712-produced and E. coli-produced BBX32 was established through 

comparison of the migration of the two proteins on SDS-PAGE, as detected by western 

blot analysis, and through bioinformatic comparison of the BBX32 coding sequence from 

the E. coli expression vector and MON 87712. The details of the materials, methods, and 

results are described in Appendix D, while the conclusions are summarized as below. 

Western blot analysis with a monoclonal antibody developed against BBX32 showed that 

the MON 87712-produced BBX32 had equivalent mobility to the E. coli-produced 

BBX32 protein on the blot, indicating the proteins from both sources were 

immunoreactive and equivalent in apparent molecular weight. These data provide a 

characterization of the BBX32 protein from MON 87712, and were used to establish its 

equivalence to the E. coli-produced BBX32 protein. 

The BBX32 coding sequence expressed in E. coli was designed to precisely match the 

amino acid coding sequence of the BBX32 gene expressed in MON 87712. Sequence 

alignment showed that translation of the BBX32 coding sequence from the vector used to 

transform conventional soybean to produce MON 87712, PV-GMAP5779, and from the 

E. coli BBX32 expression vector, pMON102114, resulted in the same amino acid 

sequence (see Appendix D). As reported in Section IV.D, the DNA sequence of the 

MON 87712 insert is identical to the corresponding T-DNA I sequence of PVGMAP5779. Thus, the pMON102114 BBX32 coding sequence is identical to the BBX32 

coding sequence in MON 87712. 

Direct assessments of BBX32 partner protein binding activity, glycosylation status, and 

N-terminal sequence , were not conducted for MON 87712-produced BBX32 due to the 

low purity of the plant-produced protein. Instead, these characteristics of BBX32 in 

MON 87712 were assessed indirectly. With respect to functional activity, the phenotypes 

observed for MON 87712 compared to conventional soybean, including discrete changes 

in gene expression (for example, Figure B-6) and increased crop yield, indicate that 

BBX32 is active in MON 87712. The E. coli-produced BBX32 was active in its partner 

protein binding assay. The N-terminal sequence of E. coli-produced BBX32 was 

evaluated and shown to match the N-terminus predicted by the BBX32 coding sequence 
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in MON 87712. Thus, the E. coli-produced BBX32 represents the full length BBX32 

protein in MON 87712. There is no predicted N-terminal targeting or signal sequence in 

the BBX32 coding sequence. The BBX32 protein sequence contains a consensus 

potential N-glycosylation site (NTT, starting at position 172), but lacks the N-terminal 

signal sequence required for transport to the endoplasmic reticulum which is the gateway 

for both N- and O-glycosylation (Pattison and Amtmann, 2009; Vitale and Denecke, 

1999). This is similar to the CP4-EPSPS, which contains potential N-glycosylation sites 

but has been shown to not be glycosylated (Harrison et al., 1996). In total, this suggests 

that MON 87712-produced BBX32 is not glycosylated. 

V.C. Expression Levels of BBX32 Protein in MON 87712 

The levels of BBX32 in various tissues of MON 87712 that are relevant to the risk 

assessment were determined by western blot analysis. For western blots, protein extracts 

are separated by gel electrophoresis and then transferred to a membrane that is probed 

with an antibody. A sensitive western blot technique was developed to separate and 

clearly differentiate the BBX32 protein present in MON 87712 tissue extracts from other 

proteins present in the tissue matrix, which allowed for the detection of this low 

expressing protein in the presence of other matrix proteins. The levels of the BBX32 

protein in various tissues of MON 87712 were estimated by densitometric analysis of Xray films exposed to immunoblots probed with BBX32 antibodies and visualized using 

chemiluminescent detection reagents (refer to Appendix E). 

Tissue samples of MON 87712 and the parental conventional control A3525 were 

collected during the 2009 growing season from eight geographically diverse field sites in 

the U.S.: Jackson County, Arkansas; Parke County, Indiana; Clinton County, Illinois; 

Madison County, Illinois; Stark County, Illinois; York County, Nebraska; Boone County, 

Indiana; and Pawnee County, Kansas. These field sites were representative of soybean 

producing regions suitable for commercial production. At each site, four replicated plots 

of MON 87712 and the conventional control A3525 were planted using a randomized 

complete block field design. Over-season leaf (OSL-1 to OSL-4), root (OSR-3), forage 

(forage-1), and harvested seed tissues were collected from each replicated plot at all field 

sites. 

Analyses of BBX32 protein levels were carried out in all seven tissue types collected as 

described in Table V-1. For leaf and root samples, densitometric analysis of the BBX32-

specific immunoblots yielded the reported quantitative values of BBX32 protein by 

interpolation from standard curves prepared using purified BBX32 protein standard. The 

results obtained from western blot analysis are summarized in Table V-1and the details of 

the materials, methods, and sample collection are described in Appendix E. 

The mean BBX32 protein levels were determined across eight sites and seven tissue 

types. BBX32 protein levels in MON 87712 across tissue types ranged from <LOD to 

110 ng/g dwt. The western blot method developed was highly sensitive as indicated by 

the low LODs established for each tissue (Table V-1). In spite of the high sensitivity of 

the western technique developed, the BBX32 protein was not detected in several samples 

including all harvested seed, forage and OSL-4 samples. The mean protein level in root 
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was 3.9 ng/g dwt, though most samples were <LOD. Expression was highest in leaf 

tissue, specifically OSL-3 (mean protein level of 35 ng/g dwt) where BBX32 was 

determined in all samples. The levels in OSL-1 and OSL-2 were slightly below those of 

OSL-3. These results indicate that the expression of BBX32 is highest in leaf. 

For seed and forage tissues the LOD of the BBX32 protein was determined by spiking 

serially diluted BBX32 protein reference standard into the conventional control soybean, 

A3525, of forage and seed tissue extracts. The LOD of BBX32 on the western blots was 

defined by the lowest amount of BBX32 protein that resulted in a visible band on the 

western blots. The LOD was determined to be 0.5 pg in forage extracts and 2.5 pg in seed 

extracts. 

Other B-box proteins, such as the Arabidopsis thaliana B-box protein, CONSTANS 

(CO), accumulate at levels too low to be detected by western blot although functional 

activity is present (Suarez-Lopez et al., 2001). Similarly to BBX32 in MON 87712, 

Valverde et al. (2004) reported that CO over-expression in Arabidopsis thaliana results in 

growth condition-dependent detection of the protein, indicating that an over-expressed 

protein can still be subject to endogenous mechanisms that regulate protein abundance. 
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Table V-1. Summary of BBX32 Protein Levels in Leaf, Root, Forage, and Seed 

from MON 87712 Grown in 2009 U.S. Field Trials 

Tissue1 Development 

Stage2

Days 

After 

Planting 

(DAP)

BBX32

Mean (SD) 

Range

(ng/g fwt)3

BBX32

Mean (SD) 

Range

(ng/g dwt)4

LOQ/LOD5

(pg)

 

OSL-1 V3-V4 24-33 3.9 (4.0) 

0.84-16 

21 (21) 

4.4-80 

0.25-1.0/0.25 

 

OSL-2 V6-V8 35-49 3.7 (1.9) 

0.43-8.0 

21 (11) 

2.7-47 

0.25-1.0/0.25 

 

OSL-3 R2 39-63 5.0 (3.7) 

1.0-14 

35 (28) 

5.5-110 

0.25-1.0/0.25 

 

OSL-4 R6 71-106 <LOD NA6 0.25-0.5/0.25 

 

OSR-3 R6 71-106 1.2 (0.15) 

1.1-1.5 

3.9 (0.54) 

3.3-5.0 

0.25-1.0/0.25 

 

Forage-1 R6 71-106 ND7 NA NA/0.5 

Seed R8 112-155 ND NA NA/2.5 

 

1

OSL = over-season leaf, OSR = over-season root. 2

The development stage each tissue was collected. Soybean plant growth stages described in Soybean 

Growth and Development (Pedersen, 2004). 

3

Protein levels are expressed as the arithmetic mean and standard deviation (SD) as nanogram (ng) of 

protein per gram (g) of tissue on a fresh weight basis (fwt). The means, SD, and ranges (minimum and 

maximum values) were calculated for each tissue across all sites. The numbers of samples (n) used in the 

calculations were as follows: OSL-1 n=29, OSL-2 n= 22, OSL-3 n=31, OSR-3 n= 12). Sample collections 

are detailed in Appendix E. 

4

Protein levels are expressed as the arithmetic mean and standard deviation (SD) as nanogram (ng) of 

protein per gram (g) of tissue on a dry weight basis (dwt). The dry weight values were calculated by 

dividing the ng/g fwt by the dry weight conversion factor obtained from moisture analysis data. 

5

LOQ=limit of quantitation; LOD=limit of detection. For leaf and root tissues; LOQ is the range of all 

lowest detectable band across all blots for the specific tissues and LOD is the lowest detectable band across 

the entire tissue type. For forage-1 and seed the LOD was the lowest detectable band when spiked into the 

corresponding tissue extracts. 

6

NA = not applicable 

7

ND = not detected 
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V.D. Assessment of Potential Allergenicity of the BBX32 Protein 

The allergenic potential of an introduced protein is assessed by comparison of the 

biochemical characteristics of the introduced protein to the biochemical characteristics of 

known allergens (Codex Alimentarius, 2003). A protein is not likely to be associated 

with allergenicity if: 1) the protein is from a non-allergenic source, 2) the protein 

represents 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. BBX32 has been 

assessed for its potential allergenicity according to these safety assessment guidelines. 

1) BBX32 originates from Arabidopsis thaliana, an organism that has not been reported 

to be a source of known allergens. 

2) BBX32 protein represents a minor component (less than 0.001%) of the total protein in 

the harvested seed of MON 87712. 

3) Bioinformatics analyses demonstrated that BBX32 does not share amino acid sequence 

similarities with known allergens and, therefore, is highly unlikely to contain 

immunologically cross-reactive allergenic epitopes. 

4) In vitro digestive fate experiments conducted with BBX32 demonstrate that the protein 

is rapidly digested in simulated gastric fluid (SGF) and in simulated intestinal fluid (SIF). 

Taken together, these data support the conclusion that BBX32 does not pose a significant 

allergenic risk to humans or animals. 

V.E. Safety Assessment Summary of BBX32 Protein in MON 87712 

V.E.1. The Donor Organism is Safe 

The gene encoding BBX32 is from Arabidopsis thaliana, a plant that has served as a 

model organism for plant biology (Meinke et al., 1998). Arabidopsis thaliana is 

generally not considered an allergenic or toxic source organism. Although Arabidopsis 

thaliana contains homologs of proteins previously described as allergens in other plant 

species (e.g., germins, lipid transfer protein, profilins, and small molecular weight 

calcium binding proteins), no Arabidopsis proteins have been reported in a peer-reviewed 

database of known allergens (FARRP, 2011). One case of occupational asthma has been 

reported in a laboratory worker due to exposure to Arabidopsis pollen (Yates et al., 

2008). 

Arabidopsis thaliana is not purposely consumed as a food source by humans, and there is 

no documented consumption by animals. However, certain populations of a close 

relative, Arabidopsis lyrata, have been reported to be subject to sheep grazing (Sandring 

et al., 2007). Arabidopsis is in the Brassicaceae family (Meinke et al., 1998), which 

contains well-known food and oilseed crops such as broccoli, cauliflower, cabbage, and 

canola/rapeseed. Camelina sativa, an emerging oilseed crop, is reported to be the 

cultivated species most closely related to Arabidopsis (Pilgeram et al., 2007). Camelina 
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sativa leaves are consumed as fresh greens by humans in the country of Georgia 

(Facciola, 1998), the meal can be used as a component of livestock feed (AAFCO, 2011), 

and the plants in a crop setting are grazed by roaming wildlife (Pilgeram et al., 2007). 

The safe consumption of near relatives of Arabidopsis thaliana by humans and animals 

supports the safety of this organism. 

V.E.2. BBX32 Protein Belongs to a Common Class of Plant Proteins

BBX32 is a member of a family of B-box-containing proteins from Arabidopsis thaliana

(Khanna et al., 2009). The B-box zinc finger family is found in many plant species 

including soybean, where the B-box family contains 61 genes (Preuss et al., submitted for 

publication). Bioinformatic searches using the BBX32 amino acid sequence as the query 

identify homologous sequences from several different plant species, including the food 

crops: citrus, grape, apple, soybean, rice, lettuce, and corn. Overall the protein sequence 

identity of BBX32 to homologs in these species range from ~31-43%. The overall 

protein sequence identity of BBX32 to its homolog in canola is 66%, indicating that 

Brassicaceae species, likely including commonly consumed species such as broccoli, 

contain proteins very similar to BBX32. The amino acid sequence alignment between 

BBX32 and its crop homologs spans the length of the proteins, with the highest identity 

found in the B-box domain. Thus BBX32 shares sequence identity and structural 

similarities with proteins present in plants currently consumed, establishing that humans 

and animals are exposed to this class of proteins and that no adverse effects have been 

attributed to this class of proteins. 

V.E.3. BBX32 Protein in MON 87712 is Not Homologous to a Known Allergen or 

Toxin 

Bioinformatics analyses were performed to assess the allergenic potential, toxicity, or 

biological activity of BBX32. The analysis demonstrated that BBX32 does not share 

amino acid sequence similarities with known allergens, gliadins, glutenins, or protein 

toxins which could have adverse effects to human or animal health. 

V.E.4. BBX32 Protein in MON 87712 is Labile in in vitro Digestion Assays 

BBX32 was readily digestible in SGF and SIF. Rapid degradation of BBX32 in SGF and 

SIF suggests that it is highly unlikely that the BBX32 in MON 87712 would be absorbed 

in the small intestine and have any adverse effects on human or animal health. 

V.E.5. BBX32 Protein in MON 87712 is Not Acutely Toxic 

An acute oral toxicology study was conducted with BBX32. Results indicate that BBX32 

did not cause any adverse effects in mice, with a No Observable Adverse Effect Level 

(NOAEL) of 29 mg/kg body weight (BW), the highest dose level tested. 

Potential human health risks from consumption of foods derived from MON 87712 were 

evaluated using a Margin of Exposure (MOE) approach. BBX32 was not detected in harvested 

seed when assayed using a sensitive immunoblot method with a LOD of 2.5 pg (Table V-1) so an 
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expression level corresponding to the LOD of BBX32 in seed was calculated by 

converting the observed LOD to a ng/g fresh weight value using the following formula: 

volume of the extract loaded per lane ( l) (buffer to tissue ratio)

the lowest spiked BBX32 standard with a visible band (pg) ng/g fwt

   3

The corresponding expression level calculated for BBX32 in seed was10 ng/g fwt. To 

generate estimates of exposure and MOE, an expression level of BBX32 in seed tissue of 

5 ng/g fwt was used. This value, which corresponds to an expression level based on onehalf the LOD of the assay used to quantify BBX32 expression in MON 87712 seed, is in 

keeping with U.S. Environmental Protection Agency guidance for calculations of acute 

human exposure to small molecules in blended commodities when the substance is not 

detectable (U.S. EPA, 2000). A MOE was calculated between the acute mouse NOAEL 

(29 mg/kg BW) for BBX32 and 95th percentile “eater-only” estimates of acute dietary 

exposure determined using the Dietary Exposure Evaluation Model (DEEM-FCID 

version 2.16, Exponent Inc.). DEEM food consumption data are obtained from the 1994-

1996 and 1998 USDA Continuing Survey of Food Intakes by Individuals (CSFII), and 

assume that 100% of soybean products consumed, excluding oil, are derived from 

MON 87712. The MOEs for acute dietary intake of BBX32 were estimated to be 

29,000,000 for the general population and 1,070,000 for non-nursing infants, the subpopulation with the highest estimated exposure. These very large MOEs, in addition to 

the above mentioned protein safety data for BBX32, support the conclusion that there is 

no meaningful risk to human health from dietary exposure to BBX32 in MON 87712. 

Potential health risks to animals from the presence of MON 87712 BBX32 in feed were 

evaluated by calculating an estimate of daily dietary intake (DDI). BBX32 was not 

detected in harvested seed or in forage (see section V.C), so expression levels 

corresponding to the LODs of BBX32 in seed and forage were calculated as described 

above. These expression levels, 10 ng/g fwt in seed and 1 ng/g fwt in forage, were used 

to calculate the DDI. In the worst case scenario, poultry, swine, and lactating dairy cattle 

would be consuming no more than 0.000003% of their total protein intake as BBX32 

protein from MON 87712. This very low level of exposure of animals to BBX32 in their 

feed, in addition to the above mentioned safety data for BBX32, supports the conclusion 

that there is no meaningful risk to animal health when MON 87712 is present in their 

diets. 

Using the guidance provided by the FDA in its 1992 Policy Statement regarding the 

evaluation of New Plant Varieties, a conclusion of “no concern” is reached for the donor 

organism and BBX32 (U.S. FDA, 1992). The food and feed products containing 

MON 87712 or derived from MON 87712 are as safe as soybean currently on the market 

for human and animal consumption. 

V.F. BBX32 Protein Characterization and Safety Conclusion 

 

3

 Buffer to tissue ratio includes the dilutions factor. For forage and seed the buffer to tissue ratio was 20 and 

40, with dilution factors of 2 and 4, respectively. 
Monsanto Company 11-SY-217U 84 of 467 

BBX32 is a transcriptional accessory protein that influences the expression of some 

diurnally regulated genes. BBX32 is derived from Arabidopsis thaliana, a member of the 

Brassicaceae family. BBX32 is a B-box-containing protein that has homologs in food 

plants with a history of safe use. BBX32 was produced in and isolated from E. coli and 

was subsequently used for the described safety studies. Expression studies using western 

blot analysis demonstrated that BBX32 was detected in leaf and root of MON 87712, and 

was expressed at levels below the limit of detection in harvested seed and forage, the 

other tissues assayed. The maximum expression level detected in leaf was 16 ng/g fwt, 

and in root the maximum expression level was 1.5 ng/g fwt; the limits of detection for 

forage and harvested seed gave expression values of 1 ng/g fwt and 10 ng/g fwt 

respectively, representing a low percentage of the total protein in soybean. 

Bioinformatics analysis determined that BBX32 does not share amino acid sequence 

similarities with known allergens, gliadins, glutenins, or protein toxins. BBX32 was 

rapidly digested in in vitro assays using simulated gastric and intestinal fluids and did not 

show any adverse effects when administered to mice via oral gavage at levels that 

resulted in large margins of exposure (MOE). 

The protein safety data presented herein support the conclusion that food and feed 

products containing MON 87712 or derived from MON 87712 are as safe as soybean 

currently on the market for human and animal consumption. 
Monsanto Company 11-SY-217U 85 of 467 

VI. COMPOSITIONAL ASSESSMENT OF MON 87712 

Safety assessments of biotechnology-derived crops follow the comparative safety 

assessment process (Codex Alimentarius, 2009) in which the composition of grain and/or 

other raw agricultural commodities of the biotechnology-derived crop is compared to the 

appropriate conventional counterpart that has a history of safe use. Compositional 

assessments are performed using the principles and analytes outlined in the OECD 

consensus documents for soybean composition (OECD, 2001). 

A recent review of compositional assessments conducted according to OECD guidelines 

that encompassed a total of seven biotechnology-derived crop varieties, nine countries, 

and eleven growing seasons concluded that incorporation of biotechnology-derived 

agronomic traits has had little impact on natural variation in crop composition; most 

compositional variation is attributable to growing region, agronomic practices and 

genetic background (Harrigan et al., 2010). Numerous scientific publications have 

further documented the extensive variability in the concentrations of crop nutrients and 

anti-nutrients that reflect the influence of environmental and genetic factors as well as 

extensive conventional breeding efforts to improve nutrition, agronomics and yield 

(Harrigan et al., 2010; Reynolds et al., 2005). Compositional equivalence between 

biotechnology-derived and conventional crops supports 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 anti-nutrients. This is based on the premise that such comprehensive and 

detailed analyses will most effectively discern any compositional changes that imply 

potential safety and nutritional concerns. Levels of the components in harvested seed and 

forage of the biotechnology-derived crop are compared to: 1) corresponding levels in a 

conventional comparator, grown concurrently, under identical field conditions, and 2) 

natural ranges generated from an evaluation of commercial reference varieties grown 

concurrently and from data published in the scientific literature. The latter comparison 

places any potential differences between the assessed crop and its comparator in the 

context of the well-documented variation in the concentrations of crop nutrients and antinutrients. 

VI.A. Compositional Equivalence of MON 87712 Seed and Forage to Conventional 

Soybean 

Harvested seed and forage samples were collected from MON 87712 and the parental 

conventional soybean control A3525 grown in a 2009 U.S. field production. Three 

different conventional commercial reference varieties were included at each site of the 

field , with a total of sixteen unique references included in this study, production to 

provide data on natural variability of each compositional component analyzed. The field 

production was conducted at eight geographically diverse sites: Jackson County, 

Arkansas (ARNE); Parke County, Indiana (INRC); Clinton County, Illinois (ILCY); 

Madison County, Illinois (ILHI); Stark County, Illinois (ILWY); Boone County, Indiana 

(INSH); Pawnee County, Kansas (KSLA); and York County, Nebraska (NEYO). All 

soybean plants including MON 87712, the conventional control A3525, and conventional 
Monsanto Company 11-SY-217U 86 of 467 

commercial reference varieties were treated with maintenance pesticides as necessary 

throughout the growing season. 

Compositional analyses were conducted to assess whether levels of key nutrients and 

anti-nutrients in MON 87712 were equivalent to levels in the conventional control A3525 

and the composition of the conventional commercial reference varieties. A description of 

nutrients and anti-nutrients present in soybean is provided in the OECD consensus 

document on compositional considerations for soybean (OECD, 2001). Nutrients 

assessed in this study included proximates, fiber, amino acids, fatty acids, and vitamin E 

in harvested seed, and proximates and fiber in forage. The anti-nutrients assessed in 

harvested seed included raffinose, stachyose, lectin, phytic acid, trypsin inhibitors, and 

isoflavones. 

In all, 63 different analytical components were measured (seven in forage and 56 in 

harvested seed). Due to statistical constraints, in order to proceed with the statistical 

analysis of any component in this study, at least 50% of the observed values for an 

analyte needed to be greater than the assay limit of quantitation (LOQ). Of the 63 

components measured, 14 had more than 50% of the observations below the assay LOQ 

and thus were excluded from statistical analysis. Therefore, 49 components were 

statistically assessed using a mixed-model analysis of variance method. Values for all 

assessed components were reported on a dry weight basis with the exception of moisture, 

which was reported as % fresh weight and fatty acids (FA), which were reported as % of 

total FA. 

For MON 87712, nine sets of statistical comparisons to the conventional control A3525 

were conducted. One comparison was based on compositional data combined across all 

eight field sites (the combined-site analysis) and eight comparisons were conducted on 

data from each of the eight individual field sites. Statistical differences were identified at 

a 5% level of significance (α=0.05). Compositional data from the conventional 

commercial reference varieties were combined across all sites and used to calculate a 

99% tolerance interval for each component to define the natural variability in soybean 

varieties that have a history of safe consumption and that were grown concurrently with 

MON 87712 and the conventional control A3525 in the same trial. 

For the combined-site analysis, significant differences in nutrient and anti-nutrient 

components were evaluated further using considerations relevant to the safety and 

nutritional quality of MON 87712 when compared to the conventional control A3525. 

The evaluation included: 1) the relative magnitude of the significant difference in the 

mean values (mean difference as % of the conventional control A3525) of nutrient and 

anti-nutrient components of MON 87712 compared to the conventional control A3525, 

relative to natural variability 2) whether the MON 87712 component mean value is 

within the range of natural variability of that component as represented by the 99% 

tolerance interval of conventional commercial reference varieties grown concurrently in 

the same trial, 3) analyses of the reproducibility of the significant combined-site 

component differences at individual sites, and 4) assessing the combined-site significant 

differences and reproducible individual site significant differences within the context of 

natural variability of commercial soybean composition published in the scientific 
Monsanto Company 11-SY-217U 87 of 467 

literature and/or in the International Life Sciences Institute (ILSI) Crop Composition 

Database (ILSI, 2010). 

This evaluation provides a comprehensive comparative assessment of the levels of key 

nutrients and anti-nutrients in harvested seed, and of key nutrients in forage of 

MON 87712 and the conventional control A3525 discussed in the context of natural 

variability in commercial conventional soybean. Results of the comparison indicate that 

the composition of the harvested seed and forage of MON 87712 is equivalent to that of 

the conventional control A3525, and within the natural variability of conventional 

commercial reference varieties. 

VI.A.1. Nutrient Levels in Harvested Soybean Seed 

In the combined-site analysis of harvested soybean seed, 22 of the 34 nutrient component 

comparisons were not significantly different between MON 87712 and the conventional 

control A3525. 

The following components showed no significant differences in mean values between 

MON 87712 and the conventional control: four proximates (ash, carbohydrates by 

calculation, moisture, and total fat), two types of fiber (ADF and NDF), nine amino acids 

(histidine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, and 

tyrosine), six fatty acids (18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic acid, and 20:0 

arachidic acid, 20:1 eicosenoic acid, and 22:0 behenic acid), and vitamin E (Table VI-2). 

The components that showed significant differences in mean values between 

MON 87712 and the conventional control A3525 were: one proximate (protein), nine 

amino acids (alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, isoleucine, 

phenylalanine, and valine), and two fatty acids (16:0 palmitic acid and 18:0 stearic acid) 

(Table VI-1 and VI-2). 

The significant differences in nutrients were further evaluated using considerations 

relevant to the safety and nutritional quality of MON 87712 when compared to the 

conventional control A3525: 

1) All nutrient component significant differences observed in the combined-site 

statistical analysis, whether reflecting increased or decreased mean values for 

MON 87712 with respect to the conventional control A3525, had small relative 

magnitudes when compared to natural variability, and therefore these differences were 

not meaningful from a food/feed nutrition or safety perspective. The relative magnitude 

of the difference for protein was 1.09%, and relative magnitudes of the differences 

ranged from 1.22 to 3.07% for amino acids; and from 1.42 to 3.04% for fatty acids. 

2) Mean values for all significantly different nutrient components from the 

combined-site analysis of MON 87712 were within the 99% tolerance interval 

established from the conventional commercial reference varieties grown concurrently in 

the same trial, and were, therefore, within the range of natural variability in commercial 

soybean varieties with a history of safe consumption. 
Monsanto Company 11-SY-217U 88 of 467 

3) Assessment of the reproducibility of the combined-site significant differences at 

the eight individual sites showed significant differences for: protein at one site; alanine, 

aspartic acid, isoleucine, and valine at two sites; arginine, glutamic acid, glycine, 

phenylalanine, and 18:0 stearic acid at three sites; and 16:0 palmitic acid at four sites. 

Cystine was not significantly different at any of the individual sites. Individual site mean 

values of MON 87712 for all nutrient components with significant differences were 

within the 99% tolerance interval established from the conventional commercial 

reference varieties grown concurrently in the same trial and were, therefore, within the 

range of natural variability in conventional commercial soybeans with a history of safe 

consumption. 

4) All combined-site mean values of MON 87712 for all nutrient components were 

within the context of the natural variability of commercial soybean composition as 

published in the scientific literature and/or available in the ILSI Crop Composition 

Database (ILSI, 2010). 

Overall, observed significant differences in protein and amino acid values between 

MON 87712 and the conventional control A3525 were not considered to be meaningful 

from a food and feed safety or nutritional perspective. Ten of the 12 significant 

differences in the harvested seed nutrient levels between MON 87712 and the 

conventional control A3525 observed in the combined-site data analysis were attributable 

to small differences in protein and nine amino acids (all expressed as % dw). The 

relative magnitude of the difference between the mean protein values for MON 87712 

and the conventional control A3525 was small (an increase of 1.09% in the combined-site 

analysis for MON 87712), and the mean protein values for MON 87712 were 

significantly different from the conventional control A3525 at only one of the eight 

individual sites. Correspondingly, the relative magnitudes of the significant differences 

for all amino acid values were small, and significant differences in the combined-site data 

analysis were not consistently observed as significant differences at all individual sites. 

Eight of the nine amino acids observed to be different in the combined-site analysis were 

increased (1.22 – 2.26%) consistent with an increase in protein, one amino acid (cystine) 

was decreased (3.07%) relative to the conventional control A3525 and, as with protein, 

significant differences between MON 87712 and the conventional control A3525 were 

not consistently observed at all sites. Cystine, the amino acid observed to be significantly 

decreased (3.07%) in MON 87712 when compared to the conventional control A3525 in 

the combined-site analysis, was not significantly different at any of the individual sites. 

Thus, observed significant differences in protein and amino acid values between 

MON 87712 and the conventional control A3525 were not considered to be meaningful 

from a food and feed safety or nutritional perspective because they were not consistently 

reproduced at the individual sites, and the mean MON 87712 values were within the 99% 

tolerance interval established by conventional commercial reference varieties grown 

concurrently in the same trial. 

Overall, observed significant differences in fatty acid values between MON 87712 and 

the conventional control A3525 were not considered to be meaningful from a food and 

feed safety and nutritional perspective. Two of the combined-site significant differences 

between MON 87712 and the conventional control A3525 were attributable to fatty acids 
Monsanto Company 11-SY-217U 89 of 467 

(expressed as % total FA). The relative magnitudes of the significant differences 

between the mean fatty acid values for MON 87712 and the conventional control A3525 

in the combined-site analysis were low with a decrease of 1.42% for 16:0 palmitic acid, 

and a decrease of 3.04% for 18:0 stearic acid. Neither of these fatty acids were 

significantly different between MON 87712 and the conventional control A3525 at more 

than four of the eight individual sites. Thus, observed significant differences in fatty acid 

values between MON 87712 and the conventional control A3525 were not considered to 

be meaningful from a food and feed safety and nutritional perspective because they were 

not consistently reproduced at the individual sites, and the mean MON 87712 values were 

within the 99% tolerance interval established by conventional commercial reference 

varieties grown concurrently in the same trial. 

In summary, statistical analyses found no consistent significant differences between the 

levels of nutrient components in harvested seed from MON 87712 and the conventional 

control A3525. These findings supported the conclusion of compositional equivalence of 

MON 87712 to conventional soybean. 

VI.A.2. Anti-Nutrient Levels in Soybean Seed 

In the combined-site analysis, no significant differences were observed for any of the 

eight anti-nutrient component comparisons (lectin, phytic acid, raffinose, stachyose, 

trypsin inhibitor, daidzein, genistein, and glycitein) between MON 87712 and the 

conventional control A3525. Thus, the evaluation of anti-nutrient components in 

harvested seed supported the conclusion that MON 87712 is compositionally equivalent 

to conventional soybean. 

VI.A.3. Nutrient Levels in Soybean Forage 

In the combined-site analysis of forage, no significant differences were observed between 

MON 87712 and the conventional control A3525 for six of the seven nutrients. No 

significant differences were observed for ash, carbohydrates, moisture, protein, ADF or 

NDF (Table VI-4). One significant difference for total fat was observed between 

MON 87712 and the conventional control. The relative magnitude of the difference, with 

respect to the conventional control, was only 11.34%, and the value was within the 99% 

tolerance interval established by conventional commercial reference varieties grown 

concurrently in the same trial. Nutritionally, only ruminants would consume soybean 

forage and the major role of fat would be as a source of energy. If the gross energy of the 

soybean forage is calculated using Atwater Values (Merrill and Watt, 1973) and the 

composition observed in the combined-site analysis, it would result in only a 1% relative 

difference. This difference is not meaningful from a nutritional perspective and, in 

addition any diet would be balanced using other feedstuffs (NRC, 2001). 

The single nutrient component significant difference between MON 87712 and the 

conventional control A3525 observed in the combined-site analysis was evaluated for 

reproducibility at the individual sites. Significant differences were observed between 

MON 87712 and the conventional control A3525 in total fat values at only two of the 

eight individual sites. The relative magnitudes of the significant differences were 12.96 
Monsanto Company 11-SY-217U 90 of 467 

and 39.10% lower, but all mean values at the individual sites that were significantly 

different were within the 99% tolerance interval established by conventional commercial 

reference varieties grown concurrently in the same trial. 

Thus, an evaluation of nutrient components in forage supported the conclusion of 

compositional equivalence of MON 87712 to conventional soybean. 
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in Combined-Site Analysis Seed Proximate (% dwt) Protein 40.93 40.49 1.09 0.005 38.87 - 42.7Seed Amino Acid (% dwt) Alanine 1.77 1.75 1.30 0.014 1.69 - 1.8Arginine 3.14 3.07 2.26 0.027 2.93 - 3.3Aspartic Acid 4.73 4.67 1.30 0.029 4.44 - 4.92 Cystine 0.59 0.61 -3.07 <0.001 0.55 - 0.63 Glutamic Acid 7.60 7.49 1.49 0.038 7.18 - 7.96 Glycine 1.79 1.77 1.22 0.020 1.70 - 1.8
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in Combined-Site Analysis Seed Amino Acid (% dwt) Isoleucine 1.90 1.87 1.57 0.032 1.78 - 1.9Phenylalanine 2.15 2.12 1.59 0.016 1.98 - 2.26 Valine 1.99 1.95 2.02 0.013 1.88 - 2.0Seed Fatty Acid (% Total FA) 16:0 Palmitic 11.48 11.64 -1.42 0.002 10.78 - 11.918:0 Stearic 4.05 4.17 -3.04 0.004 3.54 - 4.72 Forage Proximate (% dwt) Total Fat 6.23 7.03 -11.34 0.005 3.41 - 8.62 
Monsanto Company 11-SY-217U 

Table VI-1 . Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Antinutrient Raffinose (% dwt) Site ARNE 0.82 0.73 13.42 <0.001 0.77 - 0.86 Raffinose (% dwt) Site ILCY 0.78 0.70 10.76 0.006 0.72 - 0.83 Raffinose (% dwt) Site ILHI 0.78 0.90 -13.27 0.002 0.76 - 0.80 Raffinose (% dwt) Site KSLA 0.87 0.82 5.25 0.011 0.86 - 0.8Raffinose (% dwt) Site NEYO 0.66 0.71 -6.57 0.023 0.65 - 0.66 Seed Fatty Acid (% Total FA) 16:0 Palmitic Site ARNE 11.69 12.01 -2.66 0.001 11.54 - 11.16:0 Palmitic Site ILHI 11.37 11.55 -1.56 0.002 11.30 - 11.4
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Fatty Acid (% Total FA) 16:0 Palmitic Site KSLA 11.89 12.12 -1.88 0.002 11.84 - 11.916:0 Palmitic Site NEYO 11.63 11.89 -2.17 0.012 11.59 - 11.7Seed Amino Acid (% dwt) Arginine Site ILCY 3.28 3.11 5.76 0.007 3.23 - 3.3Arginine Site KSLA 3.05 2.90 5.22 0.001 3.03 - 3.0Arginine Site NEYO 3.19 3.02 5.51 <0.001 3.16 - 3.24 Glutamic Acid Site ILCY 7.78 7.57 2.76 0.042 7.63 - 7.87 Glutamic Acid Site KSLA 7.24 7.03 2.96 0.020 7.19 - 7.36 
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2 

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Amino Acid (% dwt) Glutamic Acid Site NEYO 7.68 7.41 3.66 0.007 7.60 - 7.76 Glycine Site ILCY 1.84 1.79 2.44 0.035 1.80 - 1.8Glycine Site KSLA 1.72 1.69 1.86 0.034 1.70 - 1.7Glycine Site NEYO 1.79 1.74 2.99 0.001 1.78 - 1.80 Leucine Site ILCY 3.23 3.15 2.30 0.041 3.18 - 3.27 Leucine Site KSLA 3.02 2.96 2.12 0.043 3.00 - 3.0Leucine Site NEYO 3.18 3.09 3.20 0.002 3.17 - 3.2Phenylalanine Site ILCY 2.22 2.15 3.24 0.022 2.17 - 2.26 
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87771

2 

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Amino Acid (% dwt) Phenylalanine Site KSLA 2.04 1.98 2.80 0.049 1.98 - 2.07 Phenylalanine Site NEYO 2.17 2.08 4.58 0.003 2.15 - 2.19 Seed Fatty Acid (% Total FA) 18:0 Stearic Site ARNE 3.85 4.16 -7.37 <0.001 3.78 - 3.89 18:0 Stearic Site ILCY 3.77 4.00 -5.54 0.001 3.71 - 3.918:0 Stearic Site INRC 3.67 3.96 -7.25 0.001 3.54 - 3.84 18:2 Linoleic Site ARNE 51.50 53.62 -3.96 0.003 50.26 - 52.218:2 Linoleic Site ILCY 54.93 54.34 1.09 0.045 54.84 - 55.0
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Fatty Acid (% Total FA) 18:2 Linoleic Site INSH 52.72 53.25 -0.98 0.034 52.33 - 53.2Seed Amino Acid (% dwt) Alanine Site KSLA 1.70 1.67 2.20 0.031 1.69 - 1.72 Alanine Site NEYO 1.78 1.73 3.01 0.007 1.76 - 1.80 Aspartic Acid Site KSLA 4.56 4.45 2.30 0.037 4.51 - 4.62 Aspartic Acid Site NEYO 4.77 4.64 2.75 0.005 4.74 - 4.8Histidine Site KSLA 1.04 1.01 3.05 0.021 1.03 - 1.06 Histidine Site NEYO 1.09 1.06 2.34 0.001 1.08 - 1.09 
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Amino Acid (% dwt) Isoleucine Site ILCY 1.94 1.87 3.82 0.029 1.87 - 1.97 Isoleucine Site NEYO 1.92 1.85 3.59 0.009 1.90 - 1.93 Valine Site ILCY 2.05 1.96 4.20 0.040 1.95 - 2.0Valine Site NEYO 2.03 1.95 4.29 0.013 2.00 - 2.0Seed Fatty Acid (% Total FA) 18:1 Oleic Site ARNE 24.18 21.03 14.99 0.001 23.08 - 25.718:1 Oleic Site INSH 22.26 21.25 4.76 0.001 22.07 - 22.20:0 Arachidic Site ARNE 0.29 0.32 -9.78 0.040 0.26 - 0.30 
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Seed Fatty Acid (% Total FA) 20:0 Arachidic Site KSLA 0.30 0.26 12.23 0.006 0.29 - 0.30 Seed Vitamin (mg/100g dwt) Vitamin E Site ARNE 1.78 2.16 -17.89 0.023 1.58 - 1.8Vitamin E Site NEYO 0.96 1.13 -15.03 0.027 0.94 - 0.99 Seed Secondary Metabolite (% dwt) Stachyose Site ARNE 4.13 3.80 8.77 0.021 3.94 - 4.33 Stachyose Site NEYO 4.27 4.11 3.89 0.009 4.24 - 4.29 Forage Proximate (% dwt) Moisture (% fwt) Site ARNE 75.53 72.63 3.99 0.004 74.60 - 77.
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in More than One Individual Site Forage Proximate (% dwt) Moisture (% fwt) Site ILCY 72.85 74.70 -2.48 0.006 71.80 - 74.Total Fat Site ILHI 8.15 9.36 -12.96 0.008 7.44 - 8.62 Total Fat Site KSLA 3.74 6.14 -39.10 0.001 3.41 - 4.03 Statistical Differences Observed in One Site Seed Proximate (% dwt) Carbohydrates Site ILWY 40.46 39.22 3.17 0.020 39.98 - 41.Moisture (% fwt) Site INRC 8.04 7.45 7.86 0.021 7.91 - 8.1Protein Site NEYO 41.05 40.14 2.27 <0.001 40.85 - 41.3Total Fat Site ILWY 14.35 15.35 -6.52 0.039 13.91 - 14.6
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in One Site Seed Fiber (% dwt) Acid Detergent Fiber Site ILCY 15.30 13.46 13.69 0.004 13.99 - 16.4Neutral Detergent Fiber Site ILCY 16.42 14.58 12.65 0.014 15.64 - 17.2Seed Amino Acid (% dwt) Lysine Site NEYO 2.68 2.62 2.20 0.004 2.66 - 2.6Proline Site NEYO 2.02 1.97 2.57 0.046 2.00 - 2.04 Serine Site NEYO 2.19 2.12 3.47 0.044 2.17 - 2.24 Tyrosine Site KSLA 1.45 1.40 3.39 0.035 1.41 - 1.4Seed Fatty Acid (% Total FA) 18:3 Linolenic Site ARNE 8.06 8.39 -3.86 0.031 7.87 - 8.29 
Monsanto Company 11-SY-217U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON Conventional Control (continued) 

Mean Difference 

(Test minus Control) 

Analytical Component (Units)1 

MON 87712

Mean2

Control3

Mean 

Mean 

Difference 

(% of Control) 

Significance

(p-Value) 

Test 

Range 

Statistical Differences Observed in One Site Seed Fatty Acid (% Total FA) 22:0 Behenic Site KSLA 0.29 0.23 22.35 0.017 0.28 - 0.30 Seed Antinutrient Trypsin Inhibitor (TIU/mg dwt) Site ARNE 

28.47 36.40 -21.78 0.029 24.54 - 34.9

Seed Isoflavone (μg/g dwt) Glycitein Site ILWY 117.88 96.68 21.92 0.005 112.20 - 1221dwt = dry weight; fwt = fresh weight; FA = fatty acid; TIU = Trypsin Inhibitor Units. 2Mean = least-square mean. 3Control refers to the conventional control, A3525 4With 95% confidence,the interval contains 99% of the values expressed in the population of commercial substances. Negative limits w
Monsanto Company 11-SY-217U 

Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Contro

Difference (Test minus Control) 

Analytical Component 

(Units)1 

Test2 

Mean (S.E.)3 

(Range) 

Control4

Mean (S.E.) 

(Range) 

Mean (S.E.) 

(Range) 

95% 

Confidence 

Interval 

Significa

(p-Valu

Proximate (% dwt) Ash 4.99 (0.11) 5.07 (0.11) -0.081 (0.069) -0.22, 0.055 0.238 

(4.48 - 6.36) (4.46 - 5.71) (-0.78 - 1.42) 

Carbohydrates 37.83 (0.57) 37.96 (0.57) -0.14 (0.25) -0.63, 0.35 0.582 

(34.64 - 41.50) (34.72 - 40.86) (-3.07 - 2.47) 

Moisture (% fwt) 8.01 (0.20) 7.89 (0.20) 0.12 (0.13) -0.13, 0.38 0.345 

(5.72 - 9.13) (6.44 - 10.30) (-1.69 - 1.84) 

Protein 40.93 (0.35) 40.49 (0.35) 0.44 (0.15) 0.14, 0.75 0.005 

(38.87 - 42.79) (37.73 - 42.14) (-1.65 - 2.56) 

Total Fat 16.27 (0.44) 16.48 (0.44) -0.21 (0.18) -0.58, 0.15 0.250 

(13.91 - 18.78) (13.91 - 19.05) (-2.38 - 1.47) 

Fiber (% dwt) Acid Detergent Fiber 15.54 (0.50) 15.59 (0.49) -0.048 (0.45) -1.01, 0.91 0.916 

(12.94 - 19.11) (12.41 - 20.81) (-4.82 - 2.81) 
Monsanto Company 11-SY-217U 

Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued) 

Difference (Test minus Control) 

Analytical Component 

(Units)1 

Test2 

Mean (S.E.)3 

(Range) 

Control4

Mean (S.E.) 

(Range) 

Mean (S.E.) 

(Range) 

95% 

Confidence 

Interval 

Significa

(p-Valu

Fiber (% dwt) Neutral Detergent Fiber 16.54 (0.42) 16.76 (0.42) -0.23 (0.40) -1.09, 0.63 0.579 

(13.68 - 20.74) (13.40 - 20.37) (-2.65 - 2.76) 

Amino Acid (% dwt) Alanine 1.77 (0.014) 1.75 (0.014) 0.023 (0.0081) 0.0053, 0.040 0.014 

(1.69 - 1.85) (1.65 - 1.83) (-0.055 - 0.073) 

Arginine 3.14 (0.036) 3.07 (0.036) 0.069 (0.028) 0.0089, 0.13 0.027 

(2.93 - 3.38) (2.85 - 3.31) (-0.20 - 0.23) 

Aspartic Acid 4.73 (0.043) 4.67 (0.043) 0.061 (0.025) 0.0069, 0.11 0.029 

(4.44 - 4.92) (4.36 - 4.89) (-0.16 - 0.24) 

Cystine 0.59 (0.0082) 0.61 (0.0082) -0.019 (0.0043) -0.027, -0.010 <0.001 

(0.55 - 0.63) (0.56 - 0.66) (-0.072 - 0.030) 

Glutamic Acid 7.60 (0.081) 7.49 (0.080) 0.11 (0.049) 0.0068, 0.22 0.038 

(7.18 - 7.96) (6.86 - 7.97) (-0.37 - 0.38) 
Monsanto Company 11-SY-217U 

Table VI-2 . Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Contro

Difference (Test minus Control) 

Analytical Component 

(Units)1 

Test2 

Mean (S.E.)3 

(Range) 

Control4

Mean (S.E.) 

(Range) 

Mean (S.E.) 

(Range) 

95% 

Confidence 

Interval 

Significa

(p-Valu

Amino Acid (% dwt) Glycine 1.79 (0.015) 1.77 (0.015) 0.021 (0.0082) 0.0038, 0.039 0.020 

(1.70 - 1.85) (1.66 - 1.84) (-0.041 - 0.081) 

Histidine 1.07 (0.0080) 1.06 (0.0079) 0.0089 (0.0057) -0.0033, 0.021 0.140 

(1.03 - 1.12) (1.00 - 1.12) (-0.046 - 0.051) 

Isoleucine 1.90 (0.019) 1.87 (0.019) 0.029 (0.012) 0.0028, 0.056 0.032 

(1.78 - 1.98) (1.70 - 1.97) (-0.073 - 0.13) 

Leucine 3.15 (0.029) 3.11 (0.029) 0.035 (0.017) -0.00077, 0.071 0.054 

(3.00 - 3.30) (2.90 - 3.26) (-0.12 - 0.13) 

Lysine 2.66 (0.019) 2.64 (0.019) 0.013 (0.011) -0.011, 0.038 0.263 

(2.54 - 2.74) (2.49 - 2.75) (-0.11 - 0.074) 

Methionine 0.57 (0.0037) 0.57 (0.0036) -0.0034 (0.0039) -0.012, 0.0050 0.403 

(0.52 - 0.60) (0.53 - 0.60) (-0.054 - 0.036) 
Monsanto Company 11-SY-217U 

Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued) 

Difference (Test minus Control) 

Analytical Component 

(Units)1 

Test2 

Mean (S.E.)3 

(Range) 

Control4

Mean (S.E.) 

(Range) 

Mean (S.E.) 

(Range) 

95% 

Confidence 

Interval 

Significa

(p-Valu

Amino Acid (% dwt) Phenylalanine 2.15 (0.023) 2.12 (0.023) 0.034 (0.012) 0.0071, 0.060 0.016 

(1.98 - 2.26) (1.95 - 2.24) (-0.077 - 0.15) 

Proline 2.00 (0.018) 1.99 (0.018) 0.0060 (0.014) -0.022, 0.034 0.667 

(1.83 - 2.14) (1.85 - 2.11) (-0.20 - 0.14) 

Serine 2.16 (0.019) 2.15 (0.019) 0.0080 (0.015) -0.023, 0.039 0.593 

(2.01 - 2.25) (2.01 - 2.34) (-0.16 - 0.14) 

Threonine 1.59 (0.011) 1.58 (0.011) 0.015 (0.0098) -0.0056, 0.036 0.137 

(1.51 - 1.66) (1.50 - 1.66) (-0.089 - 0.13) 

Tryptophan 0.44 (0.0052) 0.45 (0.0051) -0.0074 (0.0062) -0.021, 0.0060 0.256 

(0.36 - 0.49) (0.38 - 0.49) (-0.094 - 0.10) 

Tyrosine 1.48 (0.014) 1.46 (0.014) 0.013 (0.012) -0.010, 0.037 0.267 

(1.40 - 1.57) (1.33 - 1.56) (-0.095 - 0.18) 
Monsanto Company 11-SY-217U 

Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued) 

Difference (Test minus Control) 

Analytical Component 

(Units)1 

Test2 

Mean (S.E.)3 

(Range) 

Control4

Mean (S.E.) 

(Range) 

Mean (S.E.) 

(Range) 

95% 

Confidence 

Interval 

Significa

(p-Valu

Amino Acid (% dwt) Valine 1.99 (0.017) 1.95 (0.017) 0.039 (0.014) 0.0095, 0.069 0.013 

(1.88 - 2.08) (1.78 - 2.07) (-0.072 - 0.15) 

Fatty Acid (% Total FA) 16:0 Palmitic 11.48 (0.13) 11.64 (0.13) -0.16 (0.045) -0.26, -0.067 0.002 

(10.78 - 11.94) (11.13 - 12.23) (-0.44 - 0.091) 

18:0 Stearic 4.05 (0.099) 4.17 (0.099) -0.13 (0.037) -0.21, -0.046 0.004 

(3.54 - 4.72) (3.78 - 4.65) (-0.37 - 0.25) 

18:1 Oleic 20.82 (0.46) 20.21 (0.46) 0.61 (0.33) -0.093, 1.31 0.083 

(18.72 - 25.71) (18.72 - 21.54) (-1.57 - 4.66) 

18:2 Linoleic 54.01 (0.32) 54.18 (0.32) -0.17 (0.25) -0.71, 0.37 0.514 

(50.26 - 55.60) (53.21 - 55.02) (-3.63 - 1.50) 

18:3 Linolenic 8.95 (0.27) 9.07 (0.27) -0.12 (0.057) -0.24, 0.0016 0.052 

(7.46 - 10.22) (7.77 - 10.31) (-0.68 - 0.52) 
Monsanto Company 11-SY-217U 

Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued) 

Difference (Test minus Control) 

Analytical Component 

(Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Significa

(p-Valu

Fatty Acid (% Total FA) 20:0 Arachidic 0.29 (0.0072) 0.30 (0.0072) -0.010 (0.0060) -0.023, 0.0028 0.117 

(0.25 - 0.34) (0.25 - 0.34) (-0.077 - 0.049) 

20:1 Eicosenoic 0.14 (0.012) 0.14 (0.012) -0.0080 (0.0057) -0.019, 0.0033 0.163 

(0.075 - 0.19) (0.073 - 0.19) (-0.082 - 0.071) 

22:0 Behenic 0.26 (0.0099) 0.28 (0.0098) -0.014 (0.012) -0.039, 0.011 0.258 

(0.18 - 0.33) (0.18 - 0.32) (-0.11 - 0.096) 

Vitamin (mg/100g dwt) Vitamin E 1.24 (0.15) 1.37 (0.15) -0.13 (0.064) -0.27, 0.0025 0.053 

(0.81 - 2.05) (0.79 - 2.23) (-0.76 - 0.44) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the conventional control, A3525 5With 95% confidence, the interval contains 99% of the values expressed in the population of commercial substances. Negato zero.
Monsanto Company 11-SY-217U 

Table VI-3. Summary of Combined-site Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Co

Difference (Test minus Control) 

Analytical Component 

(Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Significa

(p-Valu

Antinutrient Lectin (H.U./mg dwt) 2.06 (0.14) 2.22 (0.13) -0.17 (0.18) -0.54, 0.21 0.363 

(1.10 - 3.41) (0.58 - 4.15) (-1.84 - 1.63) 

Phytic Acid (% dwt) 1.18 (0.077) 1.20 (0.077) -0.029 (0.043) -0.12, 0.063 0.513 

(0.65 - 1.81) (0.86 - 1.77) (-0.58 - 0.42) 

Raffinose (% dwt) 0.73 (0.030) 0.72 (0.030) 0.0095 (0.022) -0.038, 0.057 0.674 

(0.54 - 0.88) (0.52 - 0.93) (-0.17 - 0.14) 

Stachyose (% dwt) 4.28 (0.081) 4.19 (0.081) 0.092 (0.060) -0.037, 0.22 0.147 

(3.77 - 4.76) (3.52 - 4.88) (-0.41 - 0.90) 

Trypsin Inhibitor (TIU/mg dwt)

32.94 (1.09) 34.14 (1.07) -1.20 (1.53) -4.23, 1.84 0.435 (24.54 - 40.86) (20.34 - 54.88) (-23.03 - 14.32) 

Isoflavone (μg/g dwt) Daidzein 1089.62 (85.86) 1054.04 (85.80) 35.58 (30.54) -29.88, 101.04 0.263 

(549.34 -1609.37) 

(537.10 - 1533.78) 

(-187.49 - 384.84) 
Monsanto Company 11-SY-217U 

Table VI-3. Summary of Combined-site Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Co(continued) 

Difference (Test minus Control) 

Analytical Component 

(Units)1 

Test2 

Mean (S.E.)3 

(Range) 

Control4

Mean (S.E.) 

(Range) 

Mean (S.E.) 

(Range) 

95% 

Confidence 

Interval 

Significa

(p-Valu

Isoflavone (μg/g dwt) Genistein 867.61 (58.80) 832.31 (58.73) 35.30 (26.14) -20.80, 91.41 0.198 

(503.75 - 1204.29) 

(508.77 - 1175.72) 

(-219.09 - 391.63) 

Glycitein 102.13 (4.02) 100.86 (3.98) 1.27 (5.47) -10.44, 12.99 0.819 

(75.71 - 131.79) (77.28 - 162.85) (-46.46 - 33.87) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the conventional control, A3525 5With 95% confidence, the interval contains 99% of the values expressed in the population of commercial substances. Negaset to zero.
Monsanto Company 11-SY-217U 

Table VI-4. Summary of Combined-site Soybean Forage Nutrients for MON 87712 vs. Conventional Cont

Difference (Test minus Control) 

Analytical Component 

(Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Significa

(p-Valu

Proximate (% dwt) Ash 6.56 (0.31) 6.47 (0.31) 0.089 (0.17) -0.27, 0.45 0.606 

(4.55 - 8.75) (4.79 - 8.74) (-2.60 - 1.62) 

Carbohydrates 65.10 (0.78) 64.76 (0.78) 0.33 (0.34) -0.35, 1.02 0.332 

(59.41 - 70.88) (58.14 - 70.63) (-3.97 - 5.67) 

Moisture (% fwt) 72.80 (0.92) 72.01 (0.92) 0.79 (0.42) -0.099, 1.68 0.077 

(67.60 - 77.50) (66.60 - 75.50) (-3.50 - 4.70) 

Protein 22.13 (0.51) 21.75 (0.51) 0.39 (0.22) -0.058, 0.83 0.086 

(18.45 - 26.20) (17.88 - 26.12) (-2.05 - 3.29) 

Total Fat 6.23 (0.56) 7.03 (0.56) -0.80 (0.25) -1.32, -0.27 0.005 

(3.41 - 8.62) (4.57 - 9.80) (-3.15 - 1.00) 

Fiber (% dwt) Acid Detergent Fiber 29.72 (0.93) 29.05 (0.93) 0.67 (0.62) -0.67, 2.01 0.301 

(24.36 - 38.10) (24.46 - 35.21) (-5.70 - 11.81) 
Monsanto Company 11-SY-217U 

Table VI-4. Summary of Combined-site Soybean Forage Nutrients for MON 87712 vs. C(continued) 

Difference (Test minus Control) 

Analytical Component 

(Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval

Significa

(p-Valu

Fiber (% dwt) Neutral Detergent Fiber 33.30 (1.16) 32.58 (1.15) 0.72 (0.77) -0.94, 2.37 0.368 

(26.56 - 41.73) (24.92 - 41.41) (-6.68 - 8.04) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the conventional control, A3525 5With 95% confidence, the interval contains 99% of the values expressed in the population of commercial substances. Negaset to zero.
Monsanto Company 11-SY-217U 113 of 467 

Table VI-5. Literature and ILSI Database Ranges for Components in Soybean 

Forage and Seed 

Seed Tissue Components1 Literature Range2 ILSI Range3

Seed Nutrients 

Proximates (% dwt) 

Ash 4.61 – 6.32a

; 4.32 – 5.88b 3.89 – 6.99 

Carbohydrates by calculation 32.75 – 40.98a

; 29.88 – 43.48b 29.6 – 50.2 

Moisture (% fwt) 6.24 – 12.10a

; 5.44 – 11.70b 4.7 – 34.4 

Protein 34.78 – 43.35a

; 32.29 – 42.66b 33.19 – 45.48 

Total Fat 14.40 – 20.91a; 15.10 – 23.56b; 15.5 – 24.74 8.10 – 23.56 

 

Fiber (% dwt) 

Acid Detergent Fiber 9.22 – 26.26a

; 11.81 – 19.45b 7.81 – 18.61 

Neutral Detergent Fiber 10.79 – 23.90a

; 13.32 – 23.57b 8.53 – 21.25 

 

Amino Acids (% dwt) 

Alanine 1.62 – 1.89a

; 1.43 – 1.93b 1.51 – 2.10 

Arginine 2.57 – 3.34a

; 2.15 – 3.05b 2.29 – 3.40 

Aspartic acid 4.16 – 5.02a; 4.01 – 5.72b 3.81 – 5.12 

Cystine/Cysteine 0.52 – 0.69a

; 0.41 – 0.71b 0.37 – 0.81 

Glutamic acid 6.52 – 8.19a

; 5.49 – 8.72b 5.84 – 8.20 

Glycine 1.59 – 1.90a; 1.41 – 1.99b 1.46 – 2.00 

Histidine 0.96 – 1.13a

; 0.86 – 1.24b 0.88 – 1.18 

Isoleucine 1.59 – 2.00a

; 1.41 – 2.02b 1.54 – 2.08 

Leucine 2.79 – 3.42a; 2.39 – 3.32b 2.59 – 3.62 

Lysine 2.36 – 2.77a

; 2.19 – 3.15b 2.29 – 2.84 

Methionine 0.45 – 0.63a

; 0.39 – 0.65b 0.43 – 0.68 

Phenylalanine 1.82 – 2.29a; 1.62 – 2.44b 1.63 – 2.35 

Proline 1.83 – 2.23a

; 1.63 – 2.25b 1.69 – 2.28 

Serine 1.95 – 2.42a; 1.51 – 2.30b 1.11 – 2.48 

Threonine 1.44 – 1.71a; 1.23 – 1.74b 1.14 – 1.86 

Tryptophan 0.30 – 0.48a

; 0.41 – 0.56b 0.36 – 0.50 

Tyrosine 1.27 – 1.53a; 0.74 – 1.31b 1.02 – 1.61 

Valine 1.68 – 2.11a; 1.50 – 2.13b 1.60 – 2.20 

 

Fatty Acids (% total FA) 

8:0 Caprylic not available 0.148 – 0.148 

10:0 Capric 0.15 – 0.27b

 not available 

12:0 Lauric not available 0.082 – 0.132 

14:0 Myristic 0.063 – 0.11b

 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 9.80 – 12.63b

 9.55 – 15.77 

16:1 Palmitoleic 0.055 – 0.14b

 0.086 – 0.194 

17:0 Heptadecanoic 0.076 – 0.13b

 0.085 – 0.146 

17:1 Heptadecenoic 0.019 – 0.064b

 0.073 – 0.087 

18:0 Stearic 3.21 – 5.63b

 2.70 – 5.88 

18:1 Oleic 16.69 – 35.16b

 14.3 – 32.2 

18:2 Linoleic 44.17 – 57.72b

 42.3 – 58.8 

 
Monsanto Company 11-SY-217U 114 of 467 

Table VI-5. Literature and ILSI Database Ranges for Components in 

Soybean Forage and Seed (continued) 

Seed Tissue Components1 Literature Range2 ILSI Range3

18:3 Gamma Linolenic not available not available 

18:3 Linolenic 4.27 – 9.90b 3.00 – 12.52 

20:0 Arachidic 0.35 – 0.57b 0.163 – 0.482 

20:1 Eicosenoic 0.13 – 0.30b

 0.140 – 0.350 

20:2 Eicosadienoic 0.016 – 0.071b

 0.077 – 0.245 

20:3 Eicosatrienoic not available not available 

20:4 Arachidonic not available not available 

22:0 Behenic 0.35 – 0.59b

 0.277 – 0.595 

 

Vitamins (mg/100g dwt) 

Vitamin E 1.29 – 4.80a

; 1.12 – 8.08b 0.19 – 6.17 

 

Seed Anti-Nutrients 

Lectin (H.U./mg fwt) 0.45 – 10.87a

; 0.090 – 11.18b 0.09 – 8.46 

Trypsin Inhibitor (TIU/mg 

dwt) 

20.79 – 59.03a

; 18.14 – 42.51b 18.00 – 108.00 

Phytic Acid (% dwt) 0.41 – 1.92a

; 0.81 – 2.66b 0.63 – 1.96 

Raffinose (% dwt) 0.26 – 0.84a

; 0.43 – 1.85b 0.21 – 0.66 

Stachyose (% dwt) 1.53 – 3.04a; 1.97 – 6.65b 1.21 – 3.50 

 

Isoflavones (μg/g dwt) (mg/kg dwt)

Daidzein 224.03 – 1571.91a

; 198.95 – 1458.24b 60.0 – 2453.5 

Genistein 338.24 – 1488.89a

; 148.06 – 1095.57b 144.3 – 2837.2 

Glycitein 52.72 – 298.57a; 32.42 – 255.94b 15.3 – 310.0 

 

Forage Tissue Components1

 Literature Range2 ILSI Range3

Forage Nutrients 

Proximate (% dwt)

Ash 5.28 – 9.24a

; 4.77 – 8.54b 6.72 – 10.78 

Carbohydrates by calculation 62.25 – 72.30a; 60.61 – 77.26b 59.8 – 74.7 

Moisture (% fwt) 68.50 – 78.40a

; 62.76 – 80.20b 73.5 – 81.6 

Protein 16.48 – 24.29a

; 12.68 – 23.76b 14.38 – 24.71 

Total Fat 2.65 – 9.87a

; 2.96 – 7.88b 1.30 – 5.13 

 

Fiber (% dwt) 

Acid Detergent Fiber 23.86 – 50.89a

; 25.49 – 47.33b not available 

Neutral Detergent Fiber 19.61 – 43.70a

; 30.96 – 54.55b not available 1

fw=fresh weight; dwt=dry weight; H.U. = hemagglutinating unit; TIU = trypsin inhibitor unit. 2

Literature range references; a(Lundry et al., 2008); b(Berman et al., 2009). 3

(ILSI, 2010). 

4

(OECD, 2001). 

 
Monsanto Company 11-SY-217U 115 of 467 

VI.B. Compositional Assessment of MON 87712 Conclusion 

Detailed comparisons were conducted on nutrient and anti-nutrient levels identified by 

the OECD as important to understanding the safety and nutrition of soybean (OECD, 

2001) in MON 87712 to corresponding levels in the parental conventional soybean 

control A3525. These compositional comparisons were made by analyzing the seed and 

forage harvested from plants grown at each of eight field sites in the U.S. during the 2009 

field season. The composition analysis, conducted in accordance with OECD guidelines, 

also included measurement of these OECD defined soybean nutrients and anti-nutrients 

in the conventional commercial soybean varieties, to provide data on natural variability 

of each compositional component analyzed. 

For MON 87712 compared to the conventional control A3525, the combined-site analysis 

of both harvested seed and forage showed no statistically significant differences (α=0.05) 

between MON 87712 and the control for 36 (73.5%) of the 49 mean value comparisons. 

Of the significant differences observed, 12 were from the seed analysis, and one was 

from the forage analysis. No differences in mean values between MON 87712 and the 

conventional control A3525 were observed in the combined-site analysis for antinutrients. Seed nutrient component differences were observed for mean values for 

protein, amino acids (nine components), and fatty acids (two components), and the forage 

nutrient component difference was in total fat and were evaluated using considerations 

relevant to the safety and nutritional quality of MON 87712 when compared to the 

conventional control: 

1) All nutrient component differences observed in the combined-site statistical 

analysis, whether reflecting increased or decreased MON 87712 mean values with 

respect to the conventional control A3525, had small relative magnitudes when 

compared to natural variability, and therefore were not meaningful from a 

food/feed nutrition or safety perspective. The relative magnitude of the 

significant difference for protein when MON 87712 was compared to A3525 was 

1.09%, and relative magnitudes of the differences ranged from 1.22 to 3.07% for 

amino acids, from 1.42 to 3.04% for fatty acids, and a relative magnitude of the 

difference of 11.34% was observed for forage total fat. 

2) Mean values for all significantly different nutrient components from the 

combined-site analysis of MON 87712 were within the 99% tolerance interval 

established from the conventional commercial reference varieties grown 

concurrently in the same trial, and were, therefore, within the range of natural 

variability of that component in commercial soybean varieties with a history of 

safe consumption. 

3) Assessment of the reproducibility of the combined-site significant differences at 

the eight individual sites showed significant differences for: protein at one site; 

alanine, aspartic acid, isoleucine, and valine at two sites; arginine, glutamic acid, 

glycine, phenylalanine, and 18:0 stearic acid at three sites; and 16:0 palmitic acid 

at four sites. Cystine was not significantly different at any of the individual sites. 

Individual site mean values of MON 87712 for all nutrient components with 
Monsanto Company 11-SY-217U 116 of 467 

statistically significant differences were within the 99% tolerance interval 

established from the conventional commercial reference varieties grown 

concurrently in the same trial and were, therefore, within the range of natural 

variability of that component in commercial soybean with a history of safe 

consumption.. 

4) All combined-site mean values of MON 87712 for all nutrient components were 

within the context of the natural variability of commercial soybean composition 

as published in the scientific literature and/or available in the ILSI Crop 

Composition Database (ILSI, 2010). 

In summary, MON 87712 mean component values observed to be significantly different 

(α<0.05) from that of the parental conventional control A3525 were generally shown to 

have relative magnitudes of difference that were less than 12% (most were <3%) from the 

control and not reproducible across sites. For MON 87712 compared to the conventional 

control A3525, none of the 49 components assessed were observed to be significantly 

different at all eight individual sites. All MON 87712 mean component values were 

within the 99% tolerance interval established from the conventional commercial 

references varieties grown concurrently at the same field sites. Additionally, the 

combined-site component values were within the range of values reported in the scientific 

literature and/or in the ILSI Crop Composition Database. Based on these data, it is 

concluded that harvested soybean seed and soybean forage produced from MON 87712 

are compositionally equivalent to that of the conventional soybean and that the presence 

of BBX32 in MON 87712 does not have a meaningful impact on the composition and 

therefore on the food and feed safety or nutritional quality of MON 87712 compared to 

conventional soybean. In addition, these data are meaningful from a plant pest risk 

perspective because they support the concept of familiarity that the USDA recognizes as 

an important underlying concept in risk assessment. 
Monsanto Company 11-SY-217U 117 of 467 

VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT 

This section provides an assessment of the phenotypic, agronomic, and environmental 

interaction characteristics including plant-symbiont associations, and volunteer potential 

and persistence outside cultivation. These assessments were comparative between 

MON 87712 and the parental conventional control A3525, a conventional soybean 

variety that has background genetics similar to MON 87712 but does not possess the 

BBX32 gene. The data support a determination that MON 87712 is not different from 

conventional soybean as a plant pest risk or have a significant environmental impact 

compared to conventional soybean. These conclusions are based on the results of 

multiple evaluations. 

Phenotypic, agronomic, and environmental interaction characteristics of MON 87712 

were evaluated in a comparative manner to assess plant pest potential. These assessments 

included evaluation of seed germination characteristics, plant growth and development 

characteristics, observations of plant responses to abiotic stress, plant-disease and plantarthropod interactions, pollen characteristics, plant-symbiont interaction associations, and 

volunteer potential, and persitence outside of cultivation characteristics. Results from the 

phenotypic, agronomic, and environmental interactions assessment demonstrate that 

MON 87712 does not possess a) increased weediness characteristics; b) increased 

susceptibility or tolerance to specific abiotic stress, diseases, or arthropods; or c) 

characteristics that would confer a plant pest risk or a significant environmental impact 

compared to the conventional control A3525. 

VII.A. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental interactions assessment of 

MON 87712, data were collected to evaluate specific aspects of altered plant pest 

potential. A detailed description of the regulated article phenotype is requested as part of 

the petition for determination of nonregulated status in 7 CFR § 340. 6 including 

differences from the unmodified recipient organism that would “substantiate that the 

regulated article is unlikely to pose a greater plant pest risk than the unmodified organism 

from which it was derived...” As part of the characterization of MON 87712, data were 

collected to provide a detailed phenotypic, agronomic, and environmental interaction 

description of MON 87712. A subset of these data were included in an evaluation of 

specific characteristics related to altered plant pest potential and adverse environmental 

impact. 

The MON 87712 plant characterization and assessment encompassed seven general data 

categories: 1) seed germination, dormancy, and emergence; 2) vegetative growth; 3) 

reproductive development (including pollen characteristics); 4) seed retention on the 

plant and lodging; 5) plant response to abiotic stress and interactions with diseases and 

arthropods; 6) plant-symbiont interactions and 7) volunteer potential and persistence 

outside of cultivation characteristics. An overview of the characteristics assessed is 

presented in Table VII-1. 
Monsanto Company 11-SY-217U 118 of 467 

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 87712 was 

compared to an appropriate conventional control that had a genetic background similar to 

MON 87712 but did not possess the BBX32 gene. In addition, multiple commercial 

reference varieties (see Appendices G-L and Tables G-1, H-1, and J-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. Commercial reference varieties are developed through a 

process of selecting and breeding for various desirable soybean characteristics and can 

provide a range of natural variability for characteristics and context for interpreting 

experimental results. 
Monsanto Company 11-SY-217U 119 of 467 

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials, Laboratory or Greenhouse Tests 

Data 

category 

Characteristics 

measured 

(associated section 

where discussed) 

Evaluation timing (setting 

of evaluation)1

Evaluation description 

(measurement endpoints) 

Seed 

germination, 

dormancy, 

and 

emergence 

Normal germinated 

(VII.C.1) 

Day 5 and 8 (20/30°C) 

(laboratory) 

Percentage of seed producing seedlings 

exhibiting normal developmental 

characteristics 

Abnormal 

germinated (VII.C.1) 

Day 8 (20/30°C) (laboratory) Percentage of seed producing seedlings 

that could not be classified as normal 

germinated 

Germinated (VII.C.1) Day 5, 8, and 13 (10, 20, 30, 

10/20 and 10/30°C) 

(laboratory) 

Percentage of seed that had germinated 

normally or abnormally 

Dead 

(VII.C.1) 

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) (laboratory) 

Percentage of seed that had visibly 

deteriorated and become soft to the touch 

(also included non-viable hard and nonviable firm-swollen seed) 

Viable hard (VII.C.1) Day 8 (20/30°C); Day 13 

(10, 20, 30, 10/20 and 

10/30°C) (laboratory) 

Percentage of seed that did not imbibe 

water and remained hard to the touch 

(viability determined by a tetrazolium 

test2

) 

Viable firm-swollen 

(VII.C.1) 

Day 8 (20/30°C); Day 13 

(10, 20, 30, 10/20 and 

10/30°C) (laboratory) 

Percentage of seed that imbibed water and 

were firm to the touch but did not 

germinate (viability determined by a 

tetrazolium test2

) 

Early stand count 

(VII.C.2.1) 

V2 - V4 (Field) Number of emerged plants in two rows, 

standardized to 20 ft rows 

Final stand count 

(VII.C.2.1) 

Maturity, R8 (Field) Number of plants in two rows, 

standardized to 20 ft rows 

Vegetative 

growth 

Seedling vigor 

(VII.C.2.1) 

V2 - V4 (Field) Rated on a 1-9 scale, where 1 = excellent 

and 9 = poor vigor 

Growth stage 

assessment 

(VII.C.2.1) 

Every 2-3 weeks, V2-R8 

(Field) 

Average soybean plant growth stage per 

plot 

Flower color 

(VII.C.2.1) 

Flowering, R2 (Field) Color of flowers: purple, white, or mixed 

Plant pubescence 

(VII.C.2.1) 

Maturity, R8 (Field) Pubescence on plants in each plot 

categorized as hairy or hairless 

Plant height 

(VII.C.2.1) 

Maturity, R8 (Field) Distance (in) from the soil surface to the 

uppermost node on the main stem of five 

representative plants per plot 

Reproductive 

development 

Days to 50% 

flowering (VII.C.2.1) 

Flowering, R1-R2 (Field) Days after January 1 in year of planting 

until 50% of the marked plants in each plot 

were flowering 

Pollen viability 

(VII.C.3) 

Flowering, R1-R2 

(laboratory) 

Percentage of viable pollen based on pollen 

grain staining characteristics 

Pollen morphology 

(VII.C.3) 

Flowering, R1-R2 

(laboratory) 

Diameter (μm) of viable pollen grains 

Days to 50% end of 

flowering (VII.C.2.1) 

Flowering, end of (Field) Days after January 1 in year of planting 

until 50% of the marked plants in each plot 

have stopped flowering 

Days to 50% 

senescence 

(VII.C.2.1) 

R6-R7 (Field) Days after January 1 in year of planting 

until 50% of the marked plants in each plot 

have reached 50% senescence 

 
Monsanto Company 11-SY-217U 120 of 467 

Table VII-1. Phenotypic, Agronomic and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials, Laboratory or Greenhouse Tests 

(continued) 

Data 

category 

Characteristics 

measured 

(associated section 

where discussed) 

Evaluation timing (setting 

of evaluation)1

Evaluation description 

(measurement endpoints) 

Reproductive 

development 

Days to physiological 

maturity (VII.C.2.1) 

R8 (Field) Days after January 1 in year of planting 

until 50% of the marked plants in each plot 

have reach physiological maturity 

Seed moisture 

(VII.C.2.1) 

Harvest (Field) Percent moisture content of harvested seed 

100 seed weight 

(VII.C.2.1) 

Harvest (Field) Mass (g) of 100 harvested seed 

Yield 

(VII.C.2.1) 

Harvest (Field) Bushels of harvested seed per acre, 

adjusted to 13% moisture 

Seed retention 

and lodging 

Lodging 

(VII.C.2.1) 

Maturity, R8 (Field) Rated on 1-9 scale, where 1 = completely 

erect and 9 = completely flat or lodged 

Pod shattering 

(VII.C.2.1) Maturity, R8 (Field) Rated on 1-9 scale, where 1 = no shattering 

and 9 = completely shattered

Plantenvironment 

interactions 

Plant response to 

abiotic stress 

(VII.C.2.2) 

Four times per growing 

season (Field) 

Qualitative assessment of each plot, with 

rating on a 0-9 scale, where 0 = no 

symptoms and 9 = severe symptoms 

Disease damage 

(VII.C.2.2) 

Four times per growing 

season (Field) 

Qualitative assessment of each plot, with 

rating on a 0-9 scale, where 0 = no 

symptoms and 9 = severe symptoms 

Arthropod-related 

damage (VII.C.2.2) 

Four times per growing 

season (Field) 

Damage assessed on upper four nodes of 

10 representative plants per plot using 

arthropod-specific 0-5 rating scales of 

increasing severity 

Arthropod abundance 

(VII.C.2.2) 

Four times per growing 

season (Field) 

Quantitative assessment of pest and 

beneficial arthropods 

Plantsymbiont 

interactions 

Biomass 

(VII.C.4) 

6 weeks after emergence 

(Greenhouse) 

Nodule, root, and shoot dry weight 

(g/plant) 

Nodule number 

(VII.C.4) 

6 weeks after emergence 

(Greenhouse) 

Nodule number 

Total nitrogen 

(VII.C.4) 

6 weeks after emergence 

(Greenhouse) 

Shoot total nitrogen (% and g/plant) 

Volunteer 

Potential 

characteristics 

Plant Counts 

(VII.C.5) 

Every 2 weeks (Field) Number of emerged plants per plot 

Persistence 

Outside of 

Cultivation 

characteristics 

Stand Count 

(VII.C.6) 

Approximately every 14 

days (Field) 

Number of emerged plants per plot 

Growth Stage 

Monitoring (VII.C.6) 

Approximately every 14 

days (Field) 

Average soybean plant growth stage per 

plot 

Vigor Monitoring 

(VII.C.6) 

Approximately every 14 

days up to R1 growth stage 

(Field) 

Rated on a 1-9 scale, where 1 = excellent 

and 9 = poor vigor 

Number of Plants 

Producting Pods 

(VII.C.6) 

R8 (Field) Total number of plants in a plot which 

produced pods 

Number of Seeds 

Produced (VII.C.6) 

R8 (Field) Total number of seeds produced in a plot 

Seed Weight 

(VII.C.6) 

R8 (Field) Total weight of all seeds produced in a plot 

(g) 1

Soybean plant growth stages were determined using descriptions and guidelines outlined in Soybean 

Growth and Development (Pedersen, 2004). 

2

Viability of hard and firm-swollen seed were determined by a tetrazolium test (AOSA, 2000).
Monsanto Company 11-SY-217U 121 of 467 

VII.B. Interpretation of Phenotypic and Environmental Interaction Data 

Plant pest risk assessments for biotechnology-derived crops are 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, MON 87712 was compared to the conventional 

control in the assessment of phenotypic, agronomic, and environmental interaction 

characteristics. An overview of the characteristics assessed is presented in Table VII-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 of MON 87712, an element of Animal and Plant 

Health Inspection Service’s (APHIS) plant pest determination. Evaluation of 

environmental interaction characteristics (e.g., plant abiotic stress, plant-disease, plantarthropod, and plant-symbiont interactions) was also considered in the plant pest risk 

assessment. Based on all of the data collected, an assessment was made to determine if 

MON 87712 is likely to pose an increased plant pest risk compared to conventional 

soybean. Prior to analysis, the overall dataset was evaluated for evidence of biologically 

relevant changes, and for possible evidence of an unexpected plant response. No 

unexpected observations or issues were identified. 

VII.B.1. Interpretation of Detected Differences Criteria 

Comparative plant characterization data between a biotechnology-derived crop and the 

conventional 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 VII-1) or in 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 data is used for the overall risk assessment of 

differences and their significance. In detail, Figure VII-1 illustrates the stepwise 

assessment process employed: 
Monsanto Company 11-SY-217U 122 of 467 

Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or 

environmental 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 VII-1. Schematic Diagram of Agronomic and Phenotypic Data 

Interpretation Methods 

Steps 1 and 2 - Evaluate Detected Statistically Significant Differences 

Data on each measured characteristic are statistically analyzed, where appropriate, within 

each individual-site and in a combined-site analysis, in which the data are pooled among 

sites. All statistically significant differences are evaluated and considered in the context 

of a change in plant pest potential. Differences detected in individual-site analyses that 

are not consistently observed across multiple environments in the combined-site analysis 

are considered not biologically meaningful in terms of plant pest potential and, therefore, 

are not further considered in subsequent steps. Any difference detected in the combinedsite analysis is further assessed. 

Step 3 - Evaluate Differences Relative to Commercial Reference Varieties Range 

If a difference for a characteristic is detected in the combined-site analysis across 

multiple environments, then that mean value for the biotechnology-derived crop is 

assessed relative to the commercial reference varieties. 

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 references 

No 

Not adverse; the direction 

or magnitude of the 

detected difference in the 

measured characteristic 

does not contribute to a 

biological or 

environmental 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 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 

Step 3 

Step 1 
Monsanto Company 11-SY-217U 123 of 467 

Step 4 - Evaluate Differences in the Context of the Crop 

If the mean value of the biotechnology-derived crop is outside the variation of the 

commercial reference varieties (e.g., reference range), the mean value of the 

biotechnology-derived crop for the characteristic is assessed relative to known values 

common for the crop (e.g., published values). 

Step 5 - Plant Pest Potential 

If the mean value of the biotechnology-derived crop is outside the range of values 

common for the crop, the detected difference for the characteristic 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. 
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VII.C. Comparative Assessments of the Phenotypic, Agronomic, and 

Environmental Interaction Characteristics of MON 87712 

This section provides the results of comparative assessments conducted in replicated 

laboratory, greenhouse, and/or multi-site field experiments to provide a detailed 

phenotypic, agronomic, and environmental interaction description of MON 87712. The 

MON 87712 characteristics evaluated in these assessments included: seed dormancy and 

germination characteristics (Section VII.C.1), plant phenotypic, agronomic, and 

environmental interaction observations under field conditions (Section VII.C.2), pollen 

characteristics (Section VII.C.3), symbiont interactions (Section VII.C.4), volunteer 

potential characteristics (Section VII.C.5), and persistence outside of cultivation 

characteristics (Section VII.C.6). Additional details for each assessment are provided in 

Appendices G through L. 

VII.C.1. Seed Dormancy and Germination Characteristics 

USDA-APHIS considers the potential for weediness to constitute a plant pest risk (7 CFR 

§ 340.6). Seed germination and dormancy mechanisms vary among 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 weeds (Anderson, 

1996; Lingenfelter and Hartwig, 2007). However, it is important to note that it is not 

uncommon to observe low levels of hard seed in soybean (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 (AOSA), 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 87712 and the conventional control A3525. In addition, ten 

commercial reference varieties were included to provide a range of comparative values 

that are representative of existing commercial soybean varieties. The seed lots for 

MON 87712, its conventional control A3525, and the commercial reference varieties 

were produced in replicated field trials at three sites during 2009 in Illinois and Missouri, 

geographical 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 additional 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 G. 

In a combined-site analysis, in which the data were pooled among the three production 

sites, no statistically significant differences (α = 0.05) were detected between 

MON 87712 and the conventional control A3525 in any of the temperature regimes for 

any seed characteristic measured (Table VII-2). Within some temperature regimes, it was 

not possible to conduct an analysis of variance for percent viable firm swollen seed and 
Monsanto Company 11-SY-217U 125 of 467 

viable hard seed because there was no variability present in the data. For these data, the 

values of MON 87712 and the conventional control A3525 were all zero, indicating no 

biological differences. 

The biological characteristics evaluated were used to characterize MON 87712 in the 

context of plant pest risk assessment. Based on the dormancy and germination 

characteristics assessed and the results of this study, particularly the lack of increased 

hard seed, it was concluded that there were no changes indicative of increased weediness 

or plant pest potential of MON 87712 compared to conventional soybean. 
Monsanto Company 11-SY-217U 126 of 467 

Table VII-2. Germination Characteristics of MON 87712 and the Conventional 

Control A3525 

Temperature 

Regime 

Germination

Characteristic1

Mean % (S.E.)2 Reference Range3

MON 87712 Control Min. – Max.

10°C Germinated 99.8 (0.1) 99.6 (0.1) 97.5-99.8

Viable Hard† 0.0 (0.0) 0.0 (0.0) 0.0-0.0

Dead 0.3 (0.1) 0.4 (0.1) 0.3-2.5

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0-0.5

20°C Germinated 99.4 (0.2) 99.3 (0.2) 96.8-99.6

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

Dead 0.6 (0.2) 0.7 (0.2) 0.4-3.3

Viable Firm- Swollen † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

30°C Germinated 99.3 (0.3) 99.5 (0.2) 97.0-99.8

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

Dead 0.7 (0.3) 0.5 (0.2) 0.3-3.0

Viable Firm- Swollen † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

10/20°C Germinated 99.8 (0.1) 99.5 (0.2) 97.8-100.0

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

Dead 0.2 (0.1) 0.5 (0.2) 0.0-2.3

Viable Firm-Swollen † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

10/30°C Germinated 99.5 (0.2) 99.5 (0.2) 96.0-100.0

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

Dead 0.5 (0.2) 0.5 (0.2) 0.0-4.0

Viable Firm-Swollen † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

20/30°C Normal Germinated 96.8 (0.8) 96.6 (0.8) 89.8-99.0

(AOSA) Abnormal Germinated 2.6 (0.7) 3.0 (0.7) 0.8-6.3

Viable Hard† 0.0 (0.0) 0.0 (0.0) 0.0-0.0

Dead 0.7 (0.2) 0.4 (0.2) 0.1-4.0

Viable Firm-Swollen † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

 

Note: The data in this table are the combined-site results of the seeds from three 2009 field sites. 

The experimental design was a split-plot where whole-plot treatment was seed production site 

and the sub-plot treatment was seed material (i.e., MON 87712, conventional control A3525, or 

commercial reference variety). 

No statistically significant differences were detected (α=0.05) between MON 87712 and the 

conventional control A3525. S.E. = Standard Error. 

†

 No statistical comparison could be made due to lack of variability in the data. 

1

 Germinated seed in the AOSA temperature regime (20/30°C) were categorized as either normal 

germinated or abnormal germinated seed. 

2

 Means based on 12 replicates (n = 12) of 100 seeds. In some instances, the total percentage for 

both MON 87712 and the conventional control A3525 did not equal exactly 100% due to 

numerical rounding of the means. 

3

 Minimum and maximum means determined from among the commercial reference varieties. 
Monsanto Company 11-SY-217U 127 of 467 

VII.C.2. Field Phenotypic, Agronomic Characteristics, and Environmental 

Interactions 

Phenotypic, agronomic, and environmental interactions were evaluated under field 

conditions as part of the plant characterization assessment of MON 87712. These data 

were developed to provide USDA-APHIS with a detailed description of MON 87712 

relative to the parental conventional control A3525 and commercial reference varieties. 

According to 7 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 87712 and the conventional control A3525 that may impact its pest potential. 

Although all data from the plant characterization is considered in the pest potential and 

environmental impact assessments, it is important to note that with some traits, 

phenotypic differences may be detected that are consistent with the product concept or 

mode of action of the biotechnology-derived trait (e.g. increased yield with a yield trait). 

Thus, the environmental assessment takes account of this when evaluating weediness, 

pest potential, or environmental impact. Certain growth, reproduction, and pre-harvest 

seed loss characteristics (e.g., lodging, pod shattering) were used to assess whether there 

is an increase in weediness of MON 87712, an element of APHIS’s plant pest risk 

determination. Environmental interactions were also assessed on an individual site basis, 

as an indirect indicator of phenotypic changes to MON 87712 compared to the same 

comparators described above and are also considered in the plant pest risk assessment. 

Data were collected at 19 field locations in the U.S. during 2009 to evaluate phenotypic, 

agronomic, and environmental interaction characteristics. These 19 field sites provided a 

range of environmental and agronomic conditions representative of commercial soybean 

production areas in the U.S. (Table VII-3). The experimental design at each site was a 

randomized complete block with four replications. All plots of MON 87712, the parental 

conventional control A3525, and the commercial reference varieties within each site were 

uniformly managed in order to assess whether the introduction of BBX32 altered the 

phenotypic and agronomic characteristics or the environmental interactions of 

MON 87712 compared to the conventional control A3525. A description of the 

evaluated phenotypic and environmental interaction characteristics and the designated 

developmental stages when evaluations occurred are listed in Table VII-1. The methods 

and detailed results of the individual-site data comparisons are presented and discussed in 

Appendix H, while the combined-site analyses are summarized below. The results of this 

assessment demonstrated that the introduction of BBX32 protein did not alter 

MON 87712 compared to the conventional control A3525 in terms of weediness 

potential. The lack of differences in plant response to abiotic stress, disease damage, 

arthropod-related damage, and pest and beneficial arthropod abundance further support 

the conclusion that the introduction of BBX32 is not likely to result in increased plant 

pest potential or an adverse environmental impact from MON 87712 compared to 

conventional soybean. 

 
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VII.C.2.1. Field Phenotypic and Agronomic Characteristics 

A total of 13 phenotypic and agronomic characteristics were evaluated (Table VII-4 and 

Table H-4). In a combined-site analysis in which the data were pooled among the sites, 

no statistically significant differences were detected (α = 0.05) between MON 87712 and 

the conventional control A3525 for seedling vigor, days to 50% flowering, days to 50% 

end of flowering, plant height, lodging, pod shattering, grain moisture, or 100 seed 

weight (Table VII-4). Five statistically significant differences were detected between 

MON 87712 and the conventional control A3525 in the combined-site analysis. 

MON 87712 had higher early stand count (302.7 vs. 297.0 plants per plot), increased 

days to 50% senescence (267.7 vs. 265.0 days), increased days to physiological maturity 

(280.8 vs. 277.5 days), higher final stand count (296.8 vs. 286.4 plants per plot), and 

higher yield (52.6 vs. 49.0 bu/ac). Although significantly different from the conventional 

control A3525, the mean values of MON 87712 for early stand count and final stand 

count were small in magnitude and were within the range of commercial reference 

varieties for each characteristic and thus would not be adverse in terms of pest potential. 

Differences in 50% senescence, days to physiological maturity, and yield were consistent 

with the mode of action. The increase in yield is agronomically desirable and would not 

contribute to increased weediness potential of MON 87712 without changes in a 

combination of other characteristics associated with weediness (Baker, 1974). 

Furthermore, each difference, regardless of whether it is associated with the trait or not is 

assessed for pest potential and environmental impact. For each, it is unlikely that a 

difference in these characteristics (50% senescence, days of physiological maturity, and 

yield) would contribute to increased weediness potential of MON 87712 compared to the 

conventional control A3525. 

Flower color and plant growth stage data were categorical and were not statistically 

analyzed; however, at each site, all plants of MON 87712 and the conventional control 

A3525 had purple flowers as expected. Additionally, MON 87712 and the conventional 

control A3525 were within the same range of plant growth stages for all growth stage 

observations among all sites (Table H-5). Thus, there were no biologically-meaningful 

differences in plant development observed between MON 87712 and the conventional 

control A3525. 

The plant phenotypic and agronomic characteristics evaluated were used to provide a 

detailed description of MON 87712 compared to the conventional control A3525. A 

subset of these characteristics was useful to assess the weediness potential of 

MON 87712. Based on the assessed phenotypic and agronomic characteristics, the 

results support a determination that MON 87712 is no more weedy or likely to pose a 

plant pest risk or have a significant environmental impact than conventional soybean. 
Monsanto Company 11-SY-217U 129 of 467 

Table VII-3. Field Phenotypic Evaluation Sites for MON 87712 during 2009 

Location 

Location 

Code 

Site Designation in 

Statistical Report 

USDA-APHIS 

Notification Number 

Jackson County, Arkansas ARNE YARNE 09-075-110n 

Guthrie County, Iowa IAJA YIAJA 09-072-106n 

Jefferson County, Iowa IARL YIARL 09-072-106n 

Clinton County, Illinois ILCY YILCY 09-075-110n 

Effingham County, Illinois ILMS YILMS 09-075-110n 

Champaign County, Illinois ILSE YILSE 09-075-110n 

Stark County, Illinois ILWY YILWY 09-075-110n 

Parke County, Indiana INRC YINRC 09-100-102n 

Boone County, Indiana INSH YINSH 09-075-110n 

Pawnee County, Kansas KSLA YKSLA 09-075-110n 

Macon County, Missouri MOAN YMOAN 09-072-106n 

Shelby County, Missouri MOCB YMOCB 09-072-106n 

Butler County, Missouri MOFI YMOFI 09-072-106n 

Callaway County, Missouri MOKI YMOKI 09-072-106n 

St Louis County, Missouri MOSL YMOSL 09-072-106n 

Lincoln County, Missouri MOWR YMOWR 09-072-106n 

York County, Nebraska NEYO YNEYO 09-072-106n 

Berks County, Pennsylvania* PAGR YPAH2 09-075-110n 

Berks County, Pennsylvania* PAHM YPAH1 09-075-110n 

 

Note: Two additional sites, IAJE (Greene County, IA) and MOLP (Adair County, MO), 

were planted but multiple plots sustained substantial soybean plant damage due to frost 

and flood damage. 

* Two sites were planted in Berks, Pennsylvania. 
Monsanto Company 11-SY-217U 130 of 467 

Table VII-4. Combined-Site Comparison of MON 87712 to Conventional Control 

A3525 During 2009 for Phenotypic and Agronomic Characteristics 

Mean (S.E.) Reference Range1

Phenotypic 

Characteristic (units) MON 87712 Control Minimum Maximum

Early stand count (#/plot) 302.7 (5.6)* 297.0 (6.0) 239.5 345.7

Seedling vigor (1-9 scale) 2.9 (0.2) 3.0 (0.2) 1.7 4.8

Days to 50% flowering2 209.0 (1.0) 208.9 (1.0) 190.5 215.9

Days to 50% end of flowering3 231.7 (1.2) 231.5 (1.1) 215.3 239.0

Days to 50% senescence4 267.7 (0.8)* 265.0 (0.8) 251.6 270.8

Days to physiological maturity5 280.8 (0.9)* 277.5 (1.0) 262.4 281.6

Plant height (in) 31.6 (0.8) 31.1 (0.8) 23.1 34.6

Lodging (1-9 scale) 2.2 (0.2) 2.0 (0.2) 1.0 1.8

Pod shattering (1-9 scale) 1.2 (0.1) 1.3 (0.1) 1.0 2.1

Final stand count (#/plot) 296.8 (4.6)* 286.4 (4.7) 241.9 328.6

Grain moisture (%) 12.9 (0.2) 12.9 (0.2) 11.5 14.4

100 seed weight (g) 16.3 (0.2) 15.9 (0.2) 15.4 20.8

Yield (bu/ac) 52.6 (1.4)* 49.0 (1.2) 43.4 56.0

 

Note: The experimental design was a randomized complete block with four replications. S.E. = Standard 

Error. Means based on n = 76 for MON 87712 and the conventional control A3525 for all characteristics 

except for the following: for days to 50% senescence, n = 75 for MON 87712, for early stand count, n = 75 

for the conventional control A3525, for final stand count, n = 74 for MON 87712 and n = 75 for the 

conventional control A3525, for plant height, n = 64 for MON 87712 and n = 66 for the conventional 

control A3525, for seedling vigor, n = 68 for both MON 87712 and the conventional control A3525., for 

days to 50% end of flowering, n = 71 for MON 87712 and n = 72 for the conventional control A3525., for 

days to physiological maturity, n = 75 for MON 87712. For reasons of excluding data please refer to Table 

H-3. 

* Statistically significant differences were detected (α=0.05) between MON 87712 and the conventional 

soybean control A3525. 

1

 Reference range = Minimum and maximum mean values among the 18 commercial reference varieties. 

2

 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot were flowering. 3

 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot have stopped 

flowering. 

4

 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot reached 50% 

senescence. 

5

 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot reached 

physiological maturity. 

 
Monsanto Company 11-SY-217U 131 of 467 

VII.C.2.2. Environmental Interaction Characteristics 

USDA-APHIS considers the environmental interaction of the biotechnology-derived crop 

compared to its conventional control to determine the potential for increased plant pest 

characteristics. Evaluations of environmental interactions were conducted as part of the 

plant characterization for MON 87712. In the 2009 U.S. field trials conducted for 

evaluation of phenotypic and agronomic characteristics of MON 87712, data were also 

collected on plant response to abiotic stress (drought, wind, nutrient deficiencies, etc), 

disease damage, arthropod-related damage, and arthropod abundance (Tables H-6, H-7, 

H-8, H-9 and H-10). These data were used as part of the environmental consequences 

(Section IX) to assess plant pest potential and provide an indication of potential effects of 

MON 87712 on non-target organisms (NTOs) and threatened and endangered species 

compared to the conventional control. In addition, multiple commercial reference 

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-environmental 

interactions). Data was analyzed on an individual site basis; no combined site analysis 

was conducted. As expected, the results of the field evaluations showed that the presence 

of BBX32 did not meaningfully alter the assessed environmental interactions of 

MON 87712 compared to the conventional soybean A3525. The lack of significant 

biologically-meaningful differences in plant response to abiotic stress, disease damage, 

arthropod-related damage, and pest and beneficial arthropod abundance support the 

conclusion that the introduction of BBX32 protein is unlikely to result in an increased 

plant pest potential or cause an adverse environmental impact from MON 87712 

compared to conventional soybean A3525. 

In the 2009 field trials, the observations of plant response to abiotic stress, disease 

damage, and arthropod-related damage were performed four times during the growing 

season at all 19 sites, and arthropod abundance was assessed quantitatively from 

collections performed four times during the growing season at three of the 19 sites (i.e.,

ARNE, ILSE, and MOCB). The assessed stressors (abiotic, diseases, and arthropods) 

were at natural levels as no artificial infestation or imposed abiotic stress was used and 

therefore, typically varied between observations at a site and among sites. Abiotic stress 

and disease damage data were collected from each plot using a 0 – 9 scale of increasing 

severity of observed damage. Damage data were collected numerically and then placed 

into one of the following categories: none, slight, moderate, or severe. These categorical 

data were not subjected to statistical analysis. The response of MON 87712 and the 

conventional control A3525 to an abiotic stress or disease were considered different on a 

particular observation date at a site if the range of injury severity to MON 87712 did not 

overlap with the range of injury severity to the control across all three replications (e.g.,

“none” vs. “slight-moderate” rating). For each observation at a site, the range of injury 

severity across the commercial reference varieties provided assessment data that are 

representative of commercial soybean varieties. Arthropod-related damage was assessed 

from each plot on the upper four nodes of 10 representative plants using a 0 – 5 rating 

scale of increasing severity of observed damage. These numerical data along with the 

quantitative arthropod abundance data were subjected to statistical analysis. 
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In an assessment of plant response to abiotic stressors and disease damage, no statistically 

significant differences were detected between MON 87712 and the conventional control 

A3525 for any of the 384 comparisons which included 186 abiotic stressors and 198 

disease damage comparisons (Tables H-6 and H-7). 

In an assessment of arthropod-related damage, no statistically significant differences 

were detected between MON 87712 and the conventional control A3525 for 129 out of 

137 comparisons among the observations across all sites (Table H-8). In addition, no 

numerical differences were observed for the 55 comparisons for which p-value could not 

be generated due to lack of variability in the data. The mean damage ratings were 

slightly higher than the respective reference ranges for eight detected differences. 

However, the differences detected were small in magnitude and these differences were 

not consistent across observations or sites. Thus, these differences were not indicative of 

a consistent plant response associated with the trait and are unlikely to be biologically 

meaningful in terms of an adverse environmental impact of MON 87712 compared to 

conventional soybean. 

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

differences were detected between MON 87712 and the conventional control A3525 for 

106 out of 115 comparisons (including 59 pest arthropod comparisons and 56 beneficial 

arthropod comparisons) among the collections at the three sites (Tables H-9 and H-10). 

In addition, no numerical differences were observed for the eight comparisons (including 

six pest arthropod comparisons and two beneficial arthropod comparisons) for which pvalues could not be generated due to lack of variability in the data. The mean abundance 

values from MON 87712 were within the respective reference range for five of the nine 

detected differences. For the remaining four differences, the mean abundance values for 

MON 87712 were outside of the reference range; however, these differences were not 

consistent across collections or sites. Thus, these differences were not indicative of a 

consistent plant response associated with the trait and are unlikely to be biologically 

meaningful in terms of an adverse environmental impact of MON 87712 compared to 

conventional soybean. 

The results of the field evaluations showed that the presence of BBX32 did not alter the 

assessed environmental interactions of MON 87712 compared to the conventional control 

A3525. The lack of significant biological differences in plant responses to abiotic stress, 

disease damage, arthropod-related damage, and arthropod pest and beneficial insect 

abundance support the conclusion that the introduction of BBX32 in MON 87712 is 

unlikely to result in increased plant pest potential or an adverse environmental impact 

compared to conventional soybean. 

 

 
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VII.C.3. Pollen Characteristics 

USDA-APHIS considers the potential for gene flow and introgression of the 

biotechnology-derived trait into other soybean varieties and wild relatives. Pollen 

morphology and viability information are pertinent to this assessment and, therefore, 

were assessed for MON 87712. In addition, characterization of pollen produced by 

MON 87712 and the conventional control A3525 is relevant to the plant pest risk 

assessment because it adds to the detailed description of the phenotype of MON 87712 

compared to the conventional control A3525. 

The purpose of this evaluation was to assess the morphology and viability of pollen 

collected from MON 87712 compared to that of the conventional control A3525. Pollen 

was collected from MON 87712, the conventional control A3525, and four commercial 

reference varieties grown under similar agronomic conditions in a field trial in Missouri. 

The trial was arranged in a randomized completed block design with four replications. A 

minimum of twenty 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 four replications of 

MON 87712, the conventional control A3525, and the commercial reference varieties 

(see Appendix I). 

No statistically significant differences were detected between MON 87712 and the 

conventional control A3525 for percent viable pollen or pollen grain diameter (Table VII5). Furthermore, no visual differences in general pollen morphology were observed 

between MON 87712 and the conventional control A3525. These results demonstrate 

that the introduction of BBX32 did not alter the overall morphology or viability of 

MON 87712 pollen compared to the conventional control A3525. The pollen 

characterization data contribute to the detailed phenotypic description of MON 87712 

compared to the conventional control A3525. The result supports an overall conclusion 

that MON 87712 is no more likely to pose a plant pest risk than conventional soybean. 
Monsanto Company 11-SY-217U 134 of 467 

Table VII-5. Pollen Characteristics of MON 87712 Compared to the Conventional 

Control A3525 

Pollen 

Characteristic 

MON 87712 Control Reference Range2 

Mean (SE)1 Mean (SE)1 Minimum Maximum 

 

Viability (%) 98.7 (0.75) 99.4 (0.37) 98.8 99.7 

Diameter(μm) 25.4 (0.45) 24.9 (0.90) 24.8 26.1 

 

Note: No significant differences were detected between the test and the conventional 

control A3525 (α=0.05). 

1

 Mean based on n=4. SE = Standard Error. 

2 Reference range is the minimum and maximum mean value observed among the four 

commercial reference varieties. 
Monsanto Company 11-SY-217U 135 of 467 

VII.C.4. Symbiont Interactions 

As part of the plant pest risk assessment, USDA-APHIS considers the impact of the 

biotechnology-derived crop on plant pest potential and the environment as well as on 

agricultural or cultivation practices compared to its conventional counterpart. Potential 

changes in the symbiotic relationship with the rhizosphere-inhabiting bacteria 

Rhizobiaceae and Bradyrhizobiaceae could directly impact pest potential, the 

environment, or cultivation practices (i.e., the need to add additional nitrogen to sustain 

soybean production). Thus, the purpose of this evaluation was to assess whether the 

introduction of BBX32 altered the symbiotic interaction of MON 87712 with 

Bradyrhizobium japonicum compared to that of the conventional control A3525. 

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, which provide an environment in which differentiated bacteria called 

bacteroids are capable of reducing or “fixing” atmospheric nitrogen. The product of 

nitrogen fixation, ammonia, can then be utilized by the plant. As a result of this 

relationship, nitrogen inputs are typically not necessary for agricultural production of 

soybeans. 

The relative effectiveness of the symbiotic relationship between a leguminous plant and 

its rhizobial symbiont can be assessed by various methods. Measurement of nodule 

number and mass along with plant growth and nitrogen status are commonly used to 

assess differences in the symbiotic relationship 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 87712, parental conventional control A3525, and six commercial reference 

varieties were produced from seeds planted in pots containing nitrogen-deficient potting 

medium and grown in the 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 shoot and root plus nodule material were removed from the pots. Nodules 

were separated from roots prior to enumeration and determination of dry weight. 

MON 87712 was compared to the conventional control A3525 for key characteristics 

related to their association with the soybean - B. japonicum symbiosis. Detailed 

information on materials and methods used for the symbiont evaluation is presented in 

Appendix J. 

No statistically significant differences were detected (α=0.05) between MON 87712 and 

the conventional control A3525 for each measured parameter, including nodule number, 

shoot percent total nitrogen, shoot total nitrogen (g), and dry weight of nodules, shoot 

material, and root material (Table VII-6). 
Monsanto Company 11-SY-217U 136 of 467 

Based on the assessed characteristics, the results support the conclusion that the 

introduction of BBX32 does not alter the symbiotic relationship between B. japonicum

and MON 87712 compared to that of conventional soybean. Thus, these data further 

support a conclusion of no change in plant pest potential and no expected impact to 

cultivation practices relative to nitrogen inputs for MON 87712 compared to 

conventional soybean. 
Monsanto Company 11-SY-217U 137 of 467 

Table VII-6. Symbiont Interaction Assessment of MON 87712 and Conventional 

Control A3525 

Measurements

Mean (S.E.)* Reference Range1

MON 87712 A3525 p-Value Minimum Maximum

Nodule Number

(per plant) 124 (34) 110 (23) 0.5731 69 134

Nodule Dry Wt

(g/plant) 0.51 (0.06) 0.49 (0.04) 0.7954 0.41 0.59

Root Dry Wt

(g) 0.94 (0.09) 0.93 (0.07) 0.9617 0.78 1.59

Shoot Dry Wt

(g) 4.52 (0.60) 4.43 (0.36) 0.9061 4.03 6.62

Shoot Percent Total 

Nitrogen 

(% dwt)

3.07 (0.31) 3.18 (0.19) 0.5899 2.59 3.39

Shoot Total Nitrogen 

(g) 0.15 (0.03) 0.14 (0.02) 0.8043 0.13 0.18

 

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 87712 and the conventional 

control A3525 (α=0.05). 

1 Reference range is the minimum and maximum mean value observed among six 

commercial reference varieties. 

 
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VII.C.5. Volunteer Potential Assessment 

Volunteer potential can also play a role in determining whether a regulated article has 

increased weediness potential. In some crops, seed remaining in the field after harvest 

have the potential to over-winter and volunteer in the subsequent cropping season. The 

purpose of this study was to assess the volunteer potential of MON 87712 compared to 

the near isogenic conventional control A3525. In the fall of 2009, field trials were 

established at four locations to assess volunteer potential. Comparative assessments were 

conducted on MON 87712 and conventional control A3525. In addition, 4 commercial 

reference varieties were included as references at each site. It was determined that the 

seed were acceptable for evaluating volunteer potential based on standard viability 

testing. Normal seed germination rates were confirmed for MON 87712, the 

conventional control A3525, and commercial reference varieties, with two exceptions; 

two references at one site had germination rates of 42 and 31% respectively. The trials 

were established at each location as a randomized complete block design with four 

replications. Each plot was 5-8 ft wide by 20 ft long and was hand-seeded by uniformly 

scattering approximately 400 seeds on the soil surface. Seeds were then mechanically 

incorporated to a maximum depth of approximately 0.5 – 3 inches to avoid surface 

predation. Additional material and methods are provided in Appendix K. 

Agronomic practices used to prepare each study site were characteristic of each region. 

No irrigation was applied to the study areas, and no plot management was required after 

seed was incorporated into the soil. Volunteer plant counts were taken every two weeks 

after planting until the environmental conditions were no longer conducive for 

germination and emergence. Monitoring resumed in the spring of 2010, when 

environmental conditions became favorable for soybean germination and emergence. 

Volunteer counts continued approximately every two weeks until mid-June for a total of 

seven observations at each site. Data was analyzed on an individual site basis; no 

combined site analysis was conducted. 

No volunteer plants were observed at any site or observation time during the fall of 2009. 

No volunteer plants were observed at any observation time for two of the four sites 

during the spring of 2010. A small number of volunteers were observed at two of the 

four sites during the spring of 2010 (Table VII-7). However, the small number of 

volunteers observed in the study was not consistent across locations. Based on the 

assessed data, the results of this study support a conclusion that the introduction of 

BBX32 did not alter the ability of MON 87712 to volunteer compared to conventional 

soybean. Furthermore, these results demonstrate that BBX32 in MON 87712 confers no 

biologically meaningful change to the potential for soybean to persist in the environment. 
Monsanto Company 11-SY-217U 

Table VII-7. Observed Volunteer Soybean Plants of MON 87712 Compared to the Conventional References in a 2009/2010 U.S. Field TrialSite1 Season Observation Dates2

 Number of Volunteers Refer

 MON 87712 Control Min. 

ARAU Fall 12/07/09, 12/21/09 0 0 0  Spring 4/22/10, 5/06/10, 5/20/10, 6/03/10, 6/17/10 0 0 0 

ARNE Fall 12/07/09, 12/21/09 0 0 0 

 Spring 4/22/10, 5/06/10, 5/20/10, 6/03/10, 6/17/10 0 0 0 

ILWY Fall 12/07/09 0 0 0 

 Spring 4/08/10, 4/22/10, 5/06/10, 5/20/10, 

6/03/10, 6/17/10 

3 0 0 

MOAN Fall None - - - 

 Spring 3/19/10, 4/02/10, 4/16/10, 4/28/10, 

5/12/10, 5/26/10, 6/09/10 

0 1 0 

Mean Across 

 0.75 0.25 0.0 

Note: No statistical analyses were performed due to lack of variability. Numbers shown are total numbers of volunteers for each substance across all 

four replications at each specific site (n=4). 

1 ARAU = Woodruff County, AR; ARNE = Jackson County, AR; ILWY = Stark County, IL; MOAN = Shelby County, MO. 2 Observations were made approximately every two weeks. 3 Minimum and maximum number of volunteers observed from among the soybean reference varieties. 

4 Mean of volunteers observed for test control and references across all four sites and calculated using Microsoft Office Excel 2007. 
Monsanto Company 11-SY-217U 140 of 467 

VII.C.6. Persistence Outside of Cultivation Assessment 

Weediness or invasiveness may also be indicated if soybean exhibited an increased rate 

of persistence outside of cultivation. The purpose of this study was to assess the ability 

of MON 87712 to establish and persist in unmanaged, competitive environments that are 

not cultivated for agricultural production. Four sites were established in 2009. Each site 

was unmanaged and received no agricultural inputs allowing MON 87712, the 

conventional control A3525, and the commercial reference varieties (four per site) to 

compete with existing vegetation and respond to abiotic and biotic stressors present in 

each environment. Additional materials and methods are provided in Appendix L. 

Phenotypic and agronomic characteristics encompassing plant growth, development, and 

seed production were assessed for MON 87712, the parental conventional control A3525, 

and the commercial reference varieties in unmanaged environments. The experiment was 

established at each of four sites in a randomized complete block design with three 

replications. Stand count, growth stage, and plant vigor was evaluated at approximately 

14 day intervals throughout the season and the number of plants producing pods per plot, 

number of seeds produced per plot, and weight of seeds produced per plot were collected 

at the end of the season. Data was analyzed on an individual site basis; no combined site 

analysis was conducted. 

Additionally, replacement values were calculated and used to evaluate the ability of 

MON 87712, the conventional control A3525, and the commercial reference varieties to 

persist outside of cultivation. Each replacement value is the ratio of number of seeds 

produced to the number of seeds sown. A replacement value less than one means that 

fewer seeds were produced than sown. This is indicative of a population that will not 

replace itself and will not persist. A replacement value greater than one means that more 

seeds were produced than were sown and indicated a population that may potentially 

increase. 

The replacement value at three of the four sites was zero. At the end of the first growing 

season, one site had a replacement value of 2.72 for MON 87712 and 2.63 for the 

conventional control A3525, which indicated that the plots produced more seeds than 

planted. However, no plants emerged in any of the plots during the second season at this 

site and thus, the replacement value was zero. This indicated that the populations were 

all in decline and would not persist in the unmanaged environments and did not 

demonstrate a competitive advantage in this study compared to conventional soybean. 

Therefore, the presence of BBX32 in MON 87712 confers no biologically meaningful 

change to the fitness, invasiveness, or potential for soybean to persist outside of managed 

agricultural environments. 
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VII.D. 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 BBX32 altered the plant pest potential of MON 87712 compared to the 

parental conventional control A3525. Phenotypic, agronomic, and environmental 

interaction characteristics of MON 87712 were evaluated and compared to those of the 

conventional control A3525 and considered within the variation among commercial 

reference varieties. These assessments included seven general data categories: 1) seed 

germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive 

development (including pollen characteristics); 4) seed retention and lodging; 5) plant 

response to abiotic stress and interactions with diseases and arthropods; 6) plantsymbiont interactions and 7) volunteer potential and persistence outside of cultivation 

characteristics. Results from the phenotypic, agronomic, and environmental interactions 

assessment demonstrate that MON 87712 does not possess a) weediness characteristics; 

b) increased susceptibility or tolerance to specific abiotic stress, disease, or arthropods; or 

c) characteristics that would confer a plant pest risk or significant environmental impact 

compared to conventional soybean. 

 
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VIII. U.S. AGRONOMIC PRACTICES 

VIII.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 87712. Discussions include soybean production, seed 

production, plant growth and development, general management practices during the 

season, management of weeds, insects and diseases, soybean rotational crops, and 

volunteer soybean management. Information presented in Section VIII.C.2 demonstrated 

that MON 87712 produced more yield per soybean plant and has slightly delayed 

maturity compared to conventional soybean. However, MON 87712 is no more 

susceptible to diseases or pests than conventional soybean and required no additional 

inputs to produce a crop from MON 87712, and no specific impacts were noted to 

agronomic practices employed for production of soybean. In the areas where there is 

potential for impact on agronomic practices from the deregulation of MON 87712, the 

scope and magnitude of those impacts will be discussed. 

Soybean is grown as a commercial crop in over 35 countries, demonstrating its wide 

adaptation to varied 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, selection of a variety of the 

appropriate maturity that is adapted to the local environment, appropriate planting dates 

and plant population, and good integrated pest management practices are important for 

optimizing the yield potential and economic return for soybean. 

VIII.B Soybean Yield 

Improvement in grain yield remains one of the major objectives for plant breeders. Gains 

in major crop yields over the years can be attributed to genetic improvement through 

traditional breeding. Breeders have crossed plants with different genetic backgrounds 

and selected traits resulting in higher yields, compositional improvements, and desirable 

production traits. Biotechnological approaches complement traditional soybean breeding 

efforts by targeting the same major characteristics as traditional breeding. 

Crop yield results from a sequential growth and development process – first the plant 

grows vegetatively and produces photosynthetic tissue, followed by flowering and the 

production of seeds, and finally seed filling and maturation. Improvements in crop yield 

have been a primary focus of conventional breeding. The genetic changes that resulted in 

crop domestication and yield improvement in conventional varieties have been shown by 

modern molecular biology analysis to have been typically achieved through the selection 

and safe use of plant genes encoding transcriptional regulator proteins. Agricultural 

biotechnology provides the opportunity to further enhance crop yields through the 

introduction of new genetic elements that use or modify existing pathways in the plant. 
Monsanto Company 11-SY-217U 143 of 467 

World soybean production has increased steadily in the last decade, rising from 133 

million metric tons in 1996 to 258.4 million metric tons (MMT) in 2010 (ASA, 2011), 

due to higher economic values of protein and oil contents, industrial uses and medicinal 

importance. Soybean crop yields have risen consistently in North America over the past 

half-century. In the U.S., soybean yield rose at an annual average rate of 0.35bu/A 

(0.8%) between 1924 – 2010 (Specht et al., 1999), and similar yield increases have been 

reported in Canada. A survey of soybean yield in Canada between 1934 – 1992 revealed 

an annual increase in yield of 0.5%, with evidence that since 1976 the rate of genetic 

improvement of seed yield is accelerating (Morrison et al., 1999; Voldeng et al., 1997). 

Figure VIII-1. U.S. soybean yield rose at an annual average rate of 0.35 bu/A 

between 1924 – 2010. 

Linear regression analysis was conducted on data from the USDA National Agricultural 

Statistics Service (USDA-NASS, 2011b). 

Soybean yield trends in the U.S. indicate that yield growth rates have not reached a 

plateau. Average soybean yield in 2010 was 43.5 bu/A (2900 kg/ha), but record yields 

reported from yield contests in the U.S. (Iowa, Missouri and Nebraska, 1966-1998) were 

greater than 67.5 bu/A (4500 kg/ha) and in one instance reached 100 bu/A (6660 kg/ha) 

(Specht et al., 1999), demonstrating that future yield growth is possible. The concept of 

yield potential of soybean is defined as the yield of a cultivar when grown in 

environments to which it is adapted, with nutrients and water non-limiting, and with 

pests, diseases, weeds, lodging, and other stresses effectively controlled (Evans and 

Fischer, 1999; Specht et al., 1999). Conventional soybean yield potential has been Seed Yield (bu/A)

Production year
Monsanto Company 11-SY-217U 144 of 467 

estimated to be approximately 120 bu/A (8000 kg/ha) using crop simulation models 

(Specht et al., 1999). The U.S. average in 2010 of 43.5 bu/A (2900 kg/ha) was 

substantially below the current estimate of yield potential, suggesting that there is an 

opportunity to close the gap between average annual yield performance and yield 

potential. Actual yield performance is a complex outcome that is dependent on a number 

of genetic and environmental factors that influence a crop’s opportunity to realize its full 

yield potential. Growers are accustomed to experiencing field-to-field and year-to-year 

yield variation based on environmental conditions and the genetics of the varieties they 

select for planting. 

From 1924 to 2010, soybean acreage increased almost 50-fold and yield rose at an 

average annual rate of approximately 0.35 bu/A (0.8%) in the U.S. (USDA-NASS, 

2011b). Annual improvement in U.S. soybean yield is attributed to rapid producer 

adoption of agricultural improvements such as genetic and agronomic innovations that 

provide producers with means for reducing “on-farm” yield constraints (Specht et al., 

1999). Agricultural production depends on continuing infusions of genetic resources for 

yield stability and growth (Day-Rubenstein and Heisey, 2006). 

Increased soybean production in the U.S. has been accomplished by both increasing the 

area under cultivation and through yield increases per unit area. However, based on 

recent trends in farm production and land area, most OECD countries, including the U.S. 

and Canada, are predicted to face the challenge of expanding agricultural output by 

raising productivity on a stable or reduced land area (OECD-FAO, 2008). Therefore, 

much of the projected expansion in soybean production in the future is expected to come 

from increased yield rather than increased area under production (OECD-FAO, 2008). 

VIII.C. Overview of U.S. Soybean Production 

VIII.C.1. Soybean Production 

Soybean first entered North America in the 18th century (Hoeft et al., 2000b). During the 

1930s, soybean started to be processed industrially in the U.S. for edible oil and protein 

meal. In 2010, soybean represented 58% of world oilseed production, and approximately 

35% of world soybean production in 2010 were produced in the U.S. (ASA, 2011). In 

2010, the U.S. exported 1.59 billion bushels (43.27 million metric tons) of soybean, 

which accounted for 44% of the world's soybean exports (ASA, 2011). In total, the U.S. 

exported over $23 billion worth of soybean and soybean products globally in 2010 (ASA, 

2011). China was the largest export market for U.S. soybean with purchases totaling 

over $11 billion. Mexico was the second largest export market with purchases of $2.0 

billion. 

The productivity 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 Midwestern, Eastern, and portions of the Great Plains regions 

of the U.S. provide sufficient water 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., 2000b). Soil 
Monsanto Company 11-SY-217U 145 of 467 

texture and structure are key components determining water availability in soils, where 

medium-textured soils hold more water, allowing soybean roots to penetrate deeper in 

medium-textured soils than in clay soils. Irrigation is used on approximately 9% of the 

soybean acreage to supplement the water supply during dry periods in the Western and 

Southern soybean growing regions (USDA-ERS, 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 (Wilcox, 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 (Heatherly and 

Elmore, 2004). 

The U.S. soybean acreage in the past 10 years has varied from approximately 64.7 to 77.5 

million acres, with the lowest acreage recorded in 2007 and the highest in 2009 (Table 

VIII-1). Average soybean yields have varied from 33.9 to 44.0 bushels per acre over this 

same time period. Annual soybean production ranged from 2.45 to 3.19 billion bushels 

over the past ten years. According to data from USDA-NASS (2011d), soybean was 

planted on approximately 76.6 million acres in the U.S. in 2010, producing 3.32 billion 

bushels of soybean (Table VIII-1). Soybean acreage and production in 2008 was up from 

2007, mainly due to a decrease in corn acreage. The value of soybean reached $38.9 

billion in the U.S. in 2010 (USDA-NASS, 2011a). In comparison, corn and wheat values 

in 2010 were $66.65 and $12.99 billion, respectively (USDA-NASS, 2011a). 

For purposes of this agronomic practices discussion, soybean production is divided into 

three major soybean growing regions 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 

VIII-2). The vast majority of soybean was grown in the Midwest region, representing 

83.8% of the total U.S. acreage. The Southeast and Eastern Coastal regions represented 

13.5% and 2.7% of the acreage, respectively. Among the three regions, the Midwest 

region produced the highest average yield at 43.9 bushels per acre in 2010, and average 

state yields in this region ranged from 32.5 to 52.5 bushels per acre. The average yield in 

the Southeast region was 31.0 bushels per acre, with states within this region averaging 

from 26.0 to 41.0 bushels per acre. The average yield in the Eastern Coastal region was 

34.0 bushels per acre, with individual state averages ranging from 24.0 to 48.0 bushels 

per acre. 

Managing input costs is a major component to the economics of producing a soybean 

crop (Helsel and Minor, 1993). Key decisions on input costs include choosing what 

soybean varieties to plant, amounts of fertilizer to apply, and what herbicide program to 

use. The total operating cost for producing soybean in the U.S. in 2010 was $132.29 per 

acre, according to statistics compiled by the USDA-Economic Research Service (USDAERS, 2011). The value of the production less operating cost was reported to be $317.03 
Monsanto Company 11-SY-217U 146 of 467 

per acre. A summary of potential production costs and returns are presented in Table 

VIII-3. 

In the short term, individual growers’ decisions about whether to plant and which crop to 

plant are typically based on the relationship between operating costs and expected prices; 

i.e., on expected crop profitability (Ash et al., 2006). Managing input costs and managing 

the crop for yield are major components to the economics of producing a soybean crop. 

Growers’ costs include both overhead costs and operating costs. Overhead costs are 

those that are not associated with a particular crop and/or that are present whether or not a 

crop is grown, such as the cost of land and the depreciation of equipment. Operating 

costs are those associated with growing a particular crop in a given year, such as seed and 

fertilizer. A producer’s cost of growing a particular crop includes a proportional part of 

the overhead of his or her entire farming operation, plus all the operating costs associated 

with that crop. 

Figure VIII-2 shows the average per acre net value of soybean production in the U.S. 

from 1975 to 2010, based on data compiled by the USDA Economic Research Service 

(ERS). The net value is the value of the soybeans produced less all costs of production, 

both the allocated overhead and the operating costs. For comparison, corn and wheat are 

also shown. USDA’s data does not include crop subsidies. Overhead costs represent 

well over half the total costs (up to 69%), with the “opportunity cost of land (rental rate)” 

and the capital recovery cost of machinery and equipment representing the bulk of the 

overhead costs. The largest single operating cost is seed (USDA-ERS, 2011). As the 

data show, farming is often not profitable when all costs, including land value costs, are 

included. For example, USDA reports that in 2004, 70 percent of soybean-producing 

farm operations were considered profitable, not considering government payments. The 

percent profitable rises to 76 when government payments are included (Ash et al., 2006). 

While an individual grower typically makes planting decisions based on the relationship 

between operating costs and expected prices (Ash et al., 2006), many factors influence 

both operating costs and expected prices. Government price supports can have a large 

effect on costs, and supply and demand governs prices. 
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Figure VIII-2. U.S. Net Value of Soybean Production, Dollars Per Acre 

Inflation Adjusted (to 2011) Values from 1975-2009, and does not include Government Subsidies 

Sources: (USDA-ERS, 2011), U.S. Bureau of Labor Statistics 2011 

Table VIII-1. Soybean Production in the U.S, 2001 – 20101

Year 

Acres 

Planted 

(×1000) 

Acres 

Harvested 

(×1000) 

Average 

Yield 

(bushels/acre) 

Total 

Production 

(×1000 bushels) 

Value 

(billions $) 

2010 77,404 76,616 43.5 3,329,341 38.92 

2009 77,451 76,372 44.0 3,359,011 33.60 

2008 75,718 74,641 39.6 2,959,174 27.40 

2007 64,741 64,146 41.7 2,677,117 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 

Ave. 73,969 72,875 40.0 2,936,536 21.00 

1

Source is USDA-NASS(USDA-NASS, 2011c). 

 

‐$200

‐$150

‐$100

‐$50

$0

$50

$100

$150

$200

1975 1980 1985 1990 1995 2000 2005

Net Value (dollar per acre)

Year

Soybean

Corn

Wheat
Monsanto Company 11-SY-217U 148 of 467 

Table VIII-2. U.S. Soybean Production by Region and State in 2010 

Region/State

Acres Planted

(thousands)

Acres 

Harvested

(thousands)

Average Yield

(bushels/acre)

Total Production

(×1000 bushels)

Value

(billions $)

Midwest Region

Illinois 9,100 9,050 51.5 466,075 5.78

Indiana 5,350 5,330 48.5 258,505 3.05

Iowa 9,800 9,730 51.0 496,230 5.81

Kansas 4,300 4,250 32.5 138,125 1.66

Kentucky 1,400 1,390 34.0 47,260 0.57

Michigan 2,050 2,040 43.5 88,740 1.01

Minnesota 7,400 7,310 45.0 328,950 3.72

Missouri 5,150 5,070 41.5 210,405 2.55

Nebraska 5,150 5,100 52.5 267,750 3.03

North Dakota 4,100 4,070 34.0 138,380 1.56

Ohio 4,600 4,590 48.0 220,320 2.60

South Dakota 4,200 4,140 38.0 157,320 1.76

Wisconsin 1,640 1,630 50.5 82,315 0.94

Region Totals 64,240 63,700 43.9 2,900,375 34.04

Southeast Region

Alabama 350 345 26.0 8,970 0.10

Arkansas 3,190 3,150 35.0 110,250 1.25

Georgia 270 260 26.0 6,760 0.07

Louisiana 1,030 1,020 41.0 41,820 0.46

Mississippi 2,000 1,980 38.5 76,230 0.85

North Carolina 1,580 1,550 26.0 40,300 0.50

South Carolina 465 455 23.0 10,465 0.12

Tennessee 1,450 1,410 31.0 43,710 0.51

Region Totals 10,335 10,170 31.0 338,496 3.86

Eastern Coastal Region

Delaware 175 173 32.0 5,536 0.07

Maryland 470 465 34.0 15,810 0.19

New Jersey 94 92 24.0 2,208 0.02

New York 280 279 48.0 13,392 0.15

Pennsylvania 500 495 42.0 20,790 0.25

Virginia 560 540 26.0 14,040 0.17

Region Totals 2097 2044 34.0 71,776 0.85

USDA-NASS (2011d; 2011a) 
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Table VIII-3. U.S. Soybean Production Costs and Returns in 20101

Production Cost or Return 

Category Itemized Costs 

Return per 

Planted Acre 

($ USD) 

 

Total Gross Value of Production 449.32 

Operating Costs: Seed 59.2 

Fertilizer 17.87 

Chemicals 17.04 

Custom operations 6.52 

Fuel, lube and electricity 16.75 

Repairs 13.46 

Purchased irrigation water 0.14 

Interest on operating capital 1.31 

Total, operating costs 132.29 

 

Allocated overhead: Hired labor 2.11 

Opportunity cost of unpaid 

grower’s labor 

17.33 

Capital recovery of 

machinery and equipment 

77.51 

Opportunity cost of land 

(rental rate) 

148.34 

Taxes and insurance 9.41 

General farm overhead 14.86 

Total, allocated overhead 269.56 

 

Total cost listed 401.85 

Value of production less total cost 

listed 

 47.47 

Value of production less operating 

costs 

 317.03 

Supporting Information: Yield = 43 bushels/acre, Price = $10.48/bushel, Enterprise size = 

303 planted acres, Irrigated = 9%, Dry land = 91%. 

1

Source is USDA-ERS (2011). 
Monsanto Company 11-SY-217U 150 of 467 

VIII.C.2. Variety Development 

Crop domestication and improvement through breeding has been largely achieved through 

selection of genes that regulate the expression of desirable traits, such as those associated 

with higher yields or disease resistance. Once plants with the desired traits have been 

selected, a population of those plants with similar characteristics are classified as varieties.

Historically breeders have developed desirable varieties by retaining for further breeding 

those plants that possess the desirable traits, as determined by visual inspection or by 

testing. In recent years breeders have used the more direct methods of molecular breeding 

techniques, such as marker assisted breeding, to accelerate the process of identifying 

breeding lines containing a desired set of positive traits. These techniques rely on 

inventories of genomic regions or genetic markers that have been positively associated with 

the desirable traits. Once the genetic markers associated with the desired traits have been 

identified, molecular breeders can quickly select the offspring inheriting the genes for 

further development (Voosen, 2009). 

Hundreds of soybean varieties are tested each year in performance trials (variety trials) 

conducted by universities and private companies in all the major soybean growing states. 

The following information can typically be obtained from the results of variety trials: 

maturity group, disease resistance, insecticide seed treatment, yield, maturity date, percent 

lodging (plants fallen over on the ground), height, and herbicide resistance (Tylka et al., 

2010). In different parts of the country and/or in other trials, additional characteristics may 

be identified, such as iron deficiency tolerance or protein or oil content {Pedersen, 2008 

#56136}. 

Soybean is a self pollinating crop and the vast majority of commercial soybean currently 

under production was produced through a process referred to as forward breeding. Soybean 

varieties are developed by seed companies through conventional forward breeding that 

involves the following steps: 

1. Initial Crosses to generate genetic variability 

2. Advance generations through self pollination 

3. Bulk production of a segregating population of plants 

4. Selection of plants with a desired phenotype within the bulk population 

5. Harvesting of the self pollinating seed from selected plants 

6. Planting of the selected seed and bulk production through self pollination 

7. Harvest of self pollinating seed 

A typical breeding cycle takes roughly 6 generations once a desired phenotype has been 

selected to produce commercial seed. 

VIII.C.3. 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 
Monsanto Company 11-SY-217U 151 of 467 

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) was quickly identified as a major factor impacting 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 the establishment of official certifying agencies. 

Regulations first 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 (AOSCA) represents state and private seed certification organizations 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, 2009). Breeder seed is seed directly controlled by the originating 

or sponsoring plant breeding organization or firm. Foundation seed is first-generation seed 

increased from breeder seed and is handled in a manner to maintain specific levels of 

varietal purity and identity. Registered seed is the progeny of foundation seed that is 

handled to maintain satisfactory varietal purity and identity. Certified seed is the progeny of 

breeder, foundation or registered seed, and is typically two generations removed from 

foundation seed. While not all soybean seed sold to growers is officially certified, 

commercial soybean seed sold and planted for typical soybean production is produced 

predominately to meet or exceed certified seed standards. 

Soybean seed breeders and producers have put in place practical measures to assure the 

quality and genetic purity of soybean varietal seed 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 AOSCA. 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. 

U.S soybean production for all purposes has varied from approximately 64.7 to 77.5 million 

acres in the past ten years (USDA-NASS, 2011c). To plant this area of soybean acreage 

requires 105 to 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 
Monsanto Company 11-SY-217U 152 of 467 

(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. 

Certified 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 VIII.C). Seed production 

for these maturity groups is grown in the respective geographical zone for 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 a number of companies that produce and sell seed, such as 

Monsanto Company, Pioneer Hi-Bred International, Syngenta Seeds, Kruger Seed Co., and 

Becks Hybrids. In addition, certified seed is produced by toll seed producers, or tollers, 

which are companies that produce but do not directly sell certified seed, such as Remington 

Seeds LLC and Precision Soya. Seed companies and tollers in turn contract acreage with 

growers to produce the needed amount of soybean seed. Seed production or processing 

plants at these seed companies identify local soybean growers to produce the seed and also 

monitor and inspect seed fields throughout the growing season. The seed 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 operate 

using International Organization for Standardization (ISO) certification 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). 

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. 

Importantly, the seed production field should not have been planted with soybean in the 

previous crop season in order to avoid potential volunteer soybean plants (even though the 

risk of soybean volunteer plants is negligible) and to ensure genetic purity. 

Very early planting is typically 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 practices 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 
Monsanto Company 11-SY-217U 153 of 467 

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 assure genetic purity. Certain 

seed handling equipment, such as auger elevators, should be avoided because they can 

increase seed damage. 

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 visually 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 as a 

physical barrier (AOSCA, 2009). 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 the identity of the seed is maintained throughout the handling 

and storage operation. Bin inspections and sample collections are conducted at storage 

locations at the seed production 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, 2009). State seed certification standards vary slightly from state to state and can 

be more restrictive than the seed standards of AOSCA. 

When deregulated, MON 87712 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. 

VIII.D. Production Management Considerations 

VIII.D.1. Pre-Season 

Well in advance of planting a soybean crop, decisions are made regarding the planned crop 

rotation, the tillage system and row spacing that will be implemented, the planting 

equipment that will be used, the seed or variety that will be planted, and soil fertility 
Monsanto Company 11-SY-217U 154 of 467 

management requirements. 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 VIII.I). 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 on tillage 

system 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. 

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 (CTIC, 

2000). In 2004, approximately 27.5 million acres (39.6%) of soybean were planted in a notill system (CTIC, 2007). Slow soybean emergence and growth leading to 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 seedling emergence, but have little effect on soybean yield (Pedersen, 

2008a). Improved planters for establishment of good soybean populations and planting 

Roundup Ready soybean allowing the use of glyphosate to effectively control weeds in notill fields have made no-till a viable production system for soybean (Pedersen, 2008a). 

Extension specialists 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., 2000b). 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., 2000b). 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 

these bacteria are not native to U.S. soils and would not normally be found in these soils, 

inoculation of the soybean seed with these bacteria 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 decrease nodulation while increasing the plant’s 

dependency on the soil for nitrogen (Pedersen, 2008f). 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 
Monsanto Company 11-SY-217U 155 of 467 

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 in soil 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., 2000b), soil pH should be maintained at about 6.0 to 6.5 through the addition 

of limestone. 

Because soybean growth is so dependent on day/night length, different varieties are 

developed for different latitudes. In the U.S. ten geographically-designated “maturity 

groups” originally defined by Scott and Aldrich (1970) are widely used. Maturity groups 

are often designated by Arabic rather than Roman numerals, so the sequence is 00 to 8, and 

there are subdivisions within the major maturity groups. These are designated by a decimal 

value. For example, a variety with maturity group designation 2.9 would be at the northern 

end of Group II (2). These maturity groups are mapped as bands extending from north to 

south, beginning with Group 00 at the far north and ending with Group VIII in the far 

south.4

 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, which extend from approximately the northern 

border of Iowa to the southern tip of Illinois, account for approximately 76% (24%, 36%, 

and 16%, respectively) of the soybean planted in the U.S. (T. Schlueter, personal 

communication, August 2008). Because day length delays maturity, a soybean cultivar 

suited to a southern maturity group would mature too late if planted too far north. 

Conversely, a northern cultivar would mature too early if planted in the south (Heatherly 

and Elmore, 2004). 

Soybean variety selection is crucial for high yield and quality, and is the foundation of an 

effective management plan (Pedersen, 2008b). 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. 

VIII.D.2. 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, 

 

4

 They were originally designated north to south as Groups I through VIII. Groups 0 and 00 were later added 

to the north. 
Monsanto Company 11-SY-217U 156 of 467 

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, 2004). 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. 

The time of onset and the duration of the various growth stages in soybean are highly 

dependent on photoperiod (hours of daylight and darkness) and temperature (Major et al., 

1975), and therefore, for the same soybean plant grown at different latitudes, the onset and 

duration of the growth stages and the total time from planting to maturity would be 

different. Also, in contrast to most other temperate-season crops, soybean is a “short-day” 

plant, meaning that maturity is delayed by longer day length (Major, 1980). In soybeans, 

flowering is initiated only after the night is longer (and days grow shorter) than a critical 

length (Holshouser, 2010). Once flowering begins, temperature controls the duration of 

flowering time (Heatherly and Elmore, 2004). 

Adequate soil moisture and warm temperatures facilitate rapid seed germination and 

emergence. The ideal soil temperature for soybean germination and emergence is 77oF 

(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 50oF 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 

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., 2000b). Highest yields are generally obtained when planting in 

early to mid May. Yields begin to decline 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 onethird 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. 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, 2008g). 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 inches (Hoeft et al., 2000b). 
Monsanto Company 11-SY-217U 157 of 467 

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., 2000b). 

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 

fields, 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 grower 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., metalaxyl or mefenoxam) to prevent dampingoff 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 (Pedersen, 2008c). 

Annual and perennial weeds are considered to be the greatest pest problem in soybean 

production (Aref and Pike, 1998b). 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 (Section VIII.F). 

VIII.D.3. Mid to Late Season 

Ideal daytime temperatures for soybean growth are between 75oF and 85oF (Hoeft et al., 

2000b). Warmer temperatures result in larger plants and earlier flowering. Sustained 

temperatures below 75oF 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 70oF. Soybean varieties differ in their response and tolerance to 

temperatures. 

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. 

Indeterminate varieties are typically 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 
Monsanto Company 11-SY-217U 158 of 467 

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., 2000b). 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 

from 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., 2000b). 

Another critical requirement during the seed-filling stages is a high rate of photosynthesis to 

maximize yield. 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. 

VIII.D.4. 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., 2000b). 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., 2000b). 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 soybean 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. 

A larger than normal crop can stress the storage and transportation system for the crop. 

Because of the very high variability in crop production, storage facilities are not always 

adequate. Soybeans and other grain must sometimes be stored in temporary structures or in 

other existing buildings if storage facilities are overloaded, and this may result in additional 

costs for constructing or renting temporary facilities and/or potential losses from exposure 

(Dorn, 2011; Hellevang, 1998). The same conditions can result when prices are low and 

growers want to hold on to their crops in the hopes of selling at higher prices (Maier and 

Wilcke, 2011). Soybeans can also compete with corn for available storage space; and while 

corn can be stored on the ground, soybeans rarely are (Hurburgh, 2005). University 

extensions provide practical guidelines for temporary storage of soybeans and other crops 

(Dorn, 2011; Harner et al., 1998; Hellevang, 1998; Hurburgh, 2005; Maier and Wilcke, 

2011). 
Monsanto Company 11-SY-217U 159 of 467 

Growers decide where to sell their soybeans based on the cost of delivering soybeans to 

their customer, usually an elevator or processing facility (USSEC, 2009). Growers usually 

deliver their soybeans to the sale point using their own trucks. From the elevator or 

processing facility, the soybeans or oil and meal are shipped by rail, barge or truck. 

Approximately 24% of soybeans are transported by rail, although higher percentages of 

meal and soybean oil are transported by rail (STC, 2010). More than half of U.S. soybean 

exports are first shipped by barge on the Mississippi River (Ash et al., 2006). USDA 

reported in 2006 that recent record-large soybean harvests have tested the capacity of the 

U.S. bulk transportation system; however, no specifics were provided (Ash et al., 2006). 

Large crops can result in greater shipping competition and higher shipping costs, which 

translates to lower prices offered to growers (Ash et al., 2006). 

VIII.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, 1998b). Understanding the impact of insects 

on soybean growth is essential for proper management (Way, 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. 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 (Way, 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 leaf-feeding insects 

comprise the biggest group of soybean insect pests, but not necessarily the most 

economically damaging insects. Research on defoliation has determined that a major effect 

of leaf injury is to reduce light interception by the soybean canopy which in turn can have a 

significant effect on yield (Way, 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. 

VIII.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 (Bowers and Russin, 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., 2001), which equated to 16.7%, 16.0% and 18.6% of total 

soybean production, respectively. Pathogens can affect all parts of the soybean plant, 
Monsanto Company 11-SY-217U 160 of 467 

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 severity of the disease on 

individual plants, and the number of plants affected (Bowers and Russin, 1999). 

One or more diseases can generally be found in fields wherever soybean is grown (Bowers 

and Russin, 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 (SCN), Heterodera gylcines, caused the greatest soybean yield 

losses (Wrather et al., 2001). 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 (Bowers 

and Russin, 1999). Resistant varieties may have morphological or physiological 

characteristics that provide immunity, resistance, tolerance or avoidance to certain 

pathogens. Cultural practices can also play an important role in disease management by 

reducing initial inoculums or reducing the rate of disease development (Bowers and Russin, 

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 sp., 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 is one of the most damaging pathogens of soybean throughout the 

soybean growing regions of the U.S. (Pedersen, 2008d). Losses have been estimated to be 

at about $1.5 billion in the U.S. (Pedersen, 2008d). SCN can cause yield losses up to 50%, 

where this pest in 2004 alone caused an estimated loss of 50 million bushels of soybean in 

Iowa (Pedersen, 2008d). 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 sp., Pythium sp., Rhizoctonia sp., and 

Fusarium sp. (Pedersen, 2008e). Many soybean varieties demonstrate resistance to specific 
Monsanto Company 11-SY-217U 161 of 467 

taxonomic races of Phytophthora. Treating soybean seed with a fungicide (e.g., metalaxyl 

or mefenoxam) is effective against damping-off disease (seedling blight) caused by common 

soil fungi, such as Phytophthora sp. and Pythium sp. 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., 2007b). Soybean rust is caused 

by the fungus Phakopsora pachyrhizi. This disease in the U.S. was first detected in 

Louisiana in 2004 (LSU, 2010). 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 fungal diseases 

(Bowers and Russin, 1999). However, the economic return from a fungicide application 

may be limited to select soybean production systems; for example, high-yield environments 

or when producing soybean seed. According to USDA-NASS (2007) statistics, fungicides 

were applied on approximately 4% of the soybean acreage in 2006. 

VIII.G. Weed Management 

Annual weeds are perceived to be the greatest pest problem in soybean production, followed 

by perennial weeds (Aref and Pike, 1998b). 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, 2007). 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., 2001b). Generally, the 

competition between crops and weeds increases with higher levels of weed density. The 

time period that weeds compete with the soybean crop influences the level of yield loss. In 

general, early season weed competition will have the greatest negative impact on yield 

(Dalley et al., 2001b). Although, soybean plants withstand early-season weed competition 

longer than corn without affecting yield, 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. 

Crop rotations and environment have a significant impact on the adaptation and occurrence 

of weeds in soybean. Foxtail spp. (Setaria spp.), pigweed (Amaranthus spp.), velvetleaf 

(Abutilon theophrasti), lambsquarters (Chenopodium album), and cocklebur (Xanthium 

strumarium) are common weeds in Midwest corn and soybean fields. However, growers 

consider giant ragweed (Ambrosia artemisiifolia), lambsquarters, Canada thistle (Cirsium 

arvense), cocklebur, and velvetleaf to be the top five most problematic weeds in corn and 

soybean because of difficulty controlling these weeds (Nice and Johnson, 2005). In a recent 

survey of growers utilizing glyphosate-tolerant crops, pigweed, morningglory (Ipomoea 

spp.), Johnsongrass (Sorghum halepense), ragweed spp. (Ambrosia spp.), foxtail, and 

velvetleaf were mentioned as the most problematic weeds, depending on the state and 
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cropping system (Kruger et al., 2009). With the exception of morningglory and pigweed, 

the weed species identified as problematic were present and problematic before glyphosatetolerant crops were introduced, but then were to a reduced degree after implementing 

glyphosate-tolerant cropping systems (Kruger et al., 2009). Common waterhemp 

(Amaranthus rudis) and ragweed were the most frequently mentioned problematic weeds in 

glyphosate-tolerant crops in Illinois, Indiana and Iowa. 

The most frequently reported common weeds in the Southeast region were morningglory 

(Ipomoea spp.), prickly sida (Sida spinosa), johnsongrass (Sorghum halepense), sicklepod 

(Cassia obtusifolia), and broadleaf signalgrass (Brachiaria platyphylla) (Webster et al., 

2009). Morningglory, sicklepod, and pigweed are the most frequently mentioned 

problematic weeds in glyphosate-tolerant crops in Mississippi and North Carolina (Kruger 

et al., 2009). 

Cultural and mechanical weed control practices can be important components of an effective 

weed management program (Loux et al., 2010). 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-NASS, 2007). 

Herbicide-tolerant soybean was introduced to provide growers with additional options to 

improve crop safety and/or improve weed control. The Roundup Ready soybean system 

(planting Roundup Ready soybean and applying glyphosate in crop to provide primary weed 

control) was introduced in 1996 and has become the standard weed control program in U.S. 

soybean production and is utilized on 91% of U.S. soybean acreage (USDA-NASS, 2009). 

VIII.H. Crop Rotation Practices in Soybean 

The well-established farming practice of crop rotation is still a key management tool for 

growers. The purpose of growing soybean in rotation is 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 (Al-Kaisi et al., 2003; Heatherly and 

Elmore, 2004). According to the USDA Economic Research Service, 95% of the soybeanplanted 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 of 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 damage from diseases and 
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nematodes (Al-Kaisi et al., 2003; Hoeft et al., 2000b). 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. 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., 2000b). 

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 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. 

Unique to the southern portion of the Midwest and the Southeast 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., 2000b). 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. 

VIII.I. 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 small grains (e.g., wheat, barley, sorghum, and oats), 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 in each of the major soybean rotational crops. Table 

VIII-4 provides control ratings on volunteer soybean for several herbicides used in the 
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major rotational crops. These results indicate that herbicides which are effective for the 

control of volunteer soybean will control MON 87712. 

Table VIII-4. Ratings for Postemergence Control of Volunteer Soybean in Labeled 

Rotational Crops1 

Product 

Rate 

(Product/Acre) 

Soybean 

V2 – V3 

Soybean 

V4- V6 

Corn2 

AAtrex 4L (atrazine) 0.38 qts E P 

0.50 qts E F 

Hornet WDG (flumetsulam/clopyralid) 1 – 2 oz E F-G 

Widematch (clopyralid/fluroxypyr) 0.25 pt E G 

Sorghum2,4 

AAtrex 4L (atrazine) 0.38 qts E P 

0.50 qts E F 

Permit (halosulfuron) 2/3 oz E E 

Buctril® (bromoxynil) 1 pt 

Wheat, Barley & Oats2 

Buctril (bromoxynil) 1 pt E E 

Widematch (clopyralid/fluroxypyr) 0.25 pt E G 

Cotton3 

Envoke® (trifloxysulfuron) 0.1 oz E E 

Rice4 

Grandstand® CA (triclopyr) 0.5 pint E E 

Regiment® (bispyribac) 0.4 oz E E 

Grasp® SC (penoxsulam) 2 oz E E 

Permit (halosulfuron) 2/3 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

Source is Zollinger (2009). 

3

Source is York et al. (2005). 

4

Sources are Dillon et al. (2006); Bond and Walker (2009). 
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VIII.J. Stewardship of MON 87712 

Monsanto Company develops effective products and technologies and is committed to 

assuring 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 (ETS) Program5

. These policies and practices include rigorous field 

compliance and quality management systems and verification through auditing. 

Monsanto’s Stewardship Principles are also articulated in Technology Use Guides6

 that 

are distributed annually to growers who utilize Monsanto branded traits. 

As an integral action of fulfilling this commitment, Monsanto will seek biotechnology 

regulatory approvals for MON 87712 in all key soybean import countries with a 

functioning regulatory system to assure global compliance and support the flow of 

international trade. These actions will be consistent with the Biotechnology Industry 

Organization (BIO) Policy on Product Launch7. 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. 

Monsanto also commits to best industry practices on seed quality assurance and control 

to ensure the purity and integrity of MON 87712 seed. As with all of Monsanto’s 

products, before commercializing MON 87712 in any country, a MON 87712 detection 

method will be made available to soybean producers, processors, and buyers. 

VIII.K. Impact of the Introduction of MON 87712 on Agricultural Practices 

Introduction of MON 87712 is expected to have minimal impact on current cultivation 

and management practices for soybean. MON 87712 has been shown to be no different 

from conventional soybean in its agronomic, and compositional characteristics (refer to 

Sections VI and VII), and has the same levels of susceptibility to insects and diseases as 

commercial soybean. The increase in yield and small delay in senescence is expected to 

have minimal impacts if any on the agricultural practices farmers use to produce a 

soybean crop. MON 87712 did not require any additional inputs to produce a crop and 

varieties that contain MON 87712 are similarly not expected to require additional inputs. 

Farmers understand the value of increased yield for their farm’s productivity and 

profitability, and are accustomed to the incremental yield improvements for varieties 

obtained through traditional breeding. Growers are also accustomed to field-to-field or 

year-to-year yield variation based on environmental conditions and the varieties they 

 

5

 Excellence Through Stewardship Program can be found at: 

http://www.excellencethroughstewardship.org/. 6

 Monsanto Technology Use Guides can be found at: 

http://www.monsanto.com/monsanto/ag_products/pdf/stewardship/technology_use_guide.pdf. 7

 BIO’s Product Launch guidelines can be found at: 

http://www.excellencethroughstewardship.org/facts/documents/Guide%20for%20Product%20Launch%20S

tewardship.pdf. 
Monsanto Company 11-SY-217U 166 of 467 

select for planting. Therefore, farmers are capable of adjusting harvesting and storage 

equipment to handle increased yields. MON 87712 will provide another option for 

farmers to pursue better yielding varieties for their farm. MON 87712 offers the potential 

to improve productivity in the U.S. soybean production system, thereby helping to meet 

the growing global demand for soybean. 
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IX. ENVIRONMENTAL CONSEQUENCES 

IX.A. Introduction 

This section provides a brief review and assessment of the plant pest potential of 

MON 87712 and its impact on current agronomic practices. USDA-APHIS has 

responsibility, under the Plant Protection Act (PPA) (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 may be granted, thereby allowing unrestricted 

introduction of the article. 

The definition of “plant pest” in the Plant 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]). 

The regulatory endpoint under the PPA for biotechnology-derived crop products is not 

zero risk, but rather a determination that deregulation of the article in question is not 

likely to pose a plant pest risk. The approach used to assess the plant pest potential of 

MON 87712 is a weight of the evidence approach based primarily on eight lines of 

evidence: 1) insertion of a single functional copy of the BBX32 expression cassette, 2) 

characterization of BBX32 protein expressed in MON 87712, 3) safety of BBX32 in 

MON 87712, 4) compositional equivalence of harvested MON 87712 seed and forage to 

conventional soybean, 5) phenotypic, agronomic, and environmental interaction 

characteristics demonstrating no increased plant pest potential compared to conventional 

soybean, 6) negligible risk to Non Target Organisms (NTO) and threatened or 

endangered species, 7) familiarity with soybean as a cultivated crop and the inherently 

low plant pest potential of soybean, and (8) no unexpected impact to agronomic practices, 

including land use, cultivation practices, or the management of weeds, diseases, and 

insects than conventional soybean. 

Using the assessment above, the data and analysis presented in this petition lead to a 

conclusion that MON 87712 is unlikely to be a plant pest and, therefore, should no longer 

be subject to regulation under 7 CFR § 340. 

In 2008, APHIS proposed amendments to 7 CFR Part 340 that included provisions to 

utilize its noxious weed authority in regulating genetically engineered plants (73 FR 

60008). Because the data presented in this petition demonstrate that MON 87712 has no 

potential to cause injury or damage to protected interests under the noxious weed 

authority, MON 87712 would not be considered a “noxious weed” as defined by the Plant 

Protection Act. 

 
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IX.B. Plant Pest Assessment of MON 87712 Insert and Expressed Protein 

This section summarizes the details of the genetic insert, characteristics of the genetic 

modification, and safety and expression of the BBX32 protein in MON 87712 used in 

food, feed, and environmental safety evaluation of MON 87712. 

IX.B.1. Characteristics of the Genetic Insert and Expressed Protein 

IX.B.1.1. Genetic Insert 

MON 87712 was developed through Agrobacterium tumefaciens-mediated 

transformation of conventional soybean A3525 meristem tissue utilizing transformation 

plasmid vector PV-GMAP5779, a binary vector containing two transfer DNA (T-DNA I 

and T-DNA II) (Figure III-1). Each T-DNA in PV-GMAP5779 is delineated by Left and 

Right Border regions to facilitate transformation. T-DNA I contains the BBX32 coding 

sequence under regulation of the e35S promoter and the E6 3′ untranslated region while 

T-DNA II contains the cp4 epsps coding sequence under the regulation of the 

FMV/EF-1α promoter, EF-1α leader, EF-1α intron, CTP2 targeting sequence, and the E9

3′ untranslated region. During transformation, both T-DNAs were inserted into the 

soybean genome (Section III.B) where T-DNA II, containing the cp4 epsps expression 

cassette, functioned as a marker gene for the selection of transformed plantlets. 

Subsequently, conventional self-pollinated breeding methods and segregation, along with 

a combination of analytical techniques, were used to isolate those plants that contain the 

BBX32 expression cassette (T-DNA I) but not the cp4 epsps expression cassette 

(T-DNA II) resulting in the production of marker-free MON 87712. The promoter, 

leader, and border sequences of T-DNA I are not known to cause plant disease. 

Furthermore, these sequences are well characterized, are noncoding regions, and will not 

cause MON 87712 to promote plant disease. 

Molecular characterization of MON 87712 by Southern blot analyses confirmed that one 

copy of the BBX32 expression cassette was integrated into the soybean genome at a 

single locus. No T-DNA II or backbone DNA sequences from plasmid vector 

PV-GMAP5779 were detected in MON 87712. Additionally, the data confirmed the 

organization and sequence of the insert and demonstrated the stability of the insert over 

several generations. These data demonstrated that there are no unintended changes in the 

MON 87712 genome as a result of the insertion of the BBX32 expression cassette, and 

supports the overall conclusion that MON 87712 is unlikely to be a plant pest. 

IX.B.1.2. Protein Safety 

MON 87712 is a biotechnology-derived soybean that has been demonstrated to provide 

increased yield (See Appendix B), due to the insertion of the Arabidopsis thaliana BBX32

gene. Arabidopsis thaliana is generally not considered an allergenic or toxic source 

organism. Although Arabidopsis thaliana contains homologs of proteins previously 

described as allergens in other plant species (e.g., germins, lipid transfer protein, 

profilins, and small molecular weight calcium binding proteins), no Arabidopsis proteins 

have been reported in a peer-reviewed database of known allergens. 
Monsanto Company 11-SY-217U 169 of 467 

BBX32 is a member of a family of B-box-containing proteins. B-box zinc finger family 

is found in many plant species including soybean. Additionally, bioinformatic searches 

using the BBX32 amino acid sequence as a query yield homologous sequences from 

several different plant species, including the food crops citrus, grape, apple, soybean, 

rice, lettuce, and corn. Overall protein sequence identity of BBX32 to homologs in these 

food species range from ~43-31%. Further, the overall protein sequence identity of 

BBX32 to its homolog in canola is 66%, indicating that Brassicaceae species contain 

proteins very similar to BBX32. 

The levels of BBX32 in various tissues of MON 87712 that are relevant to the risk 

assessment were determined by western blot analysis. BBX32 protein levels in 

MON 87712 across tissue types ranged from <LOD to 110 ng/g dwt. The western blot 

method developed was highly sensitive as indicated by the low LODs established for 

each tissue (Table V-1). In spite of the high sensitivity of the western technique BBX32 

protein levels in many samples, including all seed, forage and OSL-4 samples, were 

<LOD or not detected, indicating that the expression levels in these tissues are very low. 

Taken together, the low level of BBX32 protein expressed in MON 87712 tissues along 

with the presence of homologous sequences of BBX32 in several plant species including 

soybean, support the conclusion that food and feed products containing or derived from 

MON 87712 are as safe for human and animal consumption as soybean currently on the 

market. Therefore, unintended environmental effects are not anticipated from dietary 

exposure to BBX32 in MON 87712, and support the overall conclusion that MON 87712 

is unlikely to be a plant pest 

IX.B.2. Compositional Characteristics 

Detailed compositional analyses in accordance with OECD guidelines were conducted to 

assess whether levels of key nutrients and anti-nutrients in MON 87712 were comparable 

to levels present in the conventional control A3525 and several conventional commercial 

reference varieties. Seed and forage were harvested from eight individual sites in which 

MON 87712, the conventional control A3525, and a range of conventional commercial 

reference varieties were concurrently grown in the same field trial. The conventional 

commercial reference varieties were used to establish a range of natural variability for the 

key nutrients and anti-nutrients in conventional soybean varieties that have a history of 

safe consumption. 

The combined site analysis was conducted to determine statistically significant 

differences (5% level of significance) between MON 87712 and the conventional control 

A3525. The results from the combined-site data were reviewed using considerations 

relevant to food and feed safety and nutritional quality including the relative magnitudes 

of the difference in the mean values of nutrient and anti-nutrient components of 

MON 87712 and the conventional control A3525, whether the MON 87712 component 

mean value was within the range of natural variability of that component as represented 

by the 99% tolerance interval of the conventional commercial reference varieties grown 

concurrently in the same field trial, and analyses of the reproducibility of the statistically 

significant combined-site component differences at individual sites. 
Monsanto Company 11-SY-217U 170 of 467 

Assessment of the analytical results confirmed that the differences observed in the 

combined-site analysis were not meaningful to food and feed safety or the nutritional 

quality of MON 87712 soybeans. In addition, the levels of assessed components in 

MON 87712 were compositionally equivalent to the conventional control A3525 and 

within the range of variability of conventional commercial soybeans that were grown 

concurrently in the same field trial. These results support the overall conclusion that 

MON 87712 is unlikely to alter plant pest potential. 

IX.B 3. Phenotypic, Agronomic, and Environmental Interaction Characteristics 

Comparative plant characterization data was used to assess whether the introduction of 

the BBX32 protein altered the plant pest potential of MON 87712 compared to the 

conventional control A3525 (Section VII). Phenotypic, agronomic, and environmental 

interaction characteristics of MON 87712 were evaluated and compared to those of the 

conventional control (Section VII.B). As described below, these assessments included: 

seed dormancy and germination characteristics; plant growth and development 

characteristics; observations for abiotic stress response, disease damage, arthropodrelated damage; pollen characteristics; arthropod abundance; plant-symbiont interaction; 

volunteer potential and persistence outside of cultivation characteristics. Results from the 

phenotypic, agronomic, and environmental interaction assessments, as well as the 

volunteer potential and persistence outside of cultivation assessments demonstrated that 

MON 87712 possess neither weedy characteristics, nor increased susceptibility or 

tolerance to specific diseases, insects, or abiotic stressors, or altered symbiont interactions 

compared to conventional soybean. Taken together, the results of the analysis support a 

determination that MON 87712 is no more likely to pose a plant pest risk or have a 

biologically meaningful change in environmental impact than conventional soybean. 

IX.B.3.1. Seed Dormancy and Germination 

Seed dormancy and germination characterization demonstrated that MON 87712 seed 

had germination characteristics not different to those of the conventional control (Section 

VII.C). 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 or plant pest 

potential of MON 87712 compared to conventional soybean. 

IX.B.3.2. 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 (Section 

VIII). No statistically significant differences were detected (α = 0.05) between 

MON 87712 and the conventional control A3525 for seedling vigor, days to 50% 

flowering, days to 50% end of flowering, plant height, lodging, pod shattering, grain 

moisture, or 100 seed weight (Table VII-4). Five statistically significant differences were 

detected between MON 87712 and the conventional control A3525 for early stand count, 

days to 50% senescence, days to physiological maturity, final stand count, and yield. 

Although significantly different from the conventional control A3525, the mean values of 

MON 87712 for early stand count and final stand count were within the range of 
Monsanto Company 11-SY-217U 171 of 467 

commercial reference varieties for each characteristic and thus would not be adverse in 

terms of pest potential. Differences in days to 50% senescence, days to physiological 

maturity, and yield were consistent with the mode of action. The increase in yield is 

agronomically desirable and would not contribute to increased weediness potential of 

MON 87712 without changes in a combination of other characteristics associated with 

weediness (Baker, 1974). Thus, the observed differences are not considered to be 

biologically meaningful in terms of increased weediness or plant pest potential of 

MON 87712 compared to conventional soybean. 

IX.B.3.3. Response to Abiotic Stressors 

No biologically meaningful differences were observed during comparative field 

observations between MON 87712 and the conventional control A3525 and responses to 

abiotic stressors, such as drought, mineral and nutrient toxicity, and temperature stress 

(Section VIII). The lack of significant biologically meaningful differences in the 

MON 87712 response to abiotic stress support the conclusion that the introduction of the 

BBX32 protein is unlikely to result in increased weediness or plant pest potential 

compared to conventional soybean. 

IX.B.3.4. Pollen Morphology and Viability 

Evaluations of pollen morphology and viability from field-grown plants provide 

information useful in a plant pest assessment as it relates to the potential for gene flow 

and introgression of the biotechnology-derived trait into other soybean varieties and wild 

relatives. Pollen morphology and viability evaluations demonstrated no statistically 

significant differences between MON 87712 and the conventional control A3525. These 

comparative assessments indicate that MON 87712 is not likely to have increased plant 

pest potential compared to conventional soybean. 

IX.B.3.5. Interactions with Non-target Organisms Including Threatened and 

Endangered Species 

Evaluation of MON 87712 for potential adverse impacts on NTOs is a component of the 

plant pest risk assessment. Since MON 87712 is a product with no pesticidal activity, all 

organisms that interact with MON 87712 are considered to be NTOs. In a 2009 U.S. 

phenotypic and agronomic assessment, observational data on environmental interactions 

were collected for MON 87712 and the conventional control A3525. In addition, 

multiple commercial reference varieties were included in the analysis to establish a range 

of natural variability for each characteristic. The environmental interactions assessment 

(Section VIII) included data collected on plant-arthropod and plant-disease interactions. 

The results of this assessment indicated that the presence of BBX32 did not meaningfully 

alter plant-arthropod interactions, including beneficial arthropods and arthropod pests, 

nor did it alter disease susceptibility of MON 87712 compared to conventional soybean. 

The lack of meaningful differences in disease damage, arthropod-related damage, and 

pest and beneficial arthropod abundance demonstrate that the introduction of the BBX32 

in MON 87712 is unlikely to be biologically meaningful in terms of increased plant pest 

potential. 
Monsanto Company 11-SY-217U 172 of 467 

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. MON 87712 was assessed for changes 

in the symbiotic relationship with B. japonicum relative to the conventional control 

A3525 by evaluating shoot total nitrogen, nodule number, and nodule, root, and shoot dry 

weights (Section VIII). No statistically significant differences were detected between 

MON 87712 and the conventional control A3525 for the parameters measured, indicating 

no impact on either the symbiotic relationship or the symbiotic nitrogen-fixing bacteria. 

These data support a conclusion of no change in plant pest potential and no expected 

impact to cultivation practices relative to nitrogen inputs for MON 87712 compared to 

conventional soybean. 

The potential for MON 87712 to affect NTOs was evaluated using a combination of 

biochemical information and experimental data. The biochemical information and 

experimental data included molecular characterization, safety assessments of BBX32, 

results from the environmental assessment described above, and the demonstration of 

compositional, agronomic and phenotypic equivalence to conventional soybean. Taken 

together, these data support the conclusion that MON 87712 is unlikely to adversely 

affect NTOs, or pose an additional risk to threatened and endangered species above those 

posed by the cultivation of conventional soybean. 

Furthermore, according to APHIS, the only listed threatened or endangered animal that 

occupies a habitat where it 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 seaboard (USDA-APHIS, 2007). It 

is known to utilize certain agricultural lands readily, but 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 (NatureServe, 2010). The safety of the BBX32 

protein in MON 87712, the compositional, agronomic and phenotypic equivalence of 

MON 87712 to conventional soybean, and the diversity of the Fox Squirrel diet, support a 

conclusion that no biologically significant changes to the habitat or diet of the Delmarva 

Peninsula Fox Squirrel are expected. Consequently, the planting of MON 87712 is not 

expected to affect the Delmarva Peninsula Fox Squirrel. 

IX.B.3.4. Volunteer Potential and Persistence Outside of Cultivation 

Evaluations of volunteer potential and persistence outside of cultivation from field-grown 

plants provide information useful in assessing potential weediness characteristics of 

MON 87712 compared to the conventional control A3525 (Section VII.C). Volunteer 

potential evaluations demonstrated no statistically significant differences between 

MON 87712 and the conventional control A3525. The persistence outside of cultivation 

evaluations demonstrated a few statistically significant differences between MON 87712 

and the conventional control A3525, however these differences were not seen across the 

individual sites and were small in magnitude. Taken together, these comparative 

assessments indicate that MON 87712 is not likely to have increased weediness or plant 

pest potential compared to conventional soybean. 
Monsanto Company 11-SY-217U 173 of 467 

IX.C. Weediness Potential of MON 87712 

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), nor is 

it present on the lists of noxious weed species distributed by the federal government (7 

CFR Part 360). Soybean does not possess any of the attributes commonly associated 

with weeds (Baker, 1974), such as 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, which is a trait that has been removed from 

soybean through commercial breeding, soybean seed can germinate quickly under 

adequate temperature and moisture conditions, and potentially grow as volunteer plants. 

However, the volunteer potential evaluation demonstrated that plants of MON 87712 

were 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 does not exist for MON 87712 to outcross to wild or weedy relatives and to 

alter their weediness potential. 

In comparative studies between MON 87712 and the conventional control A3525, 

phenotypic, agronomic, and environmental interaction data were evaluated (Section VII) 

for changes that would impact the plant pest potential and, in particular, plant weediness 

potential. Results of these evaluations show that there is no biologically meaningful 

difference between MON 87712 and the conventional control A3525 for characteristics 

potentially associated with weediness. Furthermore, comparative field observations 

between MON 87712 and its conventional control A3525 in their response to abiotic 

stressors, such as drought, mineral and nutrient toxicity, and temperature stress, indicated 

no biologically meaningful differences and, therefore, no increased weediness potential. 

Data on environmental interactions also indicate that MON 87712 does not confer any 

biologically meaningful increased susceptibility or tolerance to specific diseases or insect 

pests. Collectively, these findings support the conclusion that MON 87712 has no 

increased weediness potential compared to conventional soybean. 

IX.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 population. 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 transgene(s) inserted into the biotechnologyderived plant, and the likelihood that the presence of the transgene(s) and their 

subsequent transfer to recipient plants will result in increased plant pest potential. The 

potential for gene introgression from MON 87712 is discussed below. 
Monsanto Company 11-SY-217U 174 of 467 

The assessment for gene introgression from MON 87712 with other cultivated or wild 

relatives of soybean, discussed in detail below, indicates that MON 87712 is no more 

likely to become a weed than conventional soybean, and MON 87712 is expected to be 

similar to conventional soybean regarding its potential for and impacts from gene flow. 

Soybean lacks sexually-compatible relatives in the U.S.; therefore, the only pollenmediated gene flow would be within cultivated soybean. 

IX.D.1. Hybridization with Cultivated Soybean 

Although soybean is largely a 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 varied depending on growing season and genotype. Insect 

activity does increase the outcrossing rate, but soybean generally is not a preferred plant 

for pollinators (Abrams et al., 1978; Erickson, 1975; Jaycox, 1970a; 1970b; 1970c) . 

Numerous studies on soybean cross-pollination have been conducted, and the published 

results, with and without supplemental pollinators, are summarized in Table IX-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% 

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: 0.05% at 5.4 meters (Ray et al., 2003), 0% at 6.5 meters (Abud et al., 2003), 

0% at 10.5 meters (Yoshimura et al., 2006), and 0.004% at 13.7 meters of separation 

(Caviness, 1966). 

The potential for cross-pollination in soybean 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, 2006). 

The consequence of introgression of BBX32 from MON 87712 into other soybean is 

negligible since soybean gene flow is naturally low; therefore the presence of BBX32 

confers no increased plant pest potential to cultivated soybean. 

IX.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 

(Hymowitz, 2004; Lu, 2004). Hybridization between female G. soja and male G. max

was less successful than hybridization in the opposite direction (Dorokhov et al., 2004), 

where frequency of spontaneous cross pollination in reciprocal combinations of G. max
Monsanto Company 11-SY-217U 175 of 467 

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 were used and no gene flow from G. 

max to G. soja was detected. Many barriers to natural hybridization exist 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. 

The subgenus Soja also contains an unofficial species, G. gracilis (Hymowitz, 2004). 

Glycine 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. Glycine gracilis may be a hybrid between G. soja and G. max

(Hymowitz, 1970; Lu, 2004). 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; Singh and Hymowitz, 1989). Although hybridization 

between G. max and members of the subgenus Soja can take place, G. soja is not found in 

North or South America, and it is highly unlikely that gene transfer will occur. 

IX.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 (Hymowitz et 

al., 1992; Hymowitz and Singh, 1992). Therefore, the only opportunities for intersubgeneric hybridization would occur in areas where those species are endemic. 

Nonetheless, the likelihood of interspecific hybridization between G. max and the wild 

perennial Glycine species is extremely low because they are genomically dissimilar 

(Hymowitz, 1970; Lu, 2004) 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 slow-growing, 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). 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. 

IX.D.4. Transfer of Genetic Information to Species with which Soybean Cannot 

Interbreed (Horizontal Gene Flow) 

Monsanto is unaware 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. Therefore, potential ecological risk 
Monsanto Company 11-SY-217U 176 of 467 

associated with horizontal gene flow from MON 87712 is not expected. The 

consequence of horizontal gene flow of the BBX32 from MON 87712 into other plants 

that are sexually-incompatible is negligible since, as data presented in this petition 

confirm, the gene and trait confer no increased plant pest potential to soybean. Thus in 

the highly unlikely event that horizontal gene transfer were to occur, the presence of 

BBX32 would not be expected to increase pest potential in the recipient species. 

IX.E. Potential Impact on Soybean Agronomic Practices 

An assessment of current soybean agronomic practices was conducted to determine 

whether the cultivation of MON 87712 has the potential to impact current soybean and 

weed management practices. Soybean fields are typically highly managed agricultural 

areas that are dedicated to crop production. MON 87712 is likely to be used in common 

rotations on land previously used for agricultural purposes. Certified seed production 

will continue to use well-established industry practices to deliver high quality seed 

containing MON 87712 to growers. Cultivation of MON 87712 is not expected to differ 

from typical soybean cultivation. 

MON 87712 is comparable to conventional soybean in its agronomic, phenotypic, 

ecological, and compositional characteristics and has levels of resistance to insects and 

diseases comparable to conventional soybean. Therefore, no significant impacts on 

current cultivation and management practices for soybean are expected following the 

introduction of MON 87712. Based on this assessment, the introduction of MON 87712 

is expected to have minimal impact on current U.S. soybean cultivation practices or weed 

management practices. 

IX.F. 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 IV through 

VIII of this petition confirm that MON 87712 is not different from conventional soybean 

in terms of pest potential in its phenotypic, agronomic, and environmental interaction 

characteristics. Monsanto is not aware of any study results or observations associated 

with MON 87712 that would suggest an increased plant pest potential would result from 

its introduction. 

The plant pest assessment was based on multiple lines of evidence developed from a 

detailed characterization of MON 87712 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 BBX32 expression cassette, 2) 

characterization of BBX32 protein expressed in MON 87712, 3) safety of BBX32 in 

MON 87712, 4) compositional equivalence of harvested MON 87712 seed and forage to 

conventional soybean, 5) phenotypic, agronomic, and environmental interaction 

characteristics demonstrating no increased plant pest potential compared to conventional 

soybean, 6) negligible risk to Non Target Organisms (NTO) and threatened or 

endangered species, 7) familiarity with soybean as a cultivated crop and the inherently 
Monsanto Company 11-SY-217U 177 of 467 

low plant pest potential of soybean, and 8) no unexpected impact to 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 

conventional soybean and currently deregulated biotechnology-derived soybean, 

MON 87712 is highly unlikely to be a plant pest. Therefore, Monsanto Company 

requests a determination from APHIS that MON 87712 and any progeny derived from 

crosses between MON 87712 and other commercial soybean be granted nonregulated 

status under 7 CFR Part 340. 

 
Monsanto Company 11-SY-217U 178 of 467 

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

Distance from 

Pollen Source 

(meters) 

CrossPollination (%) Comments Reference 

0.3 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 0.07 to 0.18 Adjacent rows. Experiment conducted over two 

years. Several male and female parental varieties. 

(Garber and 

Odland, 1926) 

0.1 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 0.2 to 1.2 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 

2.7–4.6 

6.4–8.2 

10–15.5 

0.03 to 0.44 

0.007 to 0.06 

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 alfalfa leafcutting bees. 

(Abrams et al., 

1978) 

0.1–0.6 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 0.09 to 1.63 Adjacent rows. Experiment conducted over two 

years. Several male and female parental varieties. 

(Ahrent and 

Caviness, 1994) 

0.5 

1.0 

6.5 

0.44 to 0.45 

0.04 to 0.14 

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 

5.4 

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 0.65 to 6.32 

(avg. 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 

1.4 

2.1 

2.8 

3.5 

7.0 

10.5 

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) 

 
Monsanto Company 11-SY-217U 179 of 467 

X. ADVERSE CONSEQUENCES OF INTRODUCTION 

Monsanto knows of no results or observations associated with MON 87712 or the 

BBX32 protein indicating that there would be an adverse environmental consequence 

from the introduction of MON 87712. MON 87712 contains BBX32 protein that interacts 

with one or more endogenous transcription factors to regulate the plant’s day/night 

processes and results in increased availability of assimilates in the plant resulting in 

increased yield of MON 87712 when compared to a comparator without the introduced 

gene. As demonstrated by field results and laboratory tests, the difference between 

MON 87712 and conventional soybean is the increased yield opportunity in MON 87712. 

The data and information presented in this petition demonstrate that MON 87712 is 

unlikely to pose an increased plant pest risk or have an adverse environmental 

consequence compared to conventional soybean. This conclusion is 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 analyses, which confirmed the insertion 

of a single functional copy of the BBX32 expression cassette at a single locus within the 

soybean genome. In addition, protein expression analysis demonstrates that BBX32 was 

detected in leaf and root of MON 87712, and was expressed at levels below the limit of 

detection in seed and forage. The BBX32 protein produced in MON 87712 is not novel 

and it has sequence homology with several different plant species, including the food 

crops citrus, grape, apple, soybean, rice, lettuce, and corn, where a history of safe use is 

established. Compositional analysis of key nutrients and antinutrients from seed and 

forage demonstrate that MON 87712 is compositionally equivalent to conventional 

soybean. Finally, extensive characterization of the plant phenotype and environmental 

interactions indicate that MON 87712 is comparable to conventional soybean. Therefore, 

based on the lack of increased pest potential or adverse environmental consequences 

compared to conventional soybean, the risks for humans, animals, and other NTOs from 

MON 87712 are negligible under the conditions of use. Additionally, the introduction of 

MON 87712 will not adversely impact cultivation practices or the management of weeds, 

diseases, and insects in soybean production systems. Moreover, the increased yield 

performance of MON 87712 is expected to have minimal impact on any of the 

agricultural practices farmers use to produce a soybean crop. 

Introduction of MON 87712 offers the opportunity to increase soybean yield, which is 

beneficial to growers and has potential to help global efforts to provide an adequate 

supply of soybeans, and thus, help sustain a robust domestic and global livestock market 

for soybean and soybean products. 
Monsanto Company 11-SY-217U 180 of 467 

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APPENDICES 
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Appendix A: USDA Notifications 

Field trials of MON 87712 have been conducted in the U.S. since 2006. 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 

MON 87712 phenotypic assessment data, 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 2009-

2010 seasons, are still in preparation. A list of trials conducted under USDA notifications 

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 and PermitsApproved for MON 87712 and Status of 

Trials Conducted under These Notifications 

USDA No. Effective Date Release Site (State) Trial Status

2006 Field Trials

06-146-102n 6/27/2006 PR Submitted to USDA

2007 Field Trials

07-018-103n 2/17/2007 IL (8), IN(3) Submitted to USDA

07-024-101n 3/18/2007 IA (4), KS(4) Submitted to USDA

07-094-104n 5/4/2007 IA (2) Submitted to USDA

07-261-101n 10/18/2007 PR Submitted to USDA

 

2008 Field Trials

08-030-103n 2/28/2008 PR Submitted to USDA

08-039-107n 3/9/2008 IA (5), IN (2), KS (4), MO Submitted to USDA

08-043-107n 3/13/2008 IA (3), IL (9), OH, IN Submitted to USDA

07-352-101rm 3/26/2008 IA (6), IL (6), IN (3), KS (3) Submitted to USDA

08-064-105n 4/3/2008 IL, IN Submitted to USDA

08-182-101n 8/1/2008 PR Submitted to USDA

08-253-101n 10/7/2008 PR (2) Submitted to USDA

08-316-101n 12/16/2008 PR Submitted to USDA

2009 Field Trials

09-007-106n 2/25/2009 PR Submitted to USDA

08-357-101rm 3/17/2009 IA (7), IL (7), IN (3), KS (3)

09-050-116n 3/21/2009 IN, MO (2), NE Submitted to USDA

09-072-106n 4/12/2009 IA (3), MO (7), NE Submitted to USDA

09-075-110n 4/15/2009 AR, IL (4), IN, KS, PA (2) Submitted to USDA

09-090-105n 4/30/2009

AR, IA, IL (3), IN (2), MO, KS, 

NE Submitted to USDA

09-100-102n 5/10/2009 IN Submitted to USDA

09-124-102n 6/3/2009 PR Submitted to USDA

09-162-106n 7/11/2009 PR Submitted to USDA

09-162-105n 7/11/2009 PR Submitted to USDA

09-177-110n 7/26/2009 PR Submitted to USDA

09-222-101n 9/9/2009 PR (2) Submitted to USDA

09-261-105n 10/18/2009 AR (2), IL, MO Submitted to USDA

09-247-101rm 11/17/2009 PR In Progress

09-292-107rm 12/16/2009 MO In Progress

 

2010 Field Trials 

09-355-101n 1/20/2010 PR In Progress

09-351-101rm 3/10/2010

IA (8), IL (7), IN (3), MO (2), 

KS (5) In Progress

10-068-112n 3/31/2010 IA (2), IL (2), IN (3), In Progress

10-067-101n 4/4/2010 IL (2), IN, MS, NE In Progress

10-073-103n 4/9/2010

AR, IA, IL (3), IN, KS, MO, NE, 

PA In Progress
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Table A-1. USDA Notifications and PermitsApproved for MON 87712 and Status of 

Trials Conducted under These Notifications (continued) 

USDA No. Effective Date Release Site (State) Trial Status 

10-073-104n 4/10/2010 

AL (2), AR (2), KS (4), MS, 

TX In Progress 

10-074-101n 4/11/2010 IA (2) In Progress 

10-074-109n 4/14/2010 IA In Progress 

10-082-106n 4/18/2010 FL, IL (3), KY In Progress 

10-083-103n 4/22/2010 IA In Progress 

10-089-104n 4/24/2010 

AR, IA (4), IN (4), MO (2), 

NE, PA In Progress 

10-085-107n 4/25/2010 KS, MN, MO, OH In Progress 

10-089-102n 4/28/2010 IL (5), NE, OH In Progress 

10-090-102n 4/29/2010 TN In Progress 

10-091-101rm 5/21/2010 PR In Progress 

10-175-102n 7/23/2010 PR In Progress 

10-257-101rm 11/9/2010 PR In Progress 

10-334-102n 12/30/2010 PR In Progress 
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Appendix B. Mode of Action of BBX32 Protein in MON 87712 

Crop yield results from a sequential growth and development process – first the plant 

grows vegetatively and produces photosynthetic tissue, followed by flowering and the 

production of seeds, and finally seed filling and maturation (Pedersen et al., 2007). Yield 

is a complex trait that is dependent on a number of genetic and environmental factors, 

that influence a crop’s opportunity to realize its full yield potential. Improvements in crop 

yield have been a primary focus of conventional breeding. The genetic changes that 

resulted in crop domestication and yield improvement in conventional varieties have been 

shown by modern molecular biology analysis to have been typically achieved through the 

selection and safe use of plant genes encoding transcriptional regulator proteins (Doebley 

et al., 2006). Agricultural biotechnology provides the opportunity to further enhance crop 

yields through the introduction of new genetic elements that use or modify existing 

pathways in the plant. The yield increase in MON 87712 is achieved using the BBX32

gene from the plant Arabidopsis thaliana that produces a protein that interacts with one 

or more endogenous transcription factors to regulate the plant’s day/night processes and 

results in increased availability of assimilates in MON 87712 compared to a near isogenic 

comparator without this gene. Plant nutrient assimilation and utilization are known to be 

critical processes to drive yield improvement (Kumudini, 2002; Sinclair et al., 2004). 

Increased assimilate availability in MON 87712 is supported by the measurement of 

factors indicative of an extended period of photosynthetic activity in MON 87712 and 

evidence of changes in diurnal metabolism during the reproductive phase of the soybean 

plant, as well as by the significantly higher yield of MON 87712 when compared to 

control, as observed in multisite field studies in the U.S. 

To understand the mode-of-action of BBX32 protein in MON 87712, it is important to 

understand the biological processes associated with yield and the impact of diurnal 

biology on those processes in soybean. Analysis of yield improvement and yield 

determination in conventional soybean has shown that the same basic principles and 

mechanisms apply for MON 87712. Through highlighting yield limitation in 

conventional soybean as a function of carbon and nitrogen availability that are controlled 

by diurnal processes, we demonstrate how the plant BBX32 protein affects these existing 

diurnal processes in soybean to increase yield in MON 87712. 

B.1. Improvement in Grain Yield is a Major Objective for Soybean Breeders 

Soybean crop yields have risen consistently in North America since the 1920s. In the 

U.S., soybean yield rose at an average annual rate of approximately 0.35 bu/A (0.8%) 

between 1924 – 2010 (Figure B-1), and similar yield increases have been reported in 

Canada. A survey of soybean yield in Canada between 1934 – 1992 revealed an average 

increase in yield of 0.5%, with evidence that since 1976 the rate of genetic improvement 

of seed yield is accelerating (Voldeng et al. 1997; Morrison et al., 1999). Annual 

improvement in soybean yields is attributable to rapid producer adoption of repetitive 

waves of agricultural innovation in the form of genetic and agronomic improvement that 

provide producers improved means for reducing “on-farm” yield constraints (Specht et al, 

1999). 
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Figure B-1. U.S. soybean yield rose at an annual average rate of 0.35 bu/A between 

1924 – 2010. Linear regression analysis was conducted on data from the USDA National 

Agricultural Statistics Service (USDA-NASS, 2011, http://www.nass.usda.gov). 

Soybean yield trends in the U.S. indicate that yield growth rates have not reached a 

plateau. Average soybean yield in 2010 was 43.5 bu/A (2900 kg/ha), but record yields 

reported from yield contests in the U.S. (Iowa, Missouri and Nebraska, 1966-1998) were 

greater than 67.5 bu/A (4500 kg/ha) and in one instance reached 100 bu/A (6660 kg/ha) 

(Specht et al., 1999), demonstrating that future yield growth is possible. The concept of 

yield potential of soybean is defined as the yield of a cultivar when grown in 

environments where it is adapted, with nutrients and water non-limiting, and with pests, 

diseases, weeds, lodging, and other stresses effectively controlled (Evans and Fisher, 

1999; Specht et al., 1999). Conventional soybean yield potential has been estimated to be 

approximately 120 bu/A (8000 kg/ha) using crop simulation models (Specht et al., 1999). 

The U.S. average in 2010 of 43.5 bu/A (2900 kg/ha) was substantially below the current 

estimate of yield potential, suggesting that there is an opportunity to close the gap 

between average annual yield performance and yield potential. 

 Seed Yield (bu/A)

Production year
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B.2. Soybean Yield is Frequently Limited by the Availability of Assimilates 

Plants derive nutrition through critical processes such as photosynthesis (carbon 

assimilation) and absorption of raw materials such as nitrogen (nitrogen assimilation) to 

drive yield improvement (Kumudini, 2002; Sinclair et al., 2004). Soybean yield is the net 

result of metabolic assimilate availability within the plant (the source) and translocation 

of these assimilates to the developing seed (the sink) where these assimilates are used to 

synthesize storage compounds such as protein, oil and starch (Egli, 1999). Soybean yield 

can be limited by the activity of the source or by the ability of the seed to utilize the 

available assimilate produced by the source and convert into dry weight (Egli, 1999). 

This division recognizes the two major developmental processes involved in the 

accumulation of yield, the production of assimilate in the leaves (the source) and 

utilization of this assimilate by the developing seed (the sink). 

Soybean plant development can be separated into two major generally overlapping 

developmental phases: vegetative and reproductive. The duration of these phases is 

controlled primarily by genetics, temperature, and day length (Pedersen, 2007). Soybean 

producers influence the duration of these phases through variety selection, geographic 

location, and planting date. Yield production begins with vegetative growth when the 

formation of organs for nutrient absorption and photosynthesis provides the machinery to 

produce yield. The reproductive phase is typically the most important for yield 

determination and is divided into eight reproductive (R) stages (Fehr and Caviness, 1981) 

(Table B-1). Fehr and Caviness (1981) classify reproductive development based on 

flowering, pod development, seed development and plant maturation stages. The first two 

stages (R1 and R2) refer to flowering stages. The next two stages (R3 and R4) refer to 

pod development. Seed development begins when the pod approaches its maximum size. 

The R5 and R6 stages refer to seed development phases, whereas the R7 and R8 stages 

refer to phases of plant maturation. 

 
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Table B-1. Soybean reproductive stages as defined by Fehr and Caviness (1981) 

Stage ID Description of Developmental Stage 

R1 Beginning bloom — One open flower at any node on the main stem. 

R2 Full bloom — An open flower at one of the two uppermost nodes on the 

main stem with a fully developed leaf. 

R3 Beginning pod — Pods are 3/16 inch (5 mm) at one of the four uppermost 

nodes on the main stem with a fully developed leaf. 

R4 Full pod — Pods are 3/4 inch (2 cm) at one of the four uppermost nodes 

on the main stem with a fully developed leaf. 

R5 Beginning seed — Pod at one of the four uppermost nodes on the main 

stem contains seeds that are 1/8 inch (3 mm) long. 

R6 Full seed — Pod at one of the four uppermost nodes on the main stem 

contains green seeds that fill the pod cavity. 

R7 Beginning maturity — One normal pod on the main stem has reached its 

mature pod color. 

R8 Full maturity — 95 percent of the pods have reached their full mature 

color. 

Final crop yield is a function of the number and size of seeds produced (Figure B-2). The 

period from R1 to R6 stages is critical for yield, because this is when both pod and seed 

number are set. The period between the R5 stage and onset of the R7 stage is important in 

setting seed weight. Because pod development begins at the R3 stage and seed growth 

ends at the R7 stage, conditions that limit growth during this period can impact yield by 

limiting seed number, seed weight, or both (Pedersen, 2007). During the reproductive 

phase, the number and size of seeds is limited by the capacity and efficiency of the 

soybean canopies to produce and translocate assimilate (Egli, 1999). Canopy-level 

photosynthetic rates provide the best estimate of assimilate availability at a given time 

(Long, 2006). The rate of canopy photosynthesis is determined by leaf area index 

(Westgate, 2001), the photosynthetic capacity of the leaves, and environmental 

conditions. 
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Figure B-2. Determination of yield in conventional soybean. 

Soybean yield is a function of the number and size of seeds. The period from the R1 stage 

(flower initiation) to the R6 stage (full seed) is critical for yield determination, because 

this is when both seed number and seed weight are determined. The developing seed (the 

sink) receives assimilates from the plant (the source). 

Soybean plants are able to compensate for canopy gaps by increasing branch production 

per plant, improving leaf area index, therefore even large increases in plant stand may 

result in little to no yield increase (Board 2000; Carpenter and Board 1997). A wide range 

of seeding rates (100,000-200,000 plants/acre) that are used agronomically have been 

shown to have relatively little influence on soybean yield (Board 2000; Butler et al. 2010; 

De Bruin and Pedersen 2009). Specifically, in a multi-year and multi-site study, less than 

5% difference in soybean yield was reported for a final plant stand increase of 80% (De 

Bruin and Pedersen 2008). Thus, minor differences in plant stand would not be expected 

to substantially affect yield when plant stands are near agronomically acceptable levels. 

Under a broad range of environmental conditions, soybean yield is more frequently 

limited by the amount of translocated assimilates from the source than from sink 

limitation (Egli, 1999). Reducing source capacity through shading or partial defoliation 

during reproductive growth stages is known to result in decreased yield (Board and Tan, 

1995; Board et al., 1995; Egli and Zhen-wen, 1991). Reducing source capacity early in 

the reproductive growth cycle (R3 to R4) decreases seed number while reducing source 

capacity during later growth stages (R5 to R6.5) decreases individual seed size (Board 
Monsanto Company 11-SY-217U 205 of 467 

and Tan, 1995). Thus, increasing the availability of assimilates at the source is a potential 

mechanism to increase yield in soybean. 

B.3. Diurnal Processes Control Assimilate Availability in Plants 

Plant growth and development responds to the diurnal cycling of light and dark. This is 

manifested both at the physiological level, with changes in plant metabolism and 

assimilate availability, and at the molecular level, with expression of some genes 

occurring only at certain times of the day (Harmer et al, 2000; Schaffer et al, 2001). The 

day/night cycling of plant processes is called a diurnal rhythm and is achieved primarily 

by two mechanisms: first, by light, and second, by an internal circadian clock (Schaffer et 

al, 2001). Coordination of diurnal processes such as nitrogen and carbon metabolism 

which affect assimilate availability and plant growth have been shown to be necessary for 

maintaining plant productivity (Smith and Stitt, 2007; Gutierrez et al, 2008; Graf et al, 

2010). 

The first mechanism to consider in diurnal processes is light signaling. Light is one of 

the most important environmental factors for plants as it provides the source of energy for 

plant life. The ability of plants to respond to light is achieved through photoreceptors 

(Jiao et al, 2007). Plants detect a range of light intensities and wavelengths via 

photoreceptors, and subsequently convert the light signal into physiological responses 

through transcriptional regulation (Jiao et al, 2007). Light-responsive transcription 

factors, such as LONG HYPOCOTYL5 (HY5), mediate light signaling through the 

coordinated activation and repression of specific plant genes (Lee et al, 2007). The ability 

to sense a light signal allows the plant to respond to recurring diurnal cycles and thus 

control growth and development throughout vegetative and reproductive stages (Jiao et 

al, 2007). 

The second mechanism in plant diurnal processes is the circadian clock, which oscillates 

with an approximate 24-h period in the absence of external stimuli and thus allows plants 

to anticipate daily changes in the environment, such as the onset of dawn (Eckhardt, 

2005; Graf et al, 2010). Such anticipation allows plants to time internal biological 

processes to the part of the 24-h cycle that would most benefit from interaction with the 

external stimuli occurring at any given time of day (Millar, 2004). Thus, the circadian 

clock acts at an interface in the signaling network between environmental response 

pathways and internal programs (Millar, 2004). The circadian clock is continually 

modified by light via a mechanism involving the HY5 transcription factor, which helps 

the plant to regulate diurnal processes to maximize productivity in any given environment 

(Devlin and Kay, 2001; Eckhardt, 2005; Li, 2011). The core components of the circadian 

clock are two closely related transcription factor proteins CIRCADIAN CLOCK 

ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and a 

response regulator protein TIMING OF CAB EXPRESSION 1 (TOC1) (Salome and 

McClung, 2004). Diurnal regulation of CCA1, LHY and TOC1 proteins are responsible 

for generating self sustained rhythmicity in the plant. Genes regulated by diurnal output 

pathway have been shown to peak in expression at different times of the day/night and 

are associated with circadian biology, nitrogen and carbon metabolism (Harmer et al, 

2000). 
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Figure B-3. Diurnal cycling of processes such as nitrogen and carbon metabolism in 

plants is achieved through light signaling and the circadian clock 

The day/night cycling of plant processes is called a diurnal rhythm and is achieved by 

light signaling and the circadian clock. The circadian clock is continually modified by 

light input, which helps the plant optimize diurnal processes. Both light signaling and the 

circadian clock are regulated, in part, by the ELONGATED HYPOCOTYL5 (HY5) 

transcription factor. Genes regulated by the diurnal pathway have been shown to peak in 

expression at different times of the day/night and control biological processes such as 

nitrogen and carbon metabolism. 

Light and the circadian clock control diurnal processes such as carbon metabolism. 

During the day, plants absorb light and stomates open to facilitate CO2 assimilation and 

water transpiration (Blasing et al., 2005). Carbon dioxide is fixed into sugars by 

photosynthesis during the day to support metabolism, storage, and plant growth (Blasing 

et al., 2005). The entire supply of assimilated carbon is not used immediately for growth. 
Monsanto Company 11-SY-217U 207 of 467 

Newly assimilated carbon provides for both the immediate demand and, via the 

accumulation of temporary storage compounds such as starch in leaves, an anticipated 

demand during the following night (Smith and Stitt, 2007). Changes in environmental 

conditions result in adjustments of assimilation and storage that serve to maintain 

adequate carbon supply throughout the day and night (Smith and Stitt, 2007). For 

example, changes in day length result in alterations in both the partitioning of assimilate 

between starch and sucrose during the day, and the rate of starch degradation to sucrose 

for remobilization at night (Smith and Stitt, 2007). In soybean, the rate of starch 

synthesis is inversely related to day length so that the proportion of assimilate partitioned 

as starch for use at night is greater when the day length is shortened (Chatterton and 

Silvius 1979). This concept is demonstrated when soybean plants are grown under 

different light/dark cycles. Conventional soybean plants grown with a 14h light period 

partitioned 60% of assimilate into starch whereas plants grown with a 7h light period 

partitioned 90% of assimilate into starch (Chatterton and Silvius 1979). Thus, the control 

of assimilate supply for growth is a major function of diurnal mechanisms in plants. 

Soybean reproductive development responds to day/night length after flowering 

(Summerfield et al, 1998; Kantolic and Slafer, 2005). Under field conditions, Kantolic 

and Slafer (2005) showed that artificially extended day length post-flowering extends the 

R3–R6 growth phase in conventional soybean. Exposing plants to extended photoperiod 

increased the number of nodes per plant and improved node fertility, thus increasing the 

number of pods and seeds produced per unit area. Average seed weight tended to be 

reduced by 20% in plants exposed to extended photoperiod while seed number was 

increased by more than 75% (Kantolic and Slafer, 2005). The effect on seed number and 

seed size is hypothesized to include mechanisms related to carbon metabolism and 

assimilate availability (Kantolic and Slafer, 2005). Alteration of diurnal processes 

through genetic means therefore offers the potential for future yield improvements 

through optimization of post-flowering reproductive development. 

B.4. Crop Domestication and Improvement through Breeding has been Largely 

Achieved Through Selection and Safe Use of Transcriptional Regulator Proteins 

Introduced proteins that modulate plant gene expression operate through regulation of 

endogenous plant pathways and processes. Normally occurring variations in these 

pathways are likely to occur in response to genetic and environmental factors (Kier and 

Petrick, 2008). In addition, molecular biology analysis has recently shown that 

modulation of regulatory processes has been fundamental to crop domestication and 

breeding of plant varieties. Several of the genetic changes that control important 

domestication traits in maize and rice are transcription factors, as are some of the genes 

controlling varietial differences (Doebley et al, 2006). For example, conventional wheat 

varieties with specific alterations in the transcription factor Rht-1 are shorter and show 

increased yield (Peng et al., 1999). Thus, the safety of food or feed from biotechnologyderived crops that have a mechanism of action based on gene expression modulation 

should be considered in the context of the history of safe consumption of food and feed 

derived from conventionally bred plants grown under a range of environmental 

conditions (Kier and Petrick, 2008). 
Monsanto Company 11-SY-217U 208 of 467 

B.5. Plant BBX32 Protein Expression Affects Diurnal Processes in MON 87712 

Leading to Increased Yield 

The MON 87712 soybean line was produced through the insertion of the Arabidopsis 

thaliana BBX32 gene in a parental conventional soybean plant with genetic background 

A3525. BBX32 expression in Arabidopsis thaliana is known to regulate plant gene 

expression and repress plant responses to the transition from dark to light (Holtan et al, 

2011). In this section, we demonstrate how the plant BBX32 protein affects existing 

diurnal processes in soybean to increase yield in MON 87712. Mode-of-action studies 

were conducted to understand BBX32 function in MON 87712 using two controls: the 

parental conventional control soybean variety, A3525, and a near isogenic negative 

segregant control line [MON 87712(-)]. The MON 87712(-) control was included to 

allow most precise determination of a trait effect while reducing to the minimum any 

variability associated with the background genetics of A3525. In a field study across 

multiple sites in the U.S. soybean production area in 2009, a statistically significant 

increase in harvested seed weight per area was observed for MON 87712 compared to 

both controls. Given the importance of controlling variability when measuring impacts on 

yield, our primary focus for the determination of the trait effect on yield was on the 

comparison of MON 87712 with the near isogenic negative segregant control 

MON 87712(-). Furthermore, analysis of leaf area, light interception, and leaf 

photosynthetic rate demonstrates that MON 87712 has higher canopy-level assimilate 

availability during the seed-filling period. 

To examine the effect of BBX32 protein expression on diurnal processes in MON 87712, 

targeted analysis was conducted on a group of soybean genes and metabolites associated 

with diurnal biology and primary carbon and nitrogen metabolism. BBX32 protein 

expression is shown to modulate the response of MON 87712 to the plant’s transition 

from night to day by repressing light signaling. Temporal-specific differences in levels of 

metabolites involved in carbon and nitrogen metabolism, metabolites indicative of source 

capacity, and the activity of an enzyme involved in carbon metabolism were observed 

when MON 87712 was compared to A3525 and MON 87712(-) controls. Increased plant 

assimilate availability is associated with improved yield in conventional soybean and the 

mode-of-action for MON 87712 is thus similar to that observed for historical increases in 

soybean yield achieved using traditional breeding methods. 

B.5.1. MON 87712 Delivered Significantly Increased Yield when Compared to 

Conventional Soybeans of the Same Genetic Background 

Soybean seed yield is expressed as a function of the primary yield components: final 

stand count per area, seed number per plant, and individual seed weight. According to 

Mullen (1996) yield is expressed as: 

ݐ݄݁݅݃ݓ൬ Yieldݏݐ݈ܽ݊݌൬ ݐ݊ݑ݋ܿ ܽ݊݀ݐܵ ൌ ൰ ݁ܽݎܽ

ܽݎ݁ܽ ൰ ݔ Seed number per plant ൬ݏ݁݁݀ݏ

ݐ݄݁݅݃ݓ൬ weight seed Individual ݔ ൰ݐ݈ܽ݊݌

൰ ݁݁݀ݏ

Yield and yield component characteristics of MON 87712 were compared to two 

controls: the parental conventional control population, A3525, and a near isogenic 
Monsanto Company 11-SY-217U 209 of 467 

negative segregant control line [MON 87712(-)] in U.S. 2009 field trials (Table III-2 and 

Table III-3) (Appendix C). Soybean variety A3525 is the parental line to MON 87712 

and was used as the conventional soybean comparator in the safety assessment of 

MON 87712, including the plant pest risk evaluation. The MON 87712(-) control line 

was identified at the R1 generation during the MON 87712 breeding process and was 

specifically included in the 2009 study as the primary comparator for yield endpoints to 

allow determination of a trait effect while reducing the variability associated with the 

background genetics of mixed populations of A3525. 

Nineteen sites across the U.S. soybean production area were planted at a constant seeding 

rate of approximately 156,816 seeds per acre, and yield and yield components were 

measured. Three statistically significant differences were detected (p<0.05) between 

MON 87712 and the MON 87712(-) control in the combined-site analysis (Table B-2). 

Consistent with the intended phenotype, a statistically significant increase in yield was 

observed for MON 87712 compared to MON 87712(-). Corresponding increases for 

MON 87712 compared to MON 87712(-) were observed in the primary yield 

components. A statistically significant increase in final stand count and numerical 

increases in individual seed weight and seed number per plant were observed for 

MON 87712 compared to MON 87712(-). In addition, numerical increases for 

MON 87712 compared to MON 87712(-) were observed in the calculated yield 

characteristics seed number per plot and seed weight per plant. Similar results were 

observed when comparing MON 87712 and the A3525 parental conventional control 

(Table B-3). 

Results of this study across a range of sites support the conclusion that the introduced 

trait in MON 87712 significantly increased yield when compared to near isogenic control 

soybeans concurrently grown under the same conditions. Yield is defined as the seed 

weight produced per unit area, and MON 87712 produced more seed weight per unit area 

when compared to control. Corresponding numerical increases in yield components are 

consistent with the statistically significant increase in yield.
Monsanto Company 11-SY-217U 210 of 467 

Table B-2. Combined-Site Least Square Means, P-values, and Percent 

Differences of Yield and Yield Components of MON 87712 Compared to the 

Negative Segregant Control [MON 87712 (-)] from U.S. 2009 Field Trials (19 sites) 

Characteristic (units)

Least Squares Mean 

MON 

87712 MON 87712(-) P-Value % Difference

 

Yield (bu/ac) 52.6* 47.9 0.0054 11.41% 

Final Stand Count (plants/plot) 292.1* 283.2 0.0076 3.69% 

Seed Number Per Plant (seeds/plant) 69.6 66.8 0.2622 4.74% 

Individual Seed Weight (g) 0.163 0.160 0.0506 3.60% 

 

Seed Weight Per Plot (g) 3284.7 2994.5 0.0054 11.41% 

Seed Number Per Plot (seeds/plot) 20235. 18866.8 0.0627 7.02% 

Seed Weight Per Plant (g) 11.3 10.7 0.0752 8.24% 

 

* Indicates a statistically significant difference between MON 87712 and MON 87712(-) 

(α=0.05). 
Monsanto Company 11-SY-217U 211 of 467 

Table B-3. Combined-Site Least Square Means, P-values, and Percent 

Differences of Yield and Yield Components of MON 87712 Compared to the 

Conventional Control, A3525 from U.S. 2009 Field Trials (19 sites) 

Characteristic (units)

Least Squares Mean 

MON 87712 A3525 P-Value % 

Difference

 

Yield (bu/ac) 52.6* 49.0 0.0100 7.29% 

Final Stand Count (plants/plot) 292.1 288.1 0.1370 1.37% 

Seed Number Per Plant (seeds/plant) 69.6 67.7 0.3430 2.75% 

Individual Seed Weight (g) 0.163 0.159 0.0596 2.43% 

 

Seed Weight Per Plot (g) 3284.7* 3061.6 0.0100 7.29% 

Seed Number Per Plot (seeds/plot) 20235.1 19302.0 0.0635 4.83% 

Seed Weight Per Plant (g) 11.3 10.8 0.0905 5.43% 

 

* Indicates a statistically significant difference between MON 87712 and A3525 

(α=0.05). 
Monsanto Company 11-SY-217U 212 of 467 

Regarding the statistical difference in stand count observed between MON 87712 and 

controls, it is important to highlight that soybean plants are able to compensate for 

canopy gaps by increasing branch production per plant and thus that minor differences in 

soybean stand are unlikely to be a significant cause of yield differences at typical plant 

densities. Indeed, a wide range of seeding rates (100,000-200,000 plants/acre) have 

relatively little influence on soybean yield (Board 2000; Butler et al. 2010; De Bruin and 

Pedersen 2009). The means of the final stand counts of MON 87712 and the control plots 

ranged from 123,379 plants/acre (283.2 plants/plot) to 127,234 plants/acre (292.1 

plants/plot). These values are within the wide range of seeding rates and stand counts 

that have relatively little influence on soybean yield (De Bruin and Pedersen, 2009). 

Based on the compensatory aspect of soybean growth, the increase in yield for 

MON 87712 observed in the U.S. field study in 2009 was greater than would be expected 

from differences in stand count alone; thus, seed number and seed weight have clearly 

contributed to the yield increase. The data for seed number and seed weight are consistent 

with the overall increase in yield. 

As MON 87712 exhibited statistically significant differences in yield compared to control 

(Tables B-2 and B-3), an assessment was made in Section VII to evaluate whether any of 

the differences observed between MON 87712 and the conventional control A3525 

would indicate that MON 87712 may be more likely to become a weed than conventional 

soybean. The phenotypic, agronomic and environmental interactions assessment (Section 

VII) showed few statistically significant differences between MON 87712 and the 

conventional control A3525. Small increases in early plant stand (302.7 vs. 297.0 plants 

per plot) and increases in final plant stand (296.8 vs. 286.4 plants per plot) were 

observed. These differences were of small relative magnitude when compared to 

commercial conventional reference varieties grown concurrently, therefore, there were no 

changes indicative of increased weediness or plant pest potential (Baker, 1974). Not 

unexpectedly from the trait, there were statistically significant increases in yield (52.6 vs. 

49.0 bu/ac). Although the yield difference is biologically meaningful from a grower 

perspective, the increase in yield is not indicative of increased weediness or plant pest 

potential for environmental risk safety. 

B.5.2. Increased Canopy Level Source Capacity in MON 87712, Resulting in 

Increased Assimilate Availability 

Genetic improvement in soybean yield through traditional breeding methods have been 

shown to be associated with increased canopy photosynthesis (assimilate availability) 

during the reproductive period (Kumudini, 2002). The source capacity of a crop canopy 

is linearly related to the ability of a canopy to intercept solar radiation, and the ability to 

convert intercepted light into biomass (Monteith, 1977). The ability of the sink to utilize 

the assimilates produced at the source are related to the ability of the crop to partition its 

resources into seed (Beale and Long, 1995; Monteith, 1977). Light interception 

efficiency is measured directly by measuring the photosynthetic photon flux density 

above and below the canopy and calculating the proportion of light interception. Light 

interception is closely related to leaf area index, defined as the amount of leaf area per 
Monsanto Company 11-SY-217U 213 of 467 

unit land area measured in m2 leaf area per m2 land area. Conversion efficiency of 

intercepted light into biomass is typically estimated based on light-saturated 

photosynthetic rate at a single stage of crop development, however it is better to estimate 

conversion efficiency based on integrated whole-canopy photosynthesis (Long et al, 

2006). Measurement of the parameters that determine the canopy-level photosynthetic 

rate for MON 87712 and its near isogenic control provides an indirect indication of the 

difference in assimilate availability in the plant, as a result of the action of BBX32 in 

MON 87712. 

Measurements of leaf area index, light interception efficiency, and photosynthetic rate 

were collected during the seed-fill period (R4, R5 and R6 stages) in order to understand 

the canopy-level source capacity of MON 87712 compared to the conventional control, 

A3525, and to the MON 87712(-) control during the most important growth stages for 

yield determination in soybean (Figure B-4 and Figure B-5) (Appendix C). MON 87712 

had significantly higher leaf area index than the MON 87712(-) at the R4 stage, and 

significantly higher leaf area index than both controls at the R5 and R6 stages (p<0.05 in 

all cases) (Figure B-4). MON 87712 had significantly higher light interception than the 

MON 87712(-) at the R5 stage, and had higher light interception than both controls at the 

R6 stage. At the R4 growth stage, the increased leaf area index of MON 87712 is 

counteracted by a decrease in midday leaf photosynthetic rate (Figure B-5). At midday of 

the R5 growth stage, MON 87712 had significantly higher photosynthetic rate. At the R6

growth stage, MON 87712 had significantly higher photosynthetic rate than both controls 

at all of the measurement timepoints at the R6 stage (Figure B-5). 

Taken together, the increased leaf area index and light interception combined with 

increased leaf photosynthetic rate late in crop development indicate that MON 87712 had 

higher canopy-level assimilate availability during the seed-filling period (R5-R6/R7) that 

is critical to yield determination in soybean. Increased source capacity has been 

associated with improved yield in conventional soybean and the mode of action for 

MON 87712 is thus similar to that observed for historical increases in soybean yield 

achieved using traditional breeding methods (Kumudini, 2002). 

Apart from the intended effect on increased yield, increased canopy level assimilate 

availability in MON 87712 is related to two statistically significant differences observed 

between MON 87712 and the parental conventional control A3525 for phenotypic and 

agronomic data characteristics. Small increases in days to 50% senescence (267.7 vs. 

265.0 days) and increases in days to physiological maturity (280.8 vs. 277.5 days) were 

observed. Although these differences could be expected and are biologically meaningful 

with regard to the increased source capacity of MON 87712, they were of small relative 

magnitude when compared to natural variability and are therefore not indicative of 

increased weediness or plant pest potential from an environmental risk perspective, as 

discussed in Section VII. 
Monsanto Company 11-SY-217U 

Figure B-4. Leaf Area Index and Light Interception Leaf Area Index and Light Interception Efficiency data for MON 87712 (open circles), A3525 (dark circles), and (dark triangles) at the R4, R5 and R6-R7 growth stages within the thinned treatment. Subsamples of 2 measurem

collected on each of the measurement dates. Values represent arithmetic means ± 1 SE. Significant differences band MON 87712 (-) (

α=0.05) at a given measurement date are denoted by †. Significant differences between MO

(α=0.05) at a given measurement date are denoted by *. 

Growth Stage

R4 R5 R6-R7

Leaf Area Index (m2/m2)

02468

Leaf Area Index

R4 R5 R6-R7

A3525MON 87712

Light Interception MON 87712 (-

*

*

* †

†

†

†

†
Monsanto Company 11-SY-217U 

Figure B-5. Leaf Photosynthetic Rate Data Leaf photosynthetic rate of MON 87712 (open circles), parental conventional control A3525 (dark circles) and M

triangles) measured at morning, midday and afternoon timepoints at the R4, R5 and R6-R7 stages. Values repres

± 1 SE. Significant differences between MON 87712 and the MON 87712 negative segregant (p<0.05) at a give

are denoted by †. Significant differences between MON 87712 and the conventional control A3525 (p<0.05) at 

date are denoted by *. 

R4

morning midday afternoon

Photosynthetic Rate 

(mol CO2/m2/s)

0

10203040

A3525MON 87712MON 87712 (-)

*

R5

Approximate Time of Da

y

morning midday afternoon

*

R6-R7

morning midday afternoo

* * *† † †

† †
Monsanto Company 11-SY-217U 216 of 467 

B.5.3. Plant BBX32 Protein Modulates Diurnally Regulated Gene Transcription 

Plant BBX32 is a member of the B-box zinc finger family from Arabidopsis thaliana. 

This family represents a subgroup of zinc finger proteins that contain one or more B-box 

domains with specialized tertiary structures that are stabilized by binding zinc ions. The 

B-box domain is predicted to be involved in protein-protein interactions (Khanna et al., 

2009). BBX32 contains a single annotated protein domain, the B-Box B1 domain 

(Khanna et al., 2009). The B-box zinc finger family is found in many plant species; for 

example, the soybean B-box family contains 61 genes (Preuss et al., submitted for 

publication). Homologs of BBX32 are found in many agronomically-important species, 

suggesting that the function of BBX32 in its source plant, Arabidopsis thaliana, is 

conserved in other plant species. 

Transcriptional regulation of eukaryotic genes is an orchestrated process that requires the 

concerted functions of multiple proteins. In addition to the ubiquitous general 

transcription factors, such as RNA polymerase II, there are target-specific DNA-binding 

transcription regulators which modulate gene expression in response to developmental or 

environmental cues, as well as co-regulator (syn accessory) proteins that interact with 

transcription factors to affect their activity (Martinez, 2002). In Arabidopsis thaliana, 

BBX32 participates in the regulation of genes involved in plant diurnal processes. 

BBX32 protein is a repressor of light signaling in Arabidopsis thaliana, functioning as an 

accessory protein involved in plant gene transcription (Holtan, et al. 2011). 

Transcriptional accessory proteins such as BBX32 assist the function of another protein 

through protein-protein interactions, and, while not directly involved in contacting DNA, 

aid transcriptional regulation (Martin, 1991). 

Mode of action analyses in Arabidopsis thaliana show that BBX32 expression represses 

plant responses to the transition from dark to light via interaction with the HY5 

transcriptional complex (Holtan et al, 2011). The relationship between BBX32 and 

repression of the HY5 function is evidenced by the phenotypic similarity between 

Arabidopsis thaliana seedlings overexpressing BBX32 and a hy5 loss of function mutant. 

Both BBX32 overexpression lines and hy5 mutants exhibit elongated hypocotyls when 

grown in the light (Holtan et al, 2011). Expression levels of specific genes targeted by 

HY5 for induction are reduced when BBX32 is overexpressed in Arabidopsis thaliana, 

which correlates with BBX32 repression of the HY5 complex (Holtan et al. 2011). 

To investigate the function of BBX32 in MON 87712, targeted analysis of a group of 

soybean genes was conducted to determine the impact of expression of BBX32 on the 

mRNA level of certain diurnally regulated genes in MON 87712. The genes analyzed 

were selected based on their potential involvement in the mode of action for BBX32 

leading to increased yield, and included genes associated with circadian biology, carbon 

and nitrogen metabolism, and phytohormone function. Expression analysis was 

conducted on tissue samples taken from growth chamber and field grown plants, and the 

expression level of each gene in MON 87712 was compared to two soybean controls, the 

parental conventional control A3525 and the negrative segregant control MON 87712(-). 

The MON 87712(-) is a near isogenic soybean line that does not contain the MON 87712 

T-DNA insert. Sampling times in both the field and the growth chamber studies were 
Monsanto Company 11-SY-217U 217 of 467 

selected to evaluate the temporal specific impact of BBX32 protein expression on the 

expression of the analyzed soybean genes. 

The function of BBX32 protein in soybean is reflected in significant differences (p<0.05) 

in the expression level of two examples of diurnally regulated genes at dawn. For 

example, the LHY gene, encoding a component of the circadian clock, and the BBX13

gene, a B-box gene with similarity to CO-like genes in Arabidopsis thaliana (Preuss et 

al., submitted for publication), both show changes in expression in MON 87712 

compared to the controls at the onset of daylight (Figure B-6). LHY mRNA levels were 

significantly reduced (2.5 fold) one hour pre-dawn, whereas BBX13 mRNA levels were 

significantly increased (2.6 fold) one hour post-dawn in MON 87712 compared to 

controls, indicating that BBX32 activity can result in either up-regulation or downregulation of endogenous genes. These observations support a role for BBX32 in 

modulating the response of MON 87712 to the plant’s transition from night to day by 

repressing light signaling. At other time points, expression patterns of the soybean LHY

and BBX13 genes in MON 87712 were not significantly different from the expression 

pattern observed in control soybean, thus demonstrating that the introduced BBX32 

protein functions within an existing framework of gene regulation in soybean. 

 
Monsanto Company 11-SY-217U 218 of 467 

Figure B-6. Gene Expression Patterns for two Examples of Diurnally Regulated 

Genes in MON 87712, MON 87712(-) and A3525 

Gene expression pattern of (A) LHY and (B) BBX13 mRNA from V3 stage plants grown 

in the growth chamber. Time points “pre” and “post” are relative to dawn. 

B.5.4. BBX32 Impacts Existing Metabolic Processes in MON 87712, Improving 

Assimilate Availability 

The impact of BBX32 expression in MON 87712 on selected metabolite and enzyme 

activities was investigated. Leaf samples from the 2009 growing season were collected at 

two developmental stages (R1 and R6) at three different time points throughout the day: 

1 h predawn, 1 h post dawn, and 9 h post dawn. The sampling times were intended to 

understand the immediate impact of BBX32 activity and downstream differences in 

metabolite values between MON 87712 and the two controls. It was not expected that the 

timing of the differences in metabolite levels would correlate to the timing of transcript 

level differences between MON 87712 and the controls because enzyme and metabolite 

profiles represent an integration, over time, of faster but more transient transcript level 

changes (Gibon, 2006). The levels of 41 metabolites were determined for each sample, 

B

A LHY

*

p < 0.05; fold change

*

BBX13

*
Monsanto Company 11-SY-217U 219 of 467 

as well as the activities of six enzymes (Appendix D). Metabolite analyses included 

sixteen free amino acids, starch, eight organic acids, seven sugars, eight sugar 

phosphates, and two ureides. Enzyme activity analyses included allantoate 

amidohydrolase, glutamine synthetase, malate dehydrogenase, Nicotinamide Adenine 

Dinucleotide Phosphate (NADP) Malic Enzyme, Phosphoenolpyruvate (PEP) 

Carboxylase, and Sucrose Phosphate Synthase (SPS). Metabolites and enzymes selected 

for evaluation are generally associated with diurnal biology and primary carbon and 

nitrogen metabolism resulting in increased capacity for growth and reproductive 

development or correlated with yield or senescence (Morandi et al., 1990; Wingler et al., 

1998; Yazdi-Samadi et al., 1977; Rainbird et al., 1984). Apart from starch these 

metabolites and most represent a minor fraction of soybean seed biomass. A statistical 

summary of percent differences was generated, and metabolites where statistically 

significant differences (p<0.05) were observed for both comparisons (MON 87712 vs. 

MON 87712(-) and MON 87712 vs. A3525) are reported (Table B-4 and Table B-5). 
Monsanto Company 11-SY-217U 220 of 467 

Table B-4: Summary of significant differences between MON 87712 soybean and 

control (MON 87712(-) and A3525) leaf tissue at the R1 developmental stage 

Data is shown as percent change from control, and represents metabolites that exhibited 

significant differences that were consistent across comparisons to both controls. 

 MON 87712 vs. MON 87712(-) 

MON 87712 vs. 

A3525 

 R1 Leaf R1 Leaf 

1 hour 

predawn 

1 hour 

post 

dawn 

9 hours 

post 

dawn 

1 hour 

predawn

1 hour 

post dawn 

9 hours 

post dawn 

Glutamine -12.96 47.44 28.47* 149.09 28.05 47.73*

GABA 49.66* 42.54 9.04 52.12* 17.24 34.66 

Glycine 11.17 12.38 18.18* 10.78 -4.13 27.48*

Shikimic 

Acid 25.73 23.79 40.52* 17.52 47.71 121.18*

Fructose -20.91 22.25 22.63* -3.2 20.75 30.19*

Allantoic 

acid 29.12 82.35 131.38* 109.82 43.48 206.87*

* Significantly different (p < 0.05) across comparisons to both controls. 

 Shaded boxes indicate differences that were not significant across 

comparisons to both controls. 
Monsanto Company 11-SY-217U 221 of 467 

Table B-5: Summary of significant differences between MON 87712 soybean and 

control (MON 87712(-) and A3525) leaf tissue collected at the R6 developmental 

stage 

Data is shown as percent change from control, and represents enzyme/metabolites that 

exhibited significant differences that were consistent across comparisons to both controls. 

 MON 87712 vs. MON 87712(-) 

MON 87712 

vs.A3525 

 R6 Leaf R6 Leaf 

1 hour 

predawn

1 hour 

post 

dawn 

9 hours 

post 

dawn 

1 hour 

predawn 

1 hour 

post dawn 

9 

hours 

post 

dawn 

Sucrose 

Phosphate 

Synthase 55.56a 236.91a

 51.59a N/A N/A N/A 

Isoleucine -19.18 -39.02 -33.56* -21.14 -13.06 

-

28.81* 

Leucine -23.11 -29.61 -42.71* -18.55 6.38 

-

37.32* 

Valine -23.71* -18.62 -23.87 -22.98* -15.53 -13.24 

Malic Acid -23.16 -22.47* -19.79 -7.45 -21.87* -10.81 

Starch 22.46* 24.08 14.88 26.06* 11.64 12.71 

* Significantly different (p < 0.05) across comparisons to both controls. 

a

 Significantly different (p < 0.05 compared to A3525 where no data exists for 

MON 87712(-). 

 Shaded boxes indicate differences that were not significant across comparisons to 

both controls. 

N/A = Data not available 
Monsanto Company 11-SY-217U 222 of 467 

Evaluation of the overall dataset indicates temporal-specific differences in levels of 

metabolites involved in carbon and nitrogen metabolism, metabolites indicative of source 

capacity, and the activity of an enzyme involved in carbon metabolism were observed 

when MON 87712 was compared to controls. At R1, significant differences were 

observed for values of three amino acids, shikimic acid, fructose, and allantoic acid. 

Glutamine and glycine levels were higher in MON 87712 than the controls at 9 hours 

post dawn, and γ-amino butyric acid (GABA) levels were significantly higher at 1 hour 

predawn. Shikimic acid, fructose, and allantoic acid were also higher in MON 87712 

than the controls at 9 hours post dawn. Allantoic acid is a predominant form of nitrogen 

translocated from nodules to other parts of the soybean plant (Salisbury and Ross 1992), 

while glutamine is an initial product of ammonia (NH3) assimilation (Reynolds et al., 

1982). The increases in these metabolites in MON 87712 indicate changes in nitrogen 

metabolism in the soybean plant at R1, a developmental stage where increased nitrogen 

has been shown to increase yield (Gan et al., 2003). The increase in glycine levels is 

indicative of alterations in both carbon and nitrogen metabolism, as glycine is involved 

not only in protein biosynthesis, but also is one of the main sources of one-carbon units in 

higher plants, and therefore, forms the basis of carbon metabolism (Bourguignon et al., 

1999) Fructose and shikimic acid are downstream of glycine, and levels in MON 87712 

are also are likely increased as a result of alterations in carbon metabolism. GABA has 

also been hypothesized to play a role in carbon and nitrogen metabolism, possibly as 

storage and/or transport of nitrogen by assimilating carbons from glutamate to generate 

C:N fluxes that enter the tricarboxylic acid cycle, which is at the center of aerobic 

metabolism (Bouche and Fromm 2004). These temporal-specific metabolite changes at 

the R1 growth stage indicate changes in carbon and nitrogen metabolism, which are 

associated with increased seed number and increased yield. 

At R6, significant differences were observed for values of three amino acids (isoleucine, 

leucine, and valine), one organic acid (malate), sucrose phosphate synthase (SPS), and 

starch. Isoleucine and leucine were both lower in MON 87712 when compared to the 

controls at the nine hour post-dawn timepoint, while valine was lower in MON 87712 

when compared to the controls at the one hour predawn timepoint. The pathways 

synthesizing these three amino acids are considered biochemically parallel, as the 

enzymes performing the synthesis steps possess dual substrate specificities (Lea, 1997). 

These temporal-specific differences indicate changes in carbon and nitrogen metabolism, 

and support the hypothesis of increased transport of the amino acids from the source 

tissues to the sink (seed), leading to increased yield. The lower level of malate observed 

in MON 87712 compared to the controls one hour post dawn supports the observation of 

altered carbon metabolism, while the increased activitiy of SPS observed at all time 

points, coupled with higher starch levels at one hour predawn in MON 87712 compared 

to controls, relate to increased canopy carbon assimilation in MON 87712 to support the 

increased enzyme activity and production of carbon storage compounds. Taken together, 

these metabolite differences observed at R6, a critical seed-filling time for soybean, 

indicated altered carbon and nitrogen metabolism associated with increased individual 

seed weight to increased yield. 

These temporal-specific differences in levels of metabolites involved in carbon and 

nitrogen metabolism, and indicative of source capacity, and the increased activity of SPS 
Monsanto Company 11-SY-217U 223 of 467 

enzyme observed in MON 87712 are consistent with the proposed mode of action 

hypothesis for the increased yield in MON 87712 when compared to conventional 

soybean with the same genetic background. Specifically, the expression of BBX32 in 

MON 87712 increases carbon and nitrogen assimilate availability and translocation to 

reproductive parts during development stages when yield is determined. All differences 

in metabolites occurred at discrete times of day; and with the exception of SPS, no 

significant differences were present at all three sampling time points, supporting the 

proposal that these differences in carbon and nitrogen metabolites were temporalspecific. These temporal differences in metabolism observed at select timepoints during 

the plant’s reproductive phase are consistent with the ultimate increased yield phenotype 

of MON 87712 and the associated numerical increases in seed number and seed weight 

observed when compared to the conventional control (Table B-2), but they did not affect 

composition of the seed from a food and feed safety or nutritional perspective (section 

VII). Very few significant differences were observed when the composition of 

MON 87712 seed was compared to the conventional control A3525, although small 

increases in protein (1.09% relative increase), small increases in amino acids (1.22 -

2.22% relative increase), a small decrease in cystine (3.07% relative decrease), and small 

decreases in two fatty acids (palmitic acid and stearic acid, 1.42 and 3.04% relative 

decreases, respectively) were observed. These few significant differences were of small 

relative magnitude when compared to natural variability, and thus indicated that, 

although the small changes in metabolism observed in the green tissues increased seed 

number and seed weight, the changes did not affect composition of the seed from a food 

and feed safety or nutritional perspective. 

B.5.5. BBX32 Protein Function: Conclusion 

MON 87712 showed increased yield compared to its near isogenic negative segregant 

control MON 87712(-) of the same genetic background but without the introduced 

BBX32 gene. The increase in yield results from higher canopy-level assimilate 

availability, as measured by factors associated with increased photosynthetic rate at 

canopy level and a result of altered diurnal plant metabolism. Plant BBX32-mediated 

changes in soybean gene expression impact existing diurnal processes such as carbon and 

nitrogen metabolism. Increased assimilate availability for translocation to the 

reporoductive organs, with consequent increases in seed number and/or seed weight, the 

major components of soybean yield. 
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Figure B-7: BBX32 expression in MON 87712 leads to increased yield 

 
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Appendix C: Materials, Methods, and Results for Molecular Analyses of 

MON 87712 

C.1. Materials 

The genomic DNA used in molecular analyses was isolated from leaf tissue of the R3 

generation of MON 87712 and the conventional control A3525. For generational 

stability analysis, genomic DNA was extracted from leaf tissue of the R4, R5, R6, and R7 

generations of MON 87712. The reference substance, PV-GMAP5779 (Figure III-1), 

was used as a positive hybridization control in Southern blot analyses. Probe templates 

generated from PV-GMAP5779 were used as additional positive hybridization controls. 

As additional reference standards, the 1 Kb DNA Extension Ladder and λ DNA/Hind III 

Fragments from Invitrogen (Carlsbad, CA) were used for size estimations on Southern 

blots and agarose gels. The 1 Kb DNA Ladder from Invitrogen was used for size 

estimations on agarose gels for PCR analyses. 

C.2. Characterization of the Materials 

The identity of the source materials was verified by methods used in molecular 

characterization to confirm the presence or absence of MON 87712. The stability of the 

genomic DNA was confirmed by observation of interpretable signals from digested DNA 

samples on ethidium bromide stained agarose gels and/or specific PCR products, and the 

samples did not appear visibly degraded on the ethidium bromide stained gels. 

C.3. DNA Isolation for Southern Blot and PCR Analyses 

MON 87712 and conventional control genomic DNA samples were isolated from 

soybean leaf tissue. Prior to extraction, leaf tissue was processed to a fine powder by 

mortar and pestle using liquid nitrogen. Genomic DNA was extracted using a 

hexadecyltrimethylammonium bromide (CTAB) based method. Briefly, 5 ml of CTAB 

buffer (1.5% w/v CTAB, 75 mM Tris HCl, 100 mM EDTA, 1.05 M NaCl, and 

0.75% w/v PVP) and 50 μg RNase A were added to approximately 1 ml of ground leaf 

tissue and incubated at 60-70°C for 40-50 minutes with intermittent mixing. 

Approximately 5 milliliters (ml) of chloroform was added to the samples and mixed by 

hand for 2-3 minutes, then centrifuged at 10,300 × g for 8-10 minutes. The upper 

aqueous phase was put into a clean tube and the chloroform step was repeated twice. 

After the last chloroform step, the aqueous phase was put into a clean tube and the DNA 

was precipitated with approximately 4 ml of 100% ethanol. The sample was centrifuged 

at 5,100 × g for 5-7 minutes to pellet the precipitated DNA. The DNA pellets were 

washed with 10-12 ml of 70% ethanol by centrifuging the samples at 5,100 × g for 

5-7 minutes. The DNA pellets were air dried, then resuspended in 500 μl of TE buffer 

(10 mM Tris HCl, 1 mM EDTA, pH 8.0). For further purification of the DNA samples, 

polyethylene glycol (PEG) precipitation was performed. An equal volume of 

20% (w/v) PEG precipitation buffer was added to the extracted DNA sample. The 

PEG/DNA sample mixture was incubated at 37°C for ~15 minutes and then centrifuged 

at 15,000 × g for 15 minutes. After the supernatant was removed, the pellets were 

washed at least twice with 80% ethanol and then air dried. The pellets were resuspended 
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by adding ~1 ml of TE buffer and incubating at 60-70°C. To obtain the DNA, the 

samples were centrifuged at 15,000 × g for 15 minutes and the supernatant was 

transferref to a clean tube. All extracted DNA was stored in a 4°C refrigerator or a 20°C 

freezer. 

C.4. Quantification of DNA 

Genomic DNA was quantified using a DyNA Quant 200 Fluorometer (Hoefer, Inc., 

Holliston, MA). Molecular Size Marker IX (Roche, Indianapolis, IN) was used as the 

calibration standard. 

C.5. Restriction Enzyme Digestion of DNA 

Approximately 10 micrograms (μg) of genomic DNA extracted from MON 87712 and 

conventional control was digested with restriction enzyme Nco I (Fermentas, Glen 

Burnie, MD or New England Biolabs, Beverly, MA) and a combination of restriction 

enzymes EcoR I and Spe I (New England Biolabs). All Nco I digests were conducted in 

1X Tango buffer (Fernentas) and all EcoR I and Spe I digests were conducted in 

1X NEBuffer 2 (New England Biolabs.). All digests were performed at 37°C in a total 

volume of ~500 microliter (μl) with ~50 units of each restriction enzyme. For the 

purpose of running positive hybridization controls, ~10 μg of genomic DNA extracted 

from the conventional control was digested with the restriction enzyme Nco I and the 

appropriate positive hybridization control(s) were added to these digests prior to loading 

the agarose gel. 

C.6. Agarose Gel Electrophoresis 

Digested DNA was resolved on 0.8% (w/v) agarose gels. For all Southern blot analyses 

except insert stability, individual digests containing ~10 μg each of MON 87712 and 

conventional control genomic DNA were loaded on the same gel in a long run/short run 

format. The long run allows for greater resolution of large molecular weight DNA, 

whereas the short run allowed the detection of small molecular weight DNA. The 

positive hybridization controls were only run in the short run format. For the insert 

stability analysis, individual digests of ~10 μg of genomic DNA extracted from leaf 

tissue across five generations of MON 87712 were loaded on the agarose gel in a single 

run format. 

C.7. DNA Probe Preparation for Southern Blot Analyses 

Probe templates were prepared by PCR amplification using PV-GMAP5779 as the 

template. The PCR products were separated on an agarose gel by electrophoresis and 

purified from the gel using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) 

according to the manufacturer’s instruction. The probe templates were designed based on 

the nucleotide composition (% GC) of the sequence, in order to optimize the detection of 

DNA sequences during hybridization. When possible, probes possessing a similar 

melting temperature (Tm) were combined in the same Southern blot hybridization. 

Approximately 25 ng of each probe template were radiolabeled with [α32P] 

deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) using the random priming method 
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(Invitrogen). Probe locations relative to the genetic elements in PV-GMAP5779 are 

depicted in Figure III-1. 

C.8. Southern Blot Analyses of DNA 

Digested genomic DNA isolated from test and control substances was evaluated using 

Southern blot analyses (Southern, 1975). In Southern blots hybridized with a single 

probe, ~0.1 and ~1.0 genome equivalent of PV-GMAP5779 DNA previously digested 

with Nco I (Fermentas) was added to digested conventional control genomic DNA to 

serve as a positive hybridization control. In Southern blots hybridized with multiple 

probes, ~1.0 genome equivalent of the PV-GMAP5779 DNA previously digested with 

Nco I (Fermentas), as well as ~0.1 and ~1.0 genome equivalent of the appropriate probe 

templates were added to digested conventional control genomic DNA to serve as positive 

hybridization controls. The DNA was then separated by agarose gel electrophoresis and 

transferred onto a nylon membrane. Southern blots were hybridized and washed at 55°C 

or 60°C, depending on the calculated Tm of the probes used. Table C-1 lists the 

hybridization and radiolabeling conditions of the probes used in this study. Multiple 

exposures of each blot were then generated using Kodak Biomax MS film (Eastman 

Kodak, Rochester, NY) in conjunction with one Kodak Biomax MS intensifying screen 

in a -80°C freezer. 
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Table C-1. Hybridization Conditions of Utilized Probes 

Probe DNA Probe 

Probe labeled 

with dNTP (32P) 

Hybridization/ 

Wash 

Temperature (C) 

1 T-DNA I Probe 1 dCTP 60 

2 T-DNA I Probe 2 dCTP 60 

3 T-DNA II Probe 1 dCTP 55 

4 T-DNA II Probe 2 dCTP 55 

5 T-DNA II Probe 3 dCTP 55 

6 Backbone Probe 1 dCTP 60 

7 Backbone Probe 2 dCTP 60 

8 Backbone Probe 3 dCTP 60 

9 Backbone Probe 4 dCTP 60 
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C.9. DNA Sequence Analyses of the Insert 

Overlapping PCR products, denoted as Product A, Product B, and Product C, were 

generated that span the insert and adjacent 5′ and 3′ flanking DNA sequences in 

MON 87712 (Figure IV-9). These products were sequenced to determine the nucleotide 

sequence of the insert in MON 87712, as well as the nucleotide sequence of the DNA 

flanking the 5′ and 3′ ends of the insert. 

The PCR analyses for Product A, Product B, and Product C were conducted using 100 ng 

of genomic DNA template in a 50 μl reaction volume containing a final concentration of 

2 mM MgSO4, 0.2 μM of each primer, 0.2 mM of each dNTP, and 0.05 units/μl of 

Accuprime Taq DNA Polymerase High Fidelity (Invitrogen). 

The amplification of Product A and Product C were performed under the following 

cycling conditions: 1 cycle at 95°C for 30 seconds; 35 cycles at 95°C for 30 seconds, 

58°C for 30 seconds, 68°C for 2 minutes; 1 cycle at 68°C for 10 minutes. The 

amplification of Product B was performed under the following cycling conditions: 

1 cycle at 95°C for 30 seconds; 35 cycles at 95°C for 30 seconds, 60°C for 30 seconds, 

68 C for 1.5 minutes; 1 cycle at 68°C for 10 minutes. 

Aliquots of each PCR product were separated on 1.0% (w/v) agarose gels and visualized 

by ethidium staining to verify that the products were the expected size. Prior to 

sequencing, each verified PCR product was purified using the QIAquick PCR 

Purification Kit (Qiagen) according to manufacturer’s instruction. The purified PCR 

products were sequenced using multiple primers, including primers used for PCR 

amplification. All sequencing was performed by the Monsanto Genomics Sequencing 

Center using BigDye terminator chemistry (Applied Biosystems, Foster City, CA). 

A consensus sequence was generated by compiling multiple sequencing reactions 

performed on the overlapping PCR products. This consensus sequence was aligned to the 

PV-GMAP5779 sequence to determine the integrity and organization of the integrated 

DNA and the 5′ and 3′ insert-to-flank junctions in MON 87712. 

C.10. PCR and DNA Sequence Analysis to Examine the MON 87712 Insertion Site 

To examine the MON 87712 insertion site in conventional soybean, PCR and sequence 

analyses were performed on genomic DNA from both MON 87712 and conventional 

soybean (Figure IV-10). The primers used in this analysis were designed from the DNA 

sequences flanking the insert in MON 87712. A forward primer specific to the DNA 

sequence flanking the 5' end of the insert was paired with a reverse primer specific to the 

DNA sequence flanking the 3' end of the insert. 

The PCR reactions were conducted using 100 ng of genomic DNA template in a 50 μl 

reaction volume containing a final concentration of 2 mM MgSO4, 0.2 μM of each 

primer, 0.2 mM of each dNTP, and 0.05 units/μl of Accuprime Taq DNA Polymerase 

High Fidelity (Invitrogen). The amplification was performed under the following cycling 

conditions: 1 cycle at 95°C for 30 seconds; 35 cycles at 95°C for 30 seconds, 55°C for 

30 seconds, 68°C for 15 seconds; 1 cycle at 68°C for 10 minutes. 
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Aliquots of each PCR product were separated on 1.0% (w/v) agarose gels and visualized 

by ethidium staining to verify that the PCR products were the expected size prior to 

sequencing. Only the verified PCR product from the conventional control was purified 

with the QIAquick PCR Purification Kit (Qiagen) according to manufacturer’s 

instruction. The purified PCR product was sequenced using multiple primers, including 

primers used for PCR amplification. All sequencing was performed by the Monsanto 

Genomics Sequencing Center using BigDye terminator chemistry. 

A consensus sequence was generated by compiling multiple sequencing reactions 

performed on the verified PCR product. This consensus sequence was aligned to the 5' 

and 3' sequences flanking the MON 87712 insert to determine the integrity and 

organization of the insertion site. 
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References for Appendix C

Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated 

by gel electrophoresis. Journal of Molecular Biology 98:503-517. 
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Appendix D: Materials, Methods and Results for Characterization of BBX32 

Protein Produced in MON 87712 

D.1. Characterization of BBX32 Protein in MON 87712 

D.1.1. Materials 

BBX32 was isolated from MON 87712 as a component of a cation exchange (CEX)-

fractionated leaf extract (lot G-865162A), as described in Appendix D.1.3. Throughout 

this appendix, the BBX32-containing sample will be described as the MON 87712 CEX 

pool. MON 87712 leaf tissue (Orion lot 11295830) was produced under production plan 

PPN-10-450. The MON 87712 CEX pool was stored in a -80 °C freezer in a buffer 

solution containing 1 M NaCl, 40mM MES, 4mM TCEP, pH 6.2, 2 mM Benzamidine, 10 

μM Bestatin, 20μM E64, 500 μM AEBSF. The records describing the preparation of the 

MON 87712 CEX pool will be retained in the Monsanto Regulatory Archives (notebook 

G-865151). 

As a control, the CEX-enriched pool prepared from the non-transgenic A3525 leaf (lot G865172A), was also analyzed. Throughout this appendix, this sample will be described 

as the A3525 CEX pool. The A3525 leaf (Orion lot 11295829) was produced under 

production plan PPN-10-450. The A3525 CEX pool was produced and stored under the 

same conditions as the MON 87712 CEX pool. The records describing the preparation of 

the A3525 CEX pool will be retained in the Monsanto Regulatory Archives (notebook G865151). 

The E. coli-produced BBX32 reference protein (Orion lot 11267091) was purified from 

the fermentation of E. coli transformed with plasmid pMON102114. The DNA sequence 

encoding this BBX32 reference protein was confirmed both prior to and following 

fermentation of E. coli. The E. coli-produced BBX32 was previously characterized. 

Records pertaining to the purification and the characterization of this E. coli-produced 

reference protein are archived under Orion lot 11267091 in the Monsanto Regulatory 

Archives. 

D.1.2. Description of Assay Control 

Protein molecular weight standards (Precision Plus Dual Color Standards, Bio-Rad, 

Hercules, CA) were used to verify protein transfer to nitrocellulose membranes, and to 

approximate the position of BBX32 on the western blots. BSA (Pierce, Rockford, IL) 

was used to generate a standard curve for total protein determination. 

D.1.3. BBX32 Protein Purification 

The MON 87712 CEX pool was isolated from the ground and processed soy leaf of 

MON 87712 using a combination of extraction, centrifugation and filtration, diafiltration, 

and cation exchange chromatography. A brief description of the isolation process is 

below. 
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The MON 87712 and the A3525 leaf tissues were harvested and immediately transferred 

to containers and frozen on dry ice. Leaf tissue processing was carried out by the 

Monsanto Regulatory Sample Management team in St. Louis, Missouri. The frozen leaf 

tissue was ground in the presence of dry ice, and was then stored frozen at -80 °C. 

Aliquots of the ground leaf tissue were used as starting material for the isolation process. 

Approximately 135 g of processed ground MON 87712 leaf tissue was resuspended with 

1 liter of chilled (4 °C) extraction buffer [50 mM Tris-HCl, pH 8.0, 150mM NaCl, 

0.50% Igepal-630 (v/v), 4mM TCEP, 5mM sodium fluoride, 5mM sodium 

orthovanadate, 1 mM magnesium chloride, 2 mM Benzamidine, 10 μM Bestatin, 20μM 

E64, 500 μM AEBSF]. The leaf tissue suspension was thoroughly mixed by stirring for 

10 minutes with a magnetic stir bar at 4 °C. After mixing, the pH of the extract was 

adjusted to 7.5 +/- 0.1 with sodium hydroxide. The extract was then homogenized with a 

10-35GT Polytron homogenizer equipped with a medium grind probe at 12,000 rpm for 4 

min at room temperature (RT). After homogenization the extract pH was measured and 

adjusted to 7.5 +/- 0.1 with sodium hydroxide. The extract was returned to cold room 

(4 °C) and incubated for 2 h with stirring. After 2 h the extract was centrifuged at 

18,600 ×g for 45 minutes at 4 °C. The clarified extract supernatant was carefully 

decanted from pellet and the resultant supernatant poured through Miracloth (EMD 

Chemicals, Gibbstown, NJ) into a beaker chilled on ice to remove any loose pellet. The 

supernatant was then filtered through a Whatman 0.8/0.2μm 820cm2

 EFA Polycap filter 

(Whatman Inc., Piscataway, NJ). The filtered extract supernatant was kept chilled on ice. 

The filtered extract supernatant of approximately 950 ml was concentrated to 

approximately 200 ml using a Millipore Pellicon-2 unit equipped with 2 x 0.1 m2

10,000 kDa molecular weight cutoff (MWCO) cartridges (Millipore, Billerica, MA). 

Following concentration, the filtrate was diafiltered with approximately 6 volumes (1.2 

liters) of SP column buffer (40mM MES, 4mM TCEP, pH 6.2, 2 mM Benzamidine, 10 

μM Bestatin, 20μM E64, 500 μM AEBSF). This operation was performed at RT and the 

concentration unit and buffer reservoirs were chilled on ice during the operation. 

Following diafiltration and rinsing of the cartridges with SP column buffer, the 

dialfiltered extract volume was approximately 350 ml. This diafiltered extract was kept 

chilled on ice until subjected to chromatography. 

The HP SP Sepharose chromatographic step was performed in the cold room 

(4 °C). Three 5ml HiTrap SP HP Sepharose cartridges (GE Healthcare, Piscataway, NJ) 

connected in series were equilibrated with SP column buffer. The equilibrated column 

was charged with the dialfiltered extract and then washed and equilibrated with 10 

column volumes of the SP column buffer. The column was then washed with a 0.10 – 

0.50M NaCl gradient in SP column buffer over 5 column volumes to remove 

contaminating proteins. The MON 87712 BBX32 pool was then eluted with a 1M NaCl 

step gradient in SP column buffer over 5 column volumes. BBX32-containing fractions 

from the 1M elution step were pooled (approximately 20ml, based on 280 nm 

absorbance) to give the MON 87712 CEX pool. The MON 87712 CEX pool was then 

aliquoted and frozen on dry ice. The frozen aliquots were stored at -80 °C. 
Monsanto Company 11-SY-217U 239 of 467 

The A3525 CEX pool (lot G-865172A) was prepared and stored using the same methods 

used for the isolation of the MON 87712 CEX pool, except that A3525 leaf tissue was 

used as the starting material for purification. 

D.1.4. Western Blot Analysis-Immunoreactivity 

D.1.4.1. Methods 

SDS-PAGE and western blot analysis were performed to confirm the identity, and to 

estimate the molecular weight and purity of MON 87712 BBX32. 

Aliquots of the MON 87712 CEX pool (lot G-865162A), or the A3525 CEX pool 

(G-865172A), were mixed with 5 × 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] and deionized water to give a final total protein concentration of 

2 μg/μl in 1 × LB [62.4 mM Tris-HCl, 4% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, 

0.005% (w/v) bromophenol blue, 10% (v/v) glycerol, pH 6.8]. 

The E. coli-produced BBX32 reference protein (Orion lot 11267091) was serially diluted 

into matrix buffer (1.2 ml A3525 CEX pool, 0.3 ml 5 × LB) to a purity-corrected working 

concentration of 30 ng BBX32/μl, and then was further diluted in matrix buffer to 

generate a standard curve (10, 40, 80, 120 pg BBX32). All samples, including the 

MON 87712 CEX pool, the A3525 CEX pool, and the reference protein-spiked samples, 

were loaded at 40 μg of total protein per lane. Molecular weight markers (Precision Plus 

Dual Color, Bio-Rad, Hercules, CA) were loaded to estimate the size of the 

immunoreactive bands observed, and to verify electrotransfer of the proteins to the 

membrane. Samples were heated at 99°C for four minutes, and were loaded onto a precast 10% polyacrylamide Bis-Tris, 10-well gel (Invitrogen, Carlsbad CA). 

Electrophoresis was performed at a constant 170 V for 50 minutes in 1 × MES, 4 mM 

TCEP, pH 7.1 buffer. 

Electrotransfer to a 0.45 μm nitrocellulose membrane (Invitrogen) was performed for 

120 minutes at a constant 25 V in 1 × Transfer Buffer [12 mM Tris-HCl, 96 mM Glycine, 

20% (v/v) Methanol, pH 8.3] (Invitrogen). 

Western blot analysis was performed to confirm the identity of MON 87712 BBX32, to 

compare the migrations of MON 87712-produced and E. coli-produced BBX32, and to 

estimate the purity of BBX32 in the MON 87712 CEX pool. For immunodetection, the 

membrane was incubated for 14 h at 4 °C with 10% (w/v) non-fat dried milk (NFDM) in 

1 × Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBST). The membrane was 

then probed with a 1:1,000 dilution of purified mouse monoclonal anti-BBX32 antibody 

(lot A0112690) in 5% (w/v) NFDM in TBST for 90 min. Excess antibody was removed 

using six 15 min washes with TBST. The membrane was then incubated with 

horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (Vector Laboratories, 

Burlingame, CA) at a dilution of 1:15,000 in 5% (w/v) NFDM in TBST for 90 min. 

Excess HRP-conjugate was removed using six 15 min washes with TBST. All antibody 

incubations and TBST washes were performed at RT. Immunoreactive bands were 
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visualized using the ECL (Enhanced Chemiluminescence) detection system (GE 

Healthcare) and exposed to Amersham Hyperfilm (GE Healthcare). The film was 

developed using a Konica SRX-101A automated film processor (Tokyo, Japan). 

Analysis of the blot was performed using a Bio-Rad GS-800 densitometer with the 

supplied Quantity One® software (version 4.4.0). Immunoreactive bands at ~25 kDa in 

the MON 87712 CEX pool and the E. coli-produced BBX32 reference protein-spiked 

lanes were quantified using Quantity One software. The mass of BBX32 present in the 

duplicate lanes of the MON 87712 CEX pool was quantified against the standard curve of 

the E.coli-produced BBX32 reference protein using densitometric analysis of the blot 

film. The purity of the BBX32 in the MON 87712 CEX pool was calculated by dividing 

the average computed BBX32 mass in the relevant lanes by the mass of total protein 

loaded in each lane (40 μg). 

D.1.4.2. Results of MON 87712 BBX32 Western Blot Analysis - Immunoreactivity 

The relative apparent molecular weights of the BBX32 in the MON 87712 CEX pool and 

the E. coli-produced BBX32 reference protein were evaluated by western blot and shown 

to be equivalent based on visual analysis (Figure D-1). Purity of BBX32 in the 

MON 87712 CEX pool relative to total protein loaded was calculated using the mass 

computed from densitometric analysis of the western blot and the total protein loaded per 

lane (Table D-1). 

On the western blot (Figure D-1, lanes 2 and 3), the mouse anti-BBX32 antibody 

recognized two bands unique to the MON 87712 CEX pool, relative to the A3525 CEX 

pool. The major unique immunoreactive band comigrated with the spiked E. coliproduced BBX32 reference protein band at the expected apparent molecular weight of 

BBX32, ~25 kDa. The second band, which migrated at ~16 kDa, could be due to some 

degradation of the BBX32 protein. No additional bands unique to the MON 87712 CEX 

pool were observed. This western blot analysis confirmed the identity of the 

MON 87712 BBX32 protein. 
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Figure D-1. Western Blot Analysis of MON 87712- and E. coli -produced BBX32 

Proteins 

The MON 87712 CEX pool and the A3525 CEX pool were each loaded in duplicate. 

The reference protein, E. coli-produced BBX32, was added in the indicated amounts into 

the A3525 CEX pool to provide both a comparison for BBX32 migration and a standard 

curve for determining BBX32 amount in the MON 87712 CEX pool. Samples were 

separated by SDS-PAGE and electrotransferred to a nitrocellulose membrane. The 

membrane was incubated with an anti-BBX32 antibody and immunoreactive bands were 

visualized using an ECL system. Approximate MWs (in kDa) are shown on the left and 

right sides and correspond to the markers loaded in lanes 1 and 10. The 45 sec exposure 

is shown. 

Lane Sample Total Protein Load (ug)

1 Precision plus dual color MW marker N/A 

2 MON 87712 CEX pool 40 

3 MON 87712 CEX pool 40 

4 A3525 CEX pool 40 

5 A3525 CEX pool 40 

6 10 pg E.coli BBX32 in A3525 CEX pool 40 

7 40 pg E.coli BBX32 in A3525 CEX pool 40 

8 80 pg E.coli BBX32 in A3525 CEX pool 40 

9 120 pg E.coli BBX32 in A3525 CEX pool 40 

10 Precision plus dual color MW marker N/A 
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Table D-1. Purity of the MON 87712-Produced BBX32 Protein 

The purity of MON 87712-produced BBX32 was calculated using densitometric analysis 

of western blot shown in Figure D-1, and the total protein loaded. 

Total Protein 

Loaded per lane

Mass of BBX32 (pg) BBX32 Purity1

(pg BBX32/

Lane 2 Lane 3 μg total protein) 

40 μg 44.1 40.9  1

Purity of the MON 87712-produced BBX32 protein is calculated based on the average mass of 

BBX32 in the duplicate lanes of the MON 87712 CEX pool. 
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D.1.5. BBX32 Coding Sequence Comparison 

Sequence alignment showed that translation of the BBX32 coding sequence from the 

vector used to transform conventional soybean to produce MON 87712, PV-GMAP5779, 

and from the E. coli BBX32 expression vector, pMON102114, resulted in the same 

amino acid sequence (Figure D-2). As reported in Section IV.D, the DNA sequence of 

the T-DNA I region in MON 87712 is identical to the corresponding T-DNA I sequence 

of PV-GMAP5779; thus the pMON102114 BBX32 coding sequence is identical to the 

BBX32 coding sequence in MON 87712. 

Figure D-2. BBX32 Coding Sequence Comparison 

The regions of E. coli plasmid pMON102114 (ECOLI_V) and plant vector PVGMAP5779 (PLANT_V) containing the BBX32 coding sequence were translated. The 

resulting amino acid sequences were aligned. 

PLANT_V MVSFCELCGAEADLHCAADSAFLCRSCDAKFHASNFLFARHFRRVICPNC

::::::::::::::::::::::::::::::::::::::::::::::::::

ECOLI_V MVSFCELCGAEADLHCAADSAFLCRSCDAKFHASNFLFARHFRRVICPNC

PLANT_V KSLTQNFVSGPLLPWPPRTTCCSESSSSSCCSSLDCVSSSELSSTTRDVN

::::::::::::::::::::::::::::::::::::::::::::::::::

ECOLI_V KSLTQNFVSGPLLPWPPRTTCCSESSSSSCCSSLDCVSSSELSSTTRDVN

PLANT_V RARGRENRVNAKAVAVTVADGIFVNWCGKLGLNRDLTNAVVSYASLALAV

::::::::::::::::::::::::::::::::::::::::::::::::::

ECOLI_V RARGRENRVNAKAVAVTVADGIFVNWCGKLGLNRDLTNAVVSYASLALAV

PLANT_V ETRPRATKRVFLAAAFWFGVKNTTTWQNLKKVEDVTGVSAGMIRAVESKL

::::::::::::::::::::::::::::::::::::::::::::::::::

ECOLI_V ETRPRATKRVFLAAAFWFGVKNTTTWQNLKKVEDVTGVSAGMIRAVESKL

PLANT_V ARAMTQQLRRWRVDSEEGWAENDNV

:::::::::::::::::::::::::

ECOLI_V ARAMTQQLRRWRVDSEEGWAENDNV
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Appendix E: Materials and Methods Used for the Analysis of the Levels of 

BBX32 Protein in MON 87712 

E.1. Materials 

Over-season leaf (OSL-1 - OSL-4), root (OSR-3), forage (forage-1), and seed tissue 

samples from MON 87712 and the conventional control A3525 were harvested from 

eight field sites in the U.S. during the 2009 growing season from starting seed lots 

11223539 and 11223542, respectively. An E. coli-produced BBX32 protein 

(lot 11267091) was used as the analytical reference standard. 

E.2. Characterization of the Materials 

The identities of MON 87712 and the conventional control samples were confirmed by 

event specific polymerase chain reaction (PCR) analyses that were conducted on the 

harvested seed from each site. The PCR analyses and the resulting Verification of 

Identities were archived in the Monsanto Regulatory Archives under the starting seed lot 

numbers. 

E.3. Field Design and Tissue Collection 

Field trials were initiated during the 2009 planting season to generate MON 87712 

samples at various soybean growing locations in the U.S. The OSL-1 - OSL-4, OSR-3, 

forage-1 and seed tissue samples from the following field sites were analyzed: Jackson 

County, Arkansas; Parke County, Indiana; Clinton County, Illinois; Madison County, 

Illinois; Stark County, Illinois; York County, Nebraska; Boone County, Indiana; and 

Pawnee County, Kansas. These field sites were representative of soybean producing 

regions suitable for commercial production. At each site, four replicated plots of plants 

containing MON 87712 and the conventional control A3525 were planted using a 

randomized complete block field design. OSL-1 - OSL-4, OSR-3, forage-1, and seed 

samples were collected from each replicated plot at all field sites. See Table V-1 for a 

detailed description of when the samples were collected. 

E.4. Tissue Processing and Protein Extraction 

Tissue samples were shipped to Monsanto, St. Louis and prepared by the Monsanto 

Sample Management Team. The prepared tissue samples were stored in a -80 °C freezer 

until transferred on dry ice to the analytical facility, except forage conventional control 

A3525 which were stored in a -20 C freezer. 

BBX32 protein was extracted from all tissues samples at 10:1 buffer:sample ratio (v/w) 

using a Polytron (Kinematica AG, Lucerne, Switzerland) with the appropriate amount of 

BBX32 extraction buffer [50 mM Tris-HCl buffer, pH 7.5, 150 mM NaCl, 1 mM EDTA, 

0.5% (v/v) IGEPAL CA-630, 5 mM NaF, and 5 mM NaVO4, supplemented with 4 mM 

TCEP, neutralized with 0.65 M Na2HPO4 at 20:1 ratio (v/v)] containing protease 

inhibitor cocktail (1 mM Benzamidine, 0.1 μM Bestatin, 10 μM E64 and 250 μM 

AEBSF)]. After homogenization each sample had 8, 1⁄4” chrome steel beads added, and 
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was rotated at 4C for 120 minutes. Each extract was clarified by centrifugation for 20 

minutes x 10,000 × g at 4C. The supernatant was collected, aliquoted and either 

analyzed immediately, or stored at -80 C. 

E.5. BBX32 Antibody 

Production of the BBX32 monoclonal antibody was performed by Harlan Laboratories 

(Indianapolis, IN). Mouse monoclonal antibody from cell line SW2 8G7.16 (cell line 

I.D. Number 80212742, mouse IgG1 isotype), produced against the BBX32 protein, was 

purified from mouse ascites fluid using Protein G affinity chromatography and was used 

as the primary antibody in the BBX32 western blot. The antibody was then aliquoted and 

assigned lot number, G 863954. The concentration of the purified monoclonal antibody 

was determined to be 1.60 mg/ml by spectrophotometric methods. The purified antibody 

was stored in a phosphate buffered saline (pH 7.2) (1 × PBS) buffer, and 15 mM sodium 

azide. Horse anti mouse IgG conjugated to horseradish peroxidase (Cell Signaling 

Technology, Boston, MA. Lot 22) was used as the secondary antibody. 

E.6. BBX32 Western Blot Method 

Extracts were analyzed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis 

(SDS PAGE) using a Bis-Tris 10% polyacrylamide Midi Gel (Invitrogen, Carlsbad, CA, 

Cat # WG1202BOX). Prior to loading, the samples including the conventional controls 

were diluted in 2 × Laemmli Buffer (Bio Rad, Hercules, CA, Cat # 161 0737) and heated 

at 95 °C for 5 minutes. Sample extracts were loaded on the gels with the appropriate 

reference standards. The reference standards were prepared in tissue-specific 

conventional control extracts and diluted in 1 × Laemmli buffer. 

Preliminary analysis indicated that the BBX32 protein was not detectable in seed and 

forage tissues. Therefore, two different approaches for running samples and reference 

standards were taken depending on the expression level.. For tissues where the protein 

was detectable (leaf and root), the reference standards were diluted in a conventional 

tissue extract and loaded at concentrations ranging from 4 pg to 0 pg per lane. Leaf and 

root samples were loaded in triplicate and at least two bands were used for densitometry 

and quantification of the BBX32 protein. For seed and forage, only a single reference 

standard, diluted in conventional tissue extract, was loaded at 2 pg/lane for forage and 5 

pg/lane for seed. Forage and seed samples were loaded in a single well per sample. The 

conventional control extracts used for diluting the reference standard for each tissue type 

were produced from a non-systematically selected tissue specific sample. Additionally, 

the Precision Plus Protein Dual Color Standards (Bio Rad, Cat # 161 0374) was loaded 

on the gel to demonstrate the transfer of protein to the membrane and for the approximate 

molecular weight determination. Electrophoresis was conducted at 100 V for 

approximately 120 minutes in MES running buffer (Invitrogen, Cat# NP0002), pH 7.0, 

with 4 mM TCEP (Thermo Scientific, Cat # 20491). 

Proteins separated by SDS PAGE were electrophoretically transferred to 0.45 μm 

Invitrolon Polyvinylidene fluoride (PVDF) membrane (Invitrogen, Cat # LC2007) using 

1 × NuPage transfer buffer (Invitrogen, Cat # NP0006 1) containing 20% methanol at 300 
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mA for approximately one hour. After transfer, nonspecific sites on the membrane were 

blocked overnight at 4 °C using 5% (w/v) non fat dried milk (NFDM) (Bio Rad, 

Hercules, CA, Cat # 170 6404) in 1 × Phosphate Buffered Saline with 0.05% (v/v) Tween 

20 (1 × PBST). The membrane was probed for the presence of the BBX32 protein with a 

1:1000 dilution of purified mouse monoclonal antibody (lot G 863954) in 1 × PBST with 

5% (w/v) NFDM. Unbound antibody was removed by washing four times for 10 minutes 

each in 1 × PBST. Bound antibody was probed with a 1:5000 dilution of horse antimouse IgG antibody conjugated to horseradish peroxidase (Cell Signaling Technology, 

Cat # 7076, lot 22) in 1 × PBST with 5% (w/v) NFDM. Unbound antibody was removed 

by washing four times for 10 minutes each in 1 × PBST. The SuperSignal West Dura 

Extended Duration Substrate (Thermo Scientific, Rockford, IL, Cat # 34076) comprised 

of SuperSignal western Dura Luminol Enhancer solution and the SuperSignal Western 

Dura Stable Peroxide Buffer were added to the membrane at a 1:2 ratio according to the 

manufacturer’s instructions. The membrane was exposed to Amersham Hyperfilm 

enhanced chemiluninescence (ECL) (GE Healthcare, Cat # 28906839) and the film was 

developed using a Konica SRX 101A automated film processor (Tokyo, Japan). 

Different exposures were taken and a single exposure was scanned using a Bio Rad GS 

800 densitometer with the supplied Quantity One software (version 4.4.0). 

E.7. Moisture Analysis 

Tissue moisture content was determined in all tissue types that expressed BBX32 at 

detectable levels using an IR-200 Moisture Analyzer (Denver Instrument Company, 

Arvada, CO). A homogeneous tissue-specific site pool (TSSP) was prepared consisting 

of samples of a given tissue type grown at a given site. 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 െ ൬

Mean% TSSP Moisture

100 ൰

The DWCF was used to convert protein levels assessed on a ng/g fresh weight (fwt) basis 

into levels reported on a ng/g dry weight (dwt) basis using the following calculation: 

Protein Level in Dry Weight ൌ Protein Level Fresh Weight

DWCF

The protein levels that were reported to be less the limit of detection (LOD) on a fresh 

weight basis were not reported on a dry weight basis. 

 
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E.8. Data Analyses 

The standards present on the developed film were used to establish a standard curve. At 

least three standard points were utilized to generate the standard curve for sample 

quantification. Anti BBX32 antibodies detected the MON 87712 produced BBX32, a 

~25 kDa immunoreactive band not observed in the extract from the conventional control, 

A3525. For each sample where the presence of BBX32 was detected and clearly 

differentiated from the cross-reactive bands present in the conventional control, the levels 

were quantified by interpolation on the standard curve. The coefficient of variation (CV) 

met the pre-set criteria of ≤33% for the samples. Of the triplicate loads of each 

detectable sample either all three sample replicates, or a minimum of two, were used for 

densitometry and quantification. If only duplicate bands were visible for a sample they 

were used if each value was within 33% of the average. For leaf and root tissues where 

BBX32 was not detected, the levels were reported as below the limit of detection 

(<LOD). LOD is the protein amount corresponding to the lowest detectable band across 

all of the blots run for that tissue type. Thus samples reported as <LOD were not 

detected by western blot. For leaf and root tissues, LOQ corresponds to the range of all 

lowest detectable bands across all blots run for the specific tissue type. 

Following the interpolation from the standard curve, the amount of protein (pg) in the 

tissue was reported on a “ng/g fwt” basis for data that were greater than or equal to the 

lowest detectable point on the standard curve of the western blot on which it was run. 

This conversion utilized the sample dilution factor and the tissue to buffer ratio. The 

protein values in “ng/g fwt” were also converted to “ng/g dwt” by applying the DWCF. 

Microsoft Excel 2007 (Microsoft, Redmond, WA) was used to calculate the BBX32 

protein levels in soybean tissues. The sample means, standard deviations (SDs), and 

ranges were also calculated by Microsoft Excel 2007. All protein expression levels were 

rounded to two significant figures. 

For forage and seed samples each extract was loaded in a single well and a single 

reference standard was used. Western blot analysis confirmed that the BBX32 protein 

was not detected in forage and seed. Separately, the LOD of the BBX32 protein in forage 

and seed tissues was determined by serially spiking the BBX32 protein reference 

standard in conventional control forage and seed extracts. The LOD of the BBX32 

protein in MON 87712 seed and forage were analyzed using the aforementioned 

materials and methods with the exception of the standard curve preparation which was 

done as follows: Prior to loading, the samples were diluted in 2 × Laemmli Buffer (BioRad, Hercules, CA, Cat # 161-0737). The seed tissue extract was further diluted 2-fold 

with 1× Laemmli Buffer to reduce matrix effects. The BBX32 protein reference standard 

(lot 11267091) was serially diluted into the diluted extracts to generate six protein 

concentrations to be loaded on the gel of 4.0, 2.0, 1.0, 0.5, 0.25, 0 pg for forage tissue 

extracts, and 20, 10, 5.0, 2.5, 1.25, 0 pg for seed tissue extracts. The spiked protein 

standards were heated at 95 °C for 5 minutes prior to loading, cooled, and then 10 μl of 

each sample was loaded on to the gel in triplicate. The LOD of BBX32 on the western 

blots was defined by the lowest amount of BBX32 protein that resulted in a visible band 

on the western blot. 
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Appendix F: Materials, Methods, and Individual-Site Results for Compositional 

Analysis of MON 87712 Soybean Seed and Forage 

Compositional comparisons between MON 87712 and the conventional soybean control 

A3525 were 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. The compositional assessment was conducted on seed and forage 

samples harvested from a single growing season conducted in the U.S. during 2009 under 

typical agronomic practices. 

The materials and methods for compositional analysis, as well as the individual-site 

results (Tables F-4 to F-27), are provided below. 

F.1. Materials 

Forage and harvested seed from MON 87712, a conventional soybean control A3525 that 

has similar genetic background to that of MON 87712, and sixteen conventional 

commercial reference varieties were compositionally assessed. The conventional 

commercial reference varieties are listed in Table F-1. 

Table F-1. Commercial Reference Soybean Varieties 

Material Name Orion ID Field Site Codes 

Midland 363 10001570 ARNE, KSLA 

Pioneer 93B15 10001304 ARNE 

Hoegemeyer 333 10001590 ARNE 

Hoffman Seed HS387 11225760 ILCY, ILHI 

Maverick 11225761 ILCY, ILHI 

Maverick 10001507 KSLA 

Williams 82 11225762 ILCY, ILHI 

FS 3591 10001448 INRC, INSH 

Garst 3585N 10001517 INRC, INSH 

Dwight 10001434 INRC, INSH 

Crows 37003N 10001508 ILWY 

Crows C3908 10001074 ILWY 

Lewis 391 10001476 ILWY 

QualityPlus 365C 10001129 KSLA 

NuPride 3202 11225763 NEYO 

NuTech 315 11225764 NEYO 

Pioneer 93M14 11225765 NEYO 
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F.2. Characterization of the Materials 

The identities of the seed and forage samples from the T/C/R substances were verified by 

the Study Director prior to conducting the study by confirming the chain-of-custody 

documentation supplied with the seed and forage harvested from the field plots. The 

harvested seed of MON 87712, the conventional control, and the commercial reference 

varieties were characterized by event-specific polymerase chain reaction (PCR) analysis, 

for the presence or absence of the MON 87712 event. 

F.3. Field Production of the Samples 

Harvested seed and forage samples of MON 87712, the conventional control A3525, and 

conventional commercial reference varieties were collected from eight replicated sites in 

the U.S. during the 2009 growing season. The sites were: Jackson County, Arkansas 

(ARNE), Parke County, Indiana (INRC), Clinton County, Illinois (ILCY), Madison 

County, Illinois (ILHI), Stark County, Illinois (ILWY), Boone County, Indiana (INSH), 

Pawnee County, Kansas (KSLA), and York County, Nebraska (NEYO). The starting 

seeds were planted in a randomized complete block design with four blocks per site. 

MON 87712, the conventional control, and the commercial reference varieties were 

grown under normal agronomic field conditions for their respective geographic regions. 

Seed and forage samples were harvested from all plots and shipped on dry ice (forage) or 

at ambient temperature (harvested seed) to Monsanto Company, St. Louis, MO. A 

subsample for compositional analysis was obtained from each tissue sample collected. 

These subsamples were ground and stored in a freezer set to maintain 20°C until their 

shipment on dry ice to Covance Laboratories, Inc. (Madison, WI) for analysis. 

F.4. Summary of Analytical Methods and Reference Standards 

Nutrients assessed in this study included proximates (ash, carbohydrates by calculation, 

moisture, protein, and fat), fiber (ADF and NDF), amino acids (18 components), fatty 

acids (FA, C8-C22), and vitamin E (α-tochopherol) in seed, and proximates (ash, 

carbohydrates by calculation, moisture, protein, and fat) and fiber (ADF and NDF) in 

forage. Anti-nutrients assessed in seed included raffinose, stachyose, lectin, phytic acid, 

trypsin inhibitors, and isoflavones (daidzein, genistein, and glycitein). 

All compositional analyses were performed at Covance Laboratories, Inc. (Madison, WI). 

Methods for analysis were based on internationally-recognized procedures and literature 

publications. Brief descriptions of the methods utilized for the analyses are described 

below. 

F.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 (Goering and Van Soest, 1970). The limit of quantitation (LOQ) for this 

study was 0.100%. 
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F.4.2. Amino Acid Composition 

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 (AOAC-International, 2005a). The 

limit of quantitation for this study was 0.100 mg/g. 

Reference Standards: 

 ThermoScientific, Amino Acid Standard H, 2.5±0.1 mol/mL per constituent 

except cystine (1.25±0.1 mol/mL), Lot Number KG137091

 Sigma-Aldrich, L-Norvaline, 100%, Lot Number 087K1954

 Sigma-Aldrich, L-Tryptophan, 100%, Lot Number 097K0119

 Sigma-Aldrich, L-Cysteic Acid Monohydrate, 99.5%, Lot Number 1305674

 Sigma-Aldrich, L-Methionine Sulfone, 100%, Lot Number 047K1321

F.4.3. Ash 

The sample was placed in an electric furnace at 550C and ignited. The nonvolatile 

matter remaining was quantitated gravimetrically and calculated to determine percent ash 

(AOAC-International, 2005b). The limit of quantitation for this study was 0.100%. 

F.4.4. Carbohydrates 

The total carbohydrate level was calculated by difference using the fresh weight-derived 

data and the following equation (Merrill and Watt, 1973): 

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash) 

The limit of quantitation for this study was 0.100%. 

F.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 (AOACInternational, current edition). The limit of quantitation for this study was 0.100%. 

F.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 (AOAC-International, 2007a). The limit 

of quantitation for this study was 0.100%. 
Monsanto Company 11-SY-217U 251 of 467 

F.4.7. Fatty Acids 

The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The 

saponification mixture was methylated with 14% boron trifluoride in methanol. The 

resulting methyl esters were extracted with heptane containing an internal standard. The 

methyl esters of the fatty acids were analyzed by gas chromatography using external 

standards for quantitation (AOAC-International, 2005c; AOCS, 1997a). The limit of 

quantitation was 0.0200%. 

Reference Standards: 

 Component Lot Number Component Weight (%) Purity 

(%) 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 1 

MA30-U 

Methyl Octanoate 16.66 99.6 

Methyl Decanoate 16.66 99.5 

Methyl Laurate 16.66 99.8 

Methyl Myristate 16.66 99.8 

Methyl Palmitoleate 16.66 99.7 

Methyl Linolenate 16.66 99.4 

 Methyl Octanoate 16.66 99.6 

Nu-Chek Prep GLC Methyl Decanoate 16.66 99.6 

Reference Standard JY20-U Methyl Laurate 16.66 99.8 

Hazelton No. 1 Methyl Myristate 16.66 99.8 

 Methyl Palmitoleate 16.66 99.7 

 Methyl Linolenate 16.66 99.5 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 2 

AU24-T 

Methyl Arachidate 33.33 99.6 

Methyl 11-Eicosenoate 33.33 99.5 

Methyl Arachidonate 33.33 99.6 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 2 

AU16-U 

Methyl Arachidate 33.33 99.6 

Methyl 11-Eicosenoate 33.33 99.5 

Methyl Arachidonate 33.33 99.6 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 3 

JY17-T 

Methyl Myristoleate 12.5 99.6 

Methyl Pentadecanoate 12.5 99.6 

Methyl 10-Pentadecenoate 12.5 99.5 

Methyl Heptadecanoate 12.5 99.7 

Methyl 10-Heptadecenoate 12.5 99.6 

Methyl 11-14 Eicosadienoate 12.5 99.6 

Methyl Behenate 12.5 99.8 

Methyl 11-14-17 Eicosatrienoate 12.5 99.5 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 3 

JY28U 

Methyl Myristoleate 12.5 99.5 

Methyl Pentadecanoate 12.5 99.6 

Methyl 10-Pentadecenoate 12.5 99.5 

Methyl Heptadecanoate 12.5 99.6 

Methyl 10-Heptadecenoate 12.5 99.5 

Methyl 11-14 Eicosadienoate 12.5 99.6 

Methyl Behenate 12.5 99.8 

Methyl 11-14-17 Eicosatrienoate 12.5 99.5 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 4 

MA30-U 

Methyl Palmitate 27.0 99.6 

Methyl Stearate 19.0 99.5 

Methyl Oleate 27.0 99.8 

Methyl Linoleate 27.0 99.8 

 Nu Chek Prep, Methyl Gamma Linolenate, >99%, Lot Number U-63M-08-T

 Nu Chek Prep, Methyl Tridecanoate, >99%, Lot Number N-13M-MA25-T
Monsanto Company 11-SY-217U 252 of 467 

F.5.8. 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 

high-performance liquid chromatography system with ultraviolet detection and was 

compared to an external standard curve of known standards for quantitation (Pettersson 

and Kiessling, 1984; Seo and Morr, 1984). The limit of quantitation for each component 

was 10.0 ppm (μg/g). 

Reference Standards: 

 Chromadex, daidzein, 97.5%, Lot number 00004007-121 

 Indofine, glycitein, 97%, Lot number 0803103 

 Indofine, genistein, 99.35%, Lot number 0604043 

F.5.9. 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 (Klurfeld and Kritchevsky, 1987; Liener, 1955). The limit of 

quantitation for this study was 0.10 H.U./mg. 

F.5.10. 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 (AOACInternational, 2007b). The limit of quantitation for this study was 0.100%. 

F.5.11. 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 (AACC, 1998; Goering and Van 

Soest, 1970). The limit of quantitation for this study was 0.100%. 

 
Monsanto Company 11-SY-217U 253 of 467 

F.5.12. Phytic Acid 

The sample was extracted using 0.5M HCl with ultrasonication. Purification and 

concentration were accomplished on a silica-based anion-exchange column. The sample 

was analyzed on a polymer high-performance liquid chromatography column PRP-1, 

5m (150 x 4.1mm) with a refractive index detector (Lehrfeld, 1989; Lehrfeld, 1994). 

The limit of quantitation for this study was 0.100%. 

Reference Standard: 

 Sigma-Aldrich, Phytic Acid, Sodium Salt Hydrate, 96%, Lot Number 089K0159

F.5.13. Protein 

The protein and other organic nitrogen in the sample were converted to ammonia by 

digesting the sample with sulfuric acid containing a catalyst mixture. 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 (AOAC-International, 2005d; AOCS, 1997a). The limit of 

quantitation for this study was 0.100%. 

F.5.14. Raffinose and Stachyose 

Sugars in the sample are extracted with a 50:50 water:methanol solution. Aliquots are 

taken, dried under inert gas, and then reconstituted with a hydroxylamine hydrochloride 

solution in pyridine containing phenyl-β-D-glucopyranoside as the 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 (Brobst, 1972; Mason and Slover, 1971). The quantitation limit 

for this study was 0.0500%. 

Reference Standards: 

 Sigma-Aldrich, D-(+)-Raffinose Pentahydrate, 99% Lot Number 037K1059

 Sigma-Aldrich, Stachyose Hydrate, 98%, Lot Number 049K3800

F.5.15. Trypsin Inhibitor 

The sample was ground and defatted with petroleum ether. A sample of matrix was 

extracted with 0.01N 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 (Kakade et al, 1997). The limit of 

quantitation for this study was 1.00 Trypsin Inhibitor Units (TIU)/mg. 
Monsanto Company 11-SY-217U 254 of 467 

F.5.16. 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 (Cort, et al., 1983; McMurray, et al., 1980; Speek, 

et al., 1985). The limit of quantitation for this study was 0.500 mg/100g. 

Reference Standard: 

 USP, Alpha Tocopherol, 98.9%, Lot Number N0F068 

F.6. Data Processing and Statistical Analysis 

After compositional analyses were performed, data spreadsheets were forwarded to 

Monsanto Company. The data were reviewed, formatted, and sent to Certus 

International, Inc. (Chesterfield, MO) for statistical analysis. 

The following formulas were used for re-expression of soybean composition data for 

statistical analysis (Table F-2): 

Table F-2. Re-expression Formulas for Statistical Analysis of Composition Data 

Component From (X) To Formula1

Proximates (excluding Moisture), 

Fiber, Phytic Acid, Raffinose, 

Stachyose

% fwt % dwt X/d

Isoflavones g/g fwt g/g dwt X/d

Lectin H.U./mg fwt H.U./mg dwt X/d

Trypsin Inhibitor TIU/mg fwt TIU/mg dwt X/d

Vitamin E mg/100g fwt mg/100g dwt X/d

Amino Acids (AA) mg/g fwt % dwt X/(10d)

Fatty Acids (FA) % fwt % Total FA

(100)Xj/X, for 

each FAj where 

X 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 component in this study, at least 50% of 

the values for an analyte had to be greater than the assay limit of quantitation (LOQ). 

Analytes with more than 50% of observations below the assay LOQ were excluded from 

summaries and analysis. The following 14 analytes with more than 50% of observations 

below the assay LOQ were excluded from statistical analysis: 8:0 caprylic acid, 

10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 

15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 16:1 palmitoleic acid, 

17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma-linolenic acid, 

20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, and 20:4 arachidonic acid. 
Monsanto Company 11-SY-217U 255 of 467 

If less than 50% of the observations for a component were below the LOQ, individual 

analyses that were below the LOQ were assigned a value equal to one-half the LOQ. The 

following analyte was assigned a value (Table F-3): 

Table F-3. Component with Observations Below the Assay Limit of Quantification 

Not Excluded from Statistical Analysis 

Component Units Obs. Below LOQ Total

N

LOQ Value 

N (%) Assigned

Seed Fatty Acid

20:1 Eicosenoic % fwt 25 13.2 190 0.020 0.010 

The data were assessed for potential outliers using a studentized PRESS residuals 

calculation. 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 are also outside of the  6 studentized PRESS residual range are considered 

for exclusion, as outliers, from the final analyses. No values were removed from analysis 

based on this assessment. 

All soybean compositional components were statistically analyzed using a mixed model 

analysis of variance. The eight 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 site effect, B(L)jk = random block within site effect, 

LTij = random site by substance interaction effect, and eijk = residual error. 

A range of observed values from the reference varieties was determined for each 

analytical component. Additionally, the reference varieties 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. 
Monsanto Company 11-SY-217U 256 of 467 

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 reference varieties. 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. 

 

 SAS is a registered trademark of SAS Institute, Inc. 
Monsanto Company 11-SY-217U 

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.90 (0.13) 5.20 (0.13) -0.30 (0.18) -0.75, 0.15 0.152 

(4.71 - 5.24) (4.86 - 5.66) (-0.78 - -0.060) 

Carbohydrates 36.05 (0.51) 35.66 (0.51) 0.40 (0.65) -1.21, 2.00 0.566 

(34.74 - 37.51) (34.72 - 36.61) (-0.087 - 0.91) 

Moisture (% fwt) 7.57 (0.23) 7.94 (0.23) -0.37 (0.31) -1.13, 0.39 0.282 

(7.21 - 7.89) (7.54 - 8.21) (-0.68 - -0.11) 

Protein 41.06 (0.25) 40.87 (0.25) 0.19 (0.32) -0.59, 0.97 0.571 

(40.44 - 41.58) (40.39 - 41.75) (-0.42 - 1.09) 

Total Fat 17.96 (0.32) 18.28 (0.32) -0.31 (0.41) -1.32, 0.69 0.471 

(17.35 - 18.78) (17.54 - 19.05) (-0.45 - -0.19) 

Fiber (% dwt)Acid Detergent Fiber 17.99 (0.87) 18.71 (0.87) -0.72 (1.19) -3.63, 2.19 0.566 

(17.24 - 19.11) (17.80 - 19.68) (-2.40 - 0.71) 
Monsanto Company 11-SY-217U 

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 19.42 (0.54) 19.39 (0.54) 0.033 (0.76) -1.82, 1.89 0.967 

(18.32 - 20.74) (17.80 - 20.37) (-1.83 - 1.47) 

Amino Acid (% dwt)Alanine 1.77 (0.015) 1.76 (0.015) 0.0092 (0.021) -0.042, 0.060 0.672 

(1.76 - 1.79) (1.73 - 1.81) (-0.024 - 0.039) 

Arginine 3.08 (0.025) 3.03 (0.025) 0.053 (0.036) -0.035, 0.14 0.189 

(3.05 - 3.12) (2.96 - 3.11) (0.0071 - 0.16) 

Aspartic Acid 4.81 (0.037) 4.72 (0.037) 0.090 (0.053) -0.039, 0.22 0.139 

(4.73 - 4.91) (4.63 - 4.79) (-0.0027 - 0.24) 

Cystine 0.60 (0.013) 0.62 (0.013) -0.027 (0.018) -0.072, 0.018 0.199 

(0.56 - 0.63) (0.61 - 0.63) (-0.052 - -0.0029) 

Glutamic Acid 7.68 (0.062) 7.54 (0.062) 0.14 (0.088) -0.071, 0.36 0.152 

(7.53 - 7.81) (7.44 - 7.67) (0.056 - 0.36) 
Monsanto Company 11-SY-217U 

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.80 (0.012) 1.78 (0.012) 0.015 (0.017) -0.027, 0.056 0.422 

(1.77 - 1.81) (1.76 - 1.81) (-0.013 - 0.050) 

Histidine 1.07 (0.0077) 1.07 (0.0077) 0.0053 (0.011) -0.021, 0.032 0.645 

(1.06 - 1.08) (1.05 - 1.08) (-0.0058 - 0.030) 

Isoleucine 1.93 (0.025) 1.90 (0.025) 0.028 (0.035) -0.058, 0.11 0.459 

(1.91 - 1.97) (1.82 - 1.97) (-0.058 - 0.089) 

Leucine 3.16 (0.022) 3.13 (0.022) 0.031 (0.031) -0.045, 0.11 0.355 

(3.12 - 3.20) (3.08 - 3.19) (-0.023 - 0.11) 

Lysine 2.67 (0.016) 2.67 (0.016) 0.0057 (0.022) -0.049, 0.060 0.806 

(2.65 - 2.70) (2.63 - 2.70) (-0.031 - 0.069) 

Methionine 0.56 (0.012) 0.57 (0.012) -0.0071 (0.017) -0.048, 0.034 0.682 

(0.52 - 0.60) (0.56 - 0.58) (-0.031 - 0.017) 
Monsanto Company 11-SY-217U 

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.17 (0.018) 2.14 (0.018) 0.029 (0.026) -0.034, 0.093 0.301 

(2.15 - 2.19) (2.10 - 2.17) (-0.016 - 0.082) 

Proline 2.01 (0.020) 1.99 (0.020) 0.025 (0.029) -0.046, 0.095 0.423 

(1.99 - 2.06) (1.95 - 2.04) (-0.046 - 0.088) 

Serine 2.16 (0.016) 2.16 (0.016) 0.0022 (0.023) -0.053, 0.057 0.924 

(2.11 - 2.20) (2.13 - 2.20) (-0.065 - 0.071) 

Threonine 1.60 (0.016) 1.56 (0.016) 0.034 (0.023) -0.021, 0.090 0.177 

(1.57 - 1.63) (1.50 - 1.59) (-0.0077 - 0.13) 

Tryptophan 0.44 (0.0089) 0.45 (0.0089) -0.012 (0.013) -0.043, 0.019 0.367 

(0.41 - 0.46) (0.44 - 0.46) (-0.043 - 0.021) 

Tyrosine 1.48 (0.016) 1.49 (0.016) -0.014 (0.022) -0.069, 0.040 0.550 

(1.45 - 1.52) (1.47 - 1.51) (-0.045 - 0.021) 
Monsanto Company 11-SY-217U 

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 1.99 (0.029) 1.96 (0.029) 0.030 (0.041) -0.069, 0.13 0.484 

(1.95 - 2.02) (1.85 - 2.02) (-0.069 - 0.13) 

Fatty Acid (% Total FA)16:0 Palmitic 11.69 (0.043) 12.01 (0.043) -0.32 (0.057) -0.46, -0.18 0.001 

(11.54 - 11.80) (11.96 - 12.07) (-0.44 - -0.21) 

18:0 Stearic 3.85 (0.033) 4.16 (0.033) -0.31 (0.031) -0.38, -0.23 <0.00

(3.78 - 3.89) (4.07 - 4.23) (-0.34 - -0.22) 

18:1 Oleic 24.18 (0.40) 21.03 (0.40) 3.15 (0.53) 1.85, 4.45 0.001 

(23.08 - 25.71) (20.80 - 21.18) (2.07 - 4.66) 

18:2 Linoleic 51.50 (0.33) 53.62 (0.33) -2.12 (0.46) -3.25, -0.99 0.003 

(50.26 - 52.27) (53.44 - 53.90) (-3.63 - -1.28) 

18:3 Linolenic 8.06 (0.089) 8.39 (0.089) -0.32 (0.12) -0.61, -0.040 0.031 

(7.87 - 8.29) (8.17 - 8.59) (-0.68 - -0.064) 
Monsanto Company 11-SY-217U 

Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.29 (0.0084) 0.32 (0.0084) -0.031 (0.012) -0.060, -0.0018 0.040 

(0.26 - 0.30) (0.30 - 0.34) (-0.077 - -0.0061) 

20:1 Eicosenoic 0.19 (0.0040) 0.18 (0.0040) 0.0010 (0.0057) -0.013, 0.015 0.863 

(0.18 - 0.19) (0.17 - 0.19) (-0.011 - 0.019) 

22:0 Behenic 0.25 (0.019) 0.29 (0.019) -0.049 (0.026) -0.11, 0.014 0.105 

(0.22 - 0.31) (0.24 - 0.32) (-0.092 - -0.00071) 

Vitamin (mg/100g dwt)Vitamin E 1.78 (0.091) 2.16 (0.091) -0.39 (0.13) -0.70, -0.073 0.023 

(1.58 - 1.88) (2.00 - 2.23) (-0.64 - -0.12) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-5. Summary of Site ARNE Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 2.53 (0.24) 2.99 (0.24) -0.45 (0.32) -1.23, 0.32 0.204 

(2.23 - 3.17) (2.53 - 3.66) (-1.43 - 0.031) 

Phytic Acid (% dwt) 1.14 (0.087) 1.22 (0.087) -0.075 (0.12) -0.37, 0.23 0.565 

(0.86 - 1.45) (0.97 - 1.32) (-0.46 - 0.15) 

Raffinose (% dwt) 0.82 (0.016) 0.73 (0.016) 0.098 (0.0097) 0.074, 0.12 <0.00

(0.77 - 0.86) (0.69 - 0.75) (0.082 - 0.14) 

Stachyose (% dwt) 4.13 (0.078) 3.80 (0.078) 0.33 (0.11) 0.068, 0.60 0.021 

(3.94 - 4.33) (3.52 - 3.99) (0.077 - 0.53) 

Trypsin Inhibitor (TIU/mg dwt) 28.47 (2.28) 36.40 (2.28) -7.93 (2.78) -14.74, -1.11 0.029 

(24.54 - 34.92) (31.47 - 42.82) (-15.39 - -1.56) 

Isoflavone (μg/g dwt)Daidzein 603.87 (30.50) 646.83 (30.50) -42.96 (38.60) -137.42, 51.49 0.308 

(549.34 - 687.57) (537.10 - 728.12) (-178.78 - 63.51) 
Monsanto Company 11-SY-217U 

Table F-5. Summary of Site ARNE Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 532.00 (35.50) 610.70 (35.50) -78.70 (43.31) -184.67, 27.27 0.119 

(503.75 - 559.33) (508.77 - 778.42) (-219.09 - 39.80) 

Glycitein 116.05 (10.94) 116.47 (10.94) -0.42 (14.75) -36.51, 35.67 0.978 

(106.50 - 131.79) (86.28 - 162.85) (-46.46 - 23.21) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-6. Summary of Site ARNE Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 6.42 (0.38) 5.94 (0.38) 0.48 (0.43) -0.57, 1.53 0.305 

(5.35 - 7.91) (5.32 - 6.54) (-1.19 - 1.62) 

Carbohydrates 66.80 (0.56) 67.35 (0.56) -0.55 (0.79) -2.49, 1.39 0.516 

(65.75 - 67.72) (65.78 - 69.37) (-2.29 - 1.94) 

Moisture (% fwt) 75.53 (0.54) 72.63 (0.54) 2.90 (0.65) 1.30, 4.50 0.004 

(74.60 - 77.50) (71.60 - 73.70) (0.90 - 3.90) 

Protein 22.25 (0.31) 21.38 (0.31) 0.87 (0.40) -0.10, 1.85 0.071 

(21.60 - 22.99) (20.35 - 22.32) (0.12 - 1.62) 

Total Fat 4.41 (0.31) 5.39 (0.31) -0.97 (0.44) -2.05, 0.10 0.068 

(3.64 - 4.96) (5.04 - 6.10) (-2.46 - -0.076) 

Fiber (% dwt)Acid Detergent Fiber 32.48 (1.30) 31.88 (1.30) 0.60 (1.36) -2.73, 3.93 0.673 

(30.71 - 34.07) (26.14 - 35.21) (-2.21 - 6.09) 
Monsanto Company 11-SY-217U 

Table F-6. Summary of Site ARNE Soybean Forage Nutrients for MON 87712 vs. Conventional Control (c

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 37.01 (1.72) 36.71 (1.72) 0.30 (1.99) -4.56, 5.16 0.884 

(33.46 - 41.73) (33.69 - 39.79) (-4.41 - 6.39) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.86 (0.14) 5.05 (0.14) -0.19 (0.19) -0.66, 0.28 0.357 

(4.48 - 5.27) (4.64 - 5.24) (-0.73 - 0.62) 

Carbohydrates 37.26 (0.54) 37.66 (0.54) -0.40 (0.76) -2.26, 1.45 0.614 

(35.09 - 38.66) (37.17 - 38.16) (-3.07 - 1.01) 

Moisture (% fwt) 8.34 (0.27) 8.06 (0.27) 0.29 (0.38) -0.63, 1.20 0.476 

(7.78 - 9.13) (7.44 - 8.40) (-0.18 - 0.73) 

Protein 41.34 (0.37) 40.95 (0.37) 0.39 (0.51) -0.85, 1.64 0.465 

(39.62 - 42.09) (40.59 - 41.27) (-1.65 - 1.22) 

Total Fat 16.56 (0.41) 16.34 (0.41) 0.22 (0.58) -1.19, 1.63 0.717 

(14.77 - 17.60) (15.83 - 16.75) (-1.88 - 1.47) 

Fiber (% dwt)Acid Detergent Fiber 15.30 (0.51) 13.46 (0.51) 1.84 (0.42) 0.82, 2.86 0.004 

(13.99 - 16.40) (12.87 - 14.08) (1.13 - 2.47) 
Monsanto Company 11-SY-217U 

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (cont

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 16.42 (0.46) 14.58 (0.46) 1.84 (0.54) 0.52, 3.17 0.014 

(15.64 - 17.27) (13.40 - 15.37) (1.28 - 2.76) 

Amino Acid (% dwt)Alanine 1.81 (0.016) 1.76 (0.016) 0.049 (0.020) -0.00087, 0.099 0.052 

(1.78 - 1.85) (1.74 - 1.78) (0.038 - 0.069) 

Arginine 3.28 (0.034) 3.11 (0.034) 0.18 (0.045) 0.070, 0.29 0.007 

(3.23 - 3.38) (3.03 - 3.20) (0.11 - 0.23) 

Aspartic Acid 4.84 (0.035) 4.75 (0.035) 0.097 (0.049) -0.023, 0.22 0.096 

(4.77 - 4.91) (4.69 - 4.83) (0.078 - 0.13) 

Cystine 0.61 (0.0072) 0.63 (0.0072) -0.024 (0.010) -0.049, 0.0011 0.058 

(0.59 - 0.62) (0.61 - 0.66) (-0.051 - 0.00050) 

Glutamic Acid 7.78 (0.065) 7.57 (0.065) 0.21 (0.081) 0.010, 0.41 0.042 

(7.63 - 7.87) (7.46 - 7.66) (0.17 - 0.29) 
Monsanto Company 11-SY-217U 

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (cont

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.84 (0.011) 1.79 (0.011) 0.044 (0.016) 0.0041, 0.083 0.035 

(1.80 - 1.85) (1.77 - 1.81) (0.029 - 0.061) 

Histidine 1.10 (0.0083) 1.08 (0.0083) 0.024 (0.012) -0.0043, 0.053 0.082 

(1.08 - 1.12) (1.07 - 1.09) (0.011 - 0.033) 

Isoleucine 1.94 (0.018) 1.87 (0.018) 0.071 (0.025) 0.010, 0.13 0.029 

(1.87 - 1.97) (1.84 - 1.91) (0.029 - 0.13) 

Leucine 3.23 (0.021) 3.15 (0.021) 0.073 (0.028) 0.0038, 0.14 0.041 

(3.18 - 3.27) (3.12 - 3.20) (0.059 - 0.087) 

Lysine 2.71 (0.018) 2.68 (0.018) 0.036 (0.026) -0.027, 0.098 0.211 

(2.66 - 2.74) (2.66 - 2.70) (-0.0052 - 0.053) 

Methionine 0.58 (0.0049) 0.59 (0.0049) -0.0082 (0.0067) -0.025, 0.0082 0.265 

(0.57 - 0.59) (0.58 - 0.60) (-0.025 - 0.012) 
Monsanto Company 11-SY-217U 

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (cont

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.22 (0.016) 2.15 (0.016) 0.070 (0.023) 0.014, 0.13 0.022 

(2.17 - 2.26) (2.13 - 2.17) (0.039 - 0.083) 

Proline 2.04 (0.029) 2.00 (0.029) 0.036 (0.040) -0.063, 0.13 0.409 

(1.91 - 2.09) (1.95 - 2.06) (-0.15 - 0.14) 

Serine 2.22 (0.028) 2.20 (0.028) 0.018 (0.035) -0.069, 0.10 0.632 

(2.21 - 2.22) (2.18 - 2.24) (-0.024 - 0.039) 

Threonine 1.63 (0.013) 1.62 (0.013) 0.011 (0.019) -0.035, 0.056 0.591 

(1.58 - 1.66) (1.59 - 1.65) (-0.0065 - 0.040) 

Tryptophan 0.43 (0.015) 0.46 (0.015) -0.036 (0.022) -0.089, 0.017 0.146 

(0.36 - 0.47) (0.44 - 0.49) (-0.094 - 0.010) 

Tyrosine 1.50 (0.031) 1.45 (0.031) 0.057 (0.044) -0.050, 0.16 0.242 

(1.42 - 1.57) (1.40 - 1.51) (-0.090 - 0.18) 
Monsanto Company 11-SY-217U 

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (cont

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 2.05 (0.022) 1.96 (0.022) 0.083 (0.032) 0.0052, 0.16 0.040 

(1.95 - 2.08) (1.92 - 2.01) (0.018 - 0.15) 

Fatty Acid (% Total FA)16:0 Palmitic 11.79 (0.044) 11.85 (0.044) -0.065 (0.038) -0.16, 0.029 0.141 

(11.73 - 11.86) (11.76 - 11.97) (-0.17 - 0.013) 

18:0 Stearic 3.77 (0.049) 4.00 (0.049) -0.22 (0.039) -0.32, -0.13 0.001 

(3.71 - 3.91) (3.94 - 4.14) (-0.25 - -0.18) 

18:1 Oleic 19.99 (0.29) 20.31 (0.29) -0.32 (0.34) -1.15, 0.51 0.380 

(19.88 - 20.04) (19.60 - 21.45) (-1.57 - 0.44) 

18:2 Linoleic 54.93 (0.21) 54.34 (0.21) 0.59 (0.23) 0.017, 1.16 0.045 

(54.84 - 55.03) (53.49 - 54.78) (0.059 - 1.50) 

18:3 Linolenic 8.89 (0.15) 8.76 (0.15) 0.13 (0.17) -0.29, 0.55 0.469 

(8.69 - 9.13) (8.17 - 9.09) (-0.38 - 0.52) 
Monsanto Company 11-SY-217U 

Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (cont

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.27 (0.0090) 0.30 (0.0090) -0.027 (0.013) -0.058, 0.0045 0.081 

(0.25 - 0.29) (0.29 - 0.31) (-0.042 - -0.018) 

20:1 Eicosenoic 0.14 (0.012) 0.16 (0.012) -0.027 (0.017) -0.068, 0.014 0.155 

(0.077 - 0.16) (0.16 - 0.17) (-0.082 - -0.0059) 

22:0 Behenic 0.23 (0.021) 0.29 (0.021) -0.062 (0.029) -0.13, 0.0094 0.077 

(0.18 - 0.28) (0.29 - 0.30) (-0.11 - -0.012) 

Vitamin (mg/100g dwt)Vitamin E 1.03 (0.056) 1.18 (0.056) -0.15 (0.079) -0.34, 0.043 0.106 

(0.89 - 1.14) (1.09 - 1.28) (-0.28 - -0.0056) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-8. Summary of Site ILCY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 2.24 (0.44) 2.60 (0.44) -0.36 (0.39) -1.32, 0.60 0.394 

(1.71 - 2.56) (1.42 - 3.69) (-1.35 - 0.94) 

Phytic Acid (% dwt) 1.22 (0.038) 1.25 (0.038) -0.029 (0.054) -0.16, 0.10 0.610 

(1.10 - 1.28) (1.22 - 1.30) (-0.20 - 0.048) 

Raffinose (% dwt) 0.78 (0.016) 0.70 (0.016) 0.076 (0.019) 0.030, 0.12 0.006 

(0.72 - 0.83) (0.68 - 0.72) (0.036 - 0.12) 

Stachyose (% dwt) 4.02 (0.091) 4.24 (0.091) -0.22 (0.12) -0.52, 0.070 0.111 

(3.77 - 4.43) (4.18 - 4.31) (-0.41 - 0.12) 

Trypsin Inhibitor (TIU/mg dwt) 32.64 (2.52) 33.68 (2.52) -1.04 (3.56) -9.76, 7.68 0.780 

(30.15 - 36.92) (29.00 - 37.06) (-6.91 - 4.12) 

Isoflavone (μg/g dwt)Daidzein 1056.89 (32.01) 1049.27 (32.01) 7.63 (45.27) -103.14, 118.40 0.871 

(980.67 - 1126.05) (943.09 - 1120.78) (-140.11 - 182.96) 
Monsanto Company 11-SY-217U 

Table F-8. Summary of Site ILCY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 915.27 (22.00) 863.79 (22.00) 51.48 (30.69) -23.62, 126.58 0.144 

(877.25 - 957.41) (802.44 - 912.95) (-31.11 - 142.13) 

Glycitein 92.70 (6.02) 97.78 (6.02) -5.08 (8.52) -25.93, 15.77 0.573 

(82.10 - 107.24) (89.62 - 121.18) (-23.24 - 16.92) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-9. Summary of Site ILCY Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 7.68 (0.20) 7.22 (0.20) 0.47 (0.23) -0.11, 1.04 0.093 

(7.49 - 7.88) (7.10 - 7.45) (0.28 - 0.70) 

Carbohydrates 65.33 (0.77) 65.29 (0.77) 0.039 (0.91) -2.18, 2.26 0.967 

(63.14 - 66.31) (64.37 - 66.28) (-1.35 - 1.94) 

Moisture (% fwt) 72.85 (0.43) 74.70 (0.43) -1.85 (0.46) -2.97, -0.73 0.006 

(71.80 - 74.50) (73.80 - 75.50) (-3.50 - -1.00) 

Protein 22.12 (0.59) 22.05 (0.59) 0.069 (0.74) -1.74, 1.88 0.928 

(21.06 - 24.04) (21.72 - 22.49) (-1.04 - 1.55) 

Total Fat 4.83 (0.30) 5.39 (0.30) -0.56 (0.42) -1.58, 0.45 0.224 

(4.69 - 4.93) (4.57 - 6.15) (-1.33 - 0.12) 

Fiber (% dwt)Acid Detergent Fiber 29.44 (0.60) 28.68 (0.60) 0.76 (0.85) -1.31, 2.83 0.403 

(28.24 - 30.21) (28.09 - 30.32) (-0.11 - 1.68) 
Monsanto Company 11-SY-217U 

Table F-9. Summary of Site ILCY Soybean Forage Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 33.99 (1.04) 31.13 (1.04) 2.86 (1.48) -0.76, 6.47 0.101 

(33.35 - 34.71) (28.38 - 32.98) (1.20 - 5.66) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.67 (0.12) 4.80 (0.12) -0.13 (0.17) -0.54, 0.29 0.480 

(4.56 - 4.91) (4.46 - 5.34) (-0.73 - 0.22) 

Carbohydrates 35.62 (0.34) 35.84 (0.34) -0.22 (0.48) -1.39, 0.94 0.657 

(34.64 - 36.63) (35.50 - 36.10) (-1.27 - 1.13) 

Moisture (% fwt) 8.34 (0.16) 8.20 (0.16) 0.14 (0.21) -0.37, 0.64 0.536 

(8.00 - 8.49) (7.89 - 8.49) (-0.020 - 0.38) 

Protein 42.14 (0.36) 41.39 (0.36) 0.75 (0.50) -0.49, 1.98 0.188 

(40.98 - 42.79) (41.09 - 42.12) (-1.15 - 1.56) 

Total Fat 17.56 (0.23) 17.97 (0.23) -0.41 (0.22) -0.94, 0.12 0.108 

(16.83 - 18.14) (17.70 - 18.28) (-0.88 - 0.13) 

Fiber (% dwt)Acid Detergent Fiber 14.48 (0.58) 16.40 (0.58) -1.91 (0.82) -3.92, 0.10 0.059 

(13.21 - 16.72) (15.89 - 16.91) (-3.70 - 0.83) 
Monsanto Company 11-SY-217U 

Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 15.77 (0.35) 16.45 (0.35) -0.68 (0.50) -1.90, 0.53 0.218 

(14.35 - 16.83) (15.96 - 17.05) (-1.61 - 0.83) 

Amino Acid (% dwt)Alanine 1.81 (0.018) 1.79 (0.018) 0.019 (0.025) -0.043, 0.081 0.482 

(1.76 - 1.82) (1.75 - 1.80) (-0.041 - 0.073) 

Arginine 3.21 (0.060) 3.16 (0.060) 0.046 (0.084) -0.16, 0.25 0.605 

(3.05 - 3.31) (3.11 - 3.26) (-0.20 - 0.19) 

Aspartic Acid 4.84 (0.060) 4.73 (0.060) 0.11 (0.085) -0.10, 0.31 0.260 

(4.68 - 4.92) (4.65 - 4.83) (-0.15 - 0.24) 

Cystine 0.58 (0.0096) 0.59 (0.0096) -0.011 (0.011) -0.038, 0.017 0.375 

(0.57 - 0.60) (0.57 - 0.60) (-0.031 - 0.0059) 

Glutamic Acid 7.79 (0.11) 7.62 (0.11) 0.17 (0.15) -0.19, 0.54 0.291 

(7.49 - 7.96) (7.49 - 7.74) (-0.25 - 0.38) 
Monsanto Company 11-SY-217U 

Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.82 (0.018) 1.80 (0.018) 0.025 (0.025) -0.037, 0.086 0.369 

(1.77 - 1.84) (1.77 - 1.81) (-0.041 - 0.062) 

Histidine 1.10 (0.015) 1.07 (0.015) 0.021 (0.021) -0.030, 0.073 0.351 

(1.06 - 1.11) (1.05 - 1.09) (-0.024 - 0.051) 

Isoleucine 1.94 (0.025) 1.92 (0.025) 0.022 (0.035) -0.064, 0.11 0.553 

(1.88 - 1.98) (1.89 - 1.96) (-0.063 - 0.073) 

Leucine 3.23 (0.038) 3.18 (0.038) 0.046 (0.053) -0.085, 0.18 0.424 

(3.12 - 3.30) (3.12 - 3.24) (-0.12 - 0.12) 

Lysine 2.68 (0.025) 2.66 (0.025) 0.029 (0.036) -0.059, 0.12 0.454 

(2.62 - 2.72) (2.62 - 2.69) (-0.073 - 0.068) 

Methionine 0.57 (0.0074) 0.57 (0.0074) -0.00079 (0.0086) -0.022, 0.020 0.929 

(0.56 - 0.59) (0.56 - 0.58) (-0.011 - 0.012) 
Monsanto Company 11-SY-217U 

Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.22 (0.033) 2.17 (0.033) 0.044 (0.047) -0.070, 0.16 0.380 

(2.13 - 2.26) (2.14 - 2.20) (-0.073 - 0.11) 

Proline 2.03 (0.038) 2.05 (0.038) -0.013 (0.039) -0.11, 0.082 0.745 

(1.96 - 2.14) (1.99 - 2.11) (-0.095 - 0.056) 

Serine 2.21 (0.024) 2.16 (0.024) 0.044 (0.035) -0.040, 0.13 0.248 

(2.16 - 2.25) (2.11 - 2.23) (-0.019 - 0.14) 

Threonine 1.63 (0.019) 1.59 (0.019) 0.035 (0.027) -0.031, 0.10 0.239 

(1.59 - 1.65) (1.55 - 1.66) (-0.074 - 0.083) 

Tryptophan 0.46 (0.011) 0.46 (0.011) 0.00096 (0.016) -0.038, 0.040 0.954 

(0.44 - 0.49) (0.44 - 0.48) (-0.037 - 0.028) 

Tyrosine 1.49 (0.027) 1.48 (0.027) 0.010 (0.038) -0.082, 0.10 0.790 

(1.43 - 1.56) (1.43 - 1.50) (-0.063 - 0.072) 
Monsanto Company 11-SY-217U 

Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 2.03 (0.027) 2.00 (0.027) 0.028 (0.039) -0.067, 0.12 0.503 

(1.96 - 2.07) (1.97 - 2.05) (-0.063 - 0.087) 

Fatty Acid (% Total FA)16:0 Palmitic 11.37 (0.055) 11.55 (0.055) -0.18 (0.036) -0.27, -0.092 0.002 

(11.30 - 11.49) (11.46 - 11.72) (-0.23 - -0.070) 

18:0 Stearic 4.37 (0.073) 4.50 (0.073) -0.12 (0.074) -0.30, 0.058 0.147 

(4.16 - 4.58) (4.32 - 4.64) (-0.36 - 0.0014) 

18:1 Oleic 21.58 (0.25) 20.84 (0.25) 0.74 (0.34) -0.083, 1.57 0.070 

(21.05 - 21.95) (20.62 - 20.98) (0.26 - 1.32) 

18:2 Linoleic 54.24 (0.23) 54.41 (0.23) -0.16 (0.32) -0.94, 0.62 0.629 

(53.94 - 54.60) (54.19 - 54.61) (-0.44 - 0.052) 

18:3 Linolenic 7.65 (0.10) 7.92 (0.10) -0.27 (0.13) -0.59, 0.044 0.079 

(7.46 - 8.01) (7.77 - 8.17) (-0.53 - 0.23) 
Monsanto Company 11-SY-217U 

Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.32 (0.011) 0.32 (0.011) -0.0063 (0.014) -0.041, 0.028 0.674 

(0.28 - 0.34) (0.30 - 0.34) (-0.043 - 0.047) 

20:1 Eicosenoic 0.17 (0.0035) 0.17 (0.0035) 0.0014 (0.0047) -0.010, 0.013 0.780 

(0.16 - 0.18) (0.16 - 0.18) (-0.0097 - 0.019) 

22:0 Behenic 0.30 (0.017) 0.29 (0.017) 0.0039 (0.024) -0.055, 0.063 0.876 

(0.23 - 0.33) (0.23 - 0.32) (-0.073 - 0.096) 

Vitamin (mg/100g dwt)Vitamin E 1.65 (0.16) 1.85 (0.16) -0.20 (0.21) -0.73, 0.32 0.376 

(1.30 - 2.05) (1.61 - 2.06) (-0.76 - 0.44) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-11. Summary of Site ILHI Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 1.84 (0.34) 2.66 (0.34) -0.81 (0.37) -1.71, 0.080 0.067 

(1.49 - 2.46) (1.88 - 4.15) (-1.69 - 0.035) 

Phytic Acid (% dwt) 0.89 (0.072) 1.14 (0.072) -0.25 (0.10) -0.50, 0.0038 0.052 

(0.65 - 1.11) (1.02 - 1.23) (-0.58 - 0.091) 

Raffinose (% dwt) 0.78 (0.017) 0.90 (0.017) -0.12 (0.024) -0.18, -0.061 0.002 

(0.76 - 0.80) (0.84 - 0.93) (-0.17 - -0.047) 

Stachyose (% dwt) 4.50 (0.11) 4.21 (0.11) 0.29 (0.16) -0.097, 0.68 0.115 

(4.44 - 4.58) (3.69 - 4.48) (0.051 - 0.90) 

Trypsin Inhibitor (TIU/mg dwt) 35.70 (2.98) 32.16 (2.98) 3.54 (4.22) -6.79, 13.86 0.433 

(32.45 - 40.72) (26.40 - 35.84) (-3.39 - 14.32) 

Isoflavone (μg/g dwt)Daidzein 911.92 (40.49) 946.01 (40.49) -34.09 (57.27) -174.22, 106.04 0.573 

(811.93 - 1017.39) (827.27 - 1050.16) (-150.09 - 190.12) 
Monsanto Company 11-SY-217U 

Table F-11. Summary of Site ILHI Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 750.00 (30.31) 754.26 (30.31) -4.26 (37.35) -95.65, 87.13 0.912 

(679.71 - 829.24) (661.17 - 849.09) (-76.63 - 122.53) 

Glycitein 86.98 (8.44) 96.44 (8.44) -9.47 (11.94) -38.68, 19.75 0.458 

(78.13 - 99.75) (77.73 - 115.62) (-32.22 - 17.24) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-12. Summary of Site ILHI Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 5.99 (0.20) 5.55 (0.20) 0.44 (0.21) -0.081, 0.96 0.084 

(5.66 - 6.28) (4.79 - 6.11) (-0.11 - 0.87) 

Carbohydrates 63.53 (0.73) 62.90 (0.73) 0.63 (0.48) -0.54, 1.80 0.235 

(61.74 - 64.50) (61.46 - 64.97) (-0.66 - 1.52) 

Moisture (% fwt) 69.00 (0.54) 68.35 (0.54) 0.65 (0.58) -0.76, 2.06 0.302 

(67.60 - 70.20) (66.60 - 69.90) (-1.30 - 2.30) 

Protein 22.29 (0.55) 22.15 (0.55) 0.14 (0.28) -0.53, 0.82 0.626 

(21.30 - 23.56) (20.96 - 23.15) (-0.45 - 0.83) 

Total Fat 8.15 (0.27) 9.36 (0.27) -1.21 (0.32) -1.98, -0.44 0.008 

(7.44 - 8.62) (9.06 - 9.80) (-1.89 - -0.63) 

Fiber (% dwt)Acid Detergent Fiber 26.24 (0.68) 25.53 (0.68) 0.71 (0.97) -1.66, 3.07 0.492 

(24.36 - 27.87) (24.69 - 25.95) (-1.58 - 2.45) 
Monsanto Company 11-SY-217U 

Table F-12. Summary of Site ILHI Soybean Forage Nutrients for MON 87712 vs. Conventional Control (c

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 29.16 (0.84) 26.91 (0.84) 2.25 (0.93) -0.023, 4.52 0.051 

(28.30 - 30.60) (24.92 - 29.59) (-0.96 - 4.19) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 5.41 (0.22) 5.33 (0.22) 0.084 (0.31) -0.67, 0.84 0.794 

(4.98 - 6.36) (4.94 - 5.60) (-0.45 - 1.42) 

Carbohydrates 40.46 (0.46) 39.22 (0.46) 1.24 (0.40) 0.27, 2.21 0.020 

(39.98 - 41.50) (37.73 - 40.20) (-0.024 - 2.47) 

Moisture (% fwt) 8.38 (0.15) 8.16 (0.15) 0.22 (0.19) -0.25, 0.69 0.291 

(7.95 - 8.70) (7.72 - 8.55) (-0.32 - 0.94) 

Protein 39.78 (0.26) 40.09 (0.26) -0.31 (0.35) -1.17, 0.54 0.407 

(38.87 - 40.53) (39.57 - 40.42) (-1.55 - 0.39) 

Total Fat 14.35 (0.35) 15.35 (0.35) -1.00 (0.38) -1.94, -0.066 0.039 

(13.91 - 14.60) (14.41 - 16.73) (-2.38 - 0.15) 

Fiber (% dwt)Acid Detergent Fiber 16.59 (1.06) 16.54 (1.06) 0.047 (1.43) -3.44, 3.54 0.974 

(15.47 - 17.49) (14.65 - 20.81) (-4.82 - 2.76) 
Monsanto Company 11-SY-217U 

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 16.76 (0.89) 17.50 (0.89) -0.75 (1.26) -3.83, 2.34 0.576 

(15.22 - 19.93) (16.84 - 18.31) (-1.62 - 1.61) 

Amino Acid (% dwt)Alanine 1.73 (0.016) 1.73 (0.016) -0.0013 (0.015) -0.039, 0.036 0.933 

(1.72 - 1.75) (1.68 - 1.76) (-0.028 - 0.039) 

Arginine 3.03 (0.044) 3.04 (0.044) -0.015 (0.036) -0.10, 0.072 0.693 

(2.93 - 3.08) (2.96 - 3.10) (-0.065 - 0.058) 

Aspartic Acid 4.55 (0.051) 4.59 (0.051) -0.038 (0.049) -0.16, 0.082 0.463 

(4.44 - 4.64) (4.45 - 4.70) (-0.16 - 0.093) 

Cystine 0.56 (0.0064) 0.57 (0.0064) -0.015 (0.0082) -0.035, 0.0053 0.122 

(0.56 - 0.56) (0.57 - 0.58) (-0.021 - -0.0036) 

Glutamic Acid 7.34 (0.091) 7.44 (0.091) -0.10 (0.083) -0.31, 0.10 0.262 

(7.18 - 7.46) (7.18 - 7.70) (-0.37 - 0.097) 
Monsanto Company 11-SY-217U 

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.74 (0.013) 1.76 (0.013) -0.015 (0.013) -0.046, 0.016 0.279 

(1.72 - 1.76) (1.72 - 1.78) (-0.041 - 0.013) 

Histidine 1.04 (0.012) 1.06 (0.012) -0.017 (0.0098) -0.041, 0.0065 0.125 

(1.03 - 1.05) (1.03 - 1.09) (-0.045 - -0.00034) 

Isoleucine 1.82 (0.021) 1.84 (0.021) -0.020 (0.025) -0.082, 0.041 0.452 

(1.79 - 1.85) (1.80 - 1.88) (-0.072 - 0.024) 

Leucine 3.05 (0.028) 3.08 (0.028) -0.026 (0.027) -0.091, 0.040 0.378 

(3.00 - 3.09) (2.99 - 3.13) (-0.082 - 0.026) 

Lysine 2.59 (0.026) 2.62 (0.026) -0.038 (0.022) -0.092, 0.017 0.145 

(2.54 - 2.63) (2.55 - 2.69) (-0.11 - 0.0034) 

Methionine 0.55 (0.0054) 0.56 (0.0054) -0.0028 (0.0077) -0.022, 0.016 0.731 

(0.54 - 0.56) (0.55 - 0.57) (-0.026 - 0.016) 
Monsanto Company 11-SY-217U 

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.09 (0.027) 2.10 (0.027) -0.014 (0.022) -0.069, 0.040 0.547 

(2.05 - 2.11) (2.04 - 2.15) (-0.062 - 0.025) 

Proline 1.98 (0.035) 1.94 (0.035) 0.037 (0.037) -0.053, 0.13 0.350 

(1.83 - 2.04) (1.85 - 1.98) (-0.025 - 0.079) 

Serine 2.08 (0.031) 2.12 (0.031) -0.042 (0.024) -0.10, 0.017 0.135 

(2.01 - 2.13) (2.05 - 2.19) (-0.13 - 0.0028) 

Threonine 1.57 (0.022) 1.55 (0.022) 0.017 (0.026) -0.047, 0.082 0.534 

(1.51 - 1.61) (1.52 - 1.61) (-0.0063 - 0.046) 

Tryptophan 0.42 (0.010) 0.43 (0.010) -0.011 (0.015) -0.047, 0.024 0.468 

(0.39 - 0.44) (0.40 - 0.46) (-0.047 - 0.042) 

Tyrosine 1.45 (0.027) 1.44 (0.027) 0.0089 (0.037) -0.081, 0.098 0.815 

(1.40 - 1.47) (1.37 - 1.49) (-0.027 - 0.080) 
Monsanto Company 11-SY-217U 

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 1.93 (0.023) 1.95 (0.023) -0.017 (0.027) -0.083, 0.049 0.548 

(1.89 - 1.96) (1.91 - 2.00) (-0.072 - 0.035) 

Fatty Acid (% Total FA)16:0 Palmitic 11.23 (0.031) 11.28 (0.031) -0.049 (0.044) -0.16, 0.059 0.306 

(11.21 - 11.26) (11.22 - 11.33) (-0.12 - -0.0020) 

18:0 Stearic 4.09 (0.062) 4.27 (0.062) -0.18 (0.075) -0.36, 0.0023 0.052 

(3.99 - 4.21) (4.11 - 4.44) (-0.29 - -0.075) 

18:1 Oleic 20.03 (0.21) 19.84 (0.21) 0.19 (0.30) -0.54, 0.92 0.549 

(19.88 - 20.16) (19.41 - 20.17) (-0.0059 - 0.54) 

18:2 Linoleic 54.42 (0.19) 54.42 (0.19) 0.0039 (0.26) -0.64, 0.65 0.988 

(54.31 - 54.55) (53.98 - 54.81) (-0.26 - 0.32) 

18:3 Linolenic 9.61 (0.078) 9.50 (0.078) 0.11 (0.11) -0.15, 0.37 0.327 

(9.45 - 9.78) (9.34 - 9.71) (-0.087 - 0.44) 
Monsanto Company 11-SY-217U 

Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.29 (0.0070) 0.30 (0.0070) -0.014 (0.0099) -0.038, 0.010 0.203 

(0.27 - 0.31) (0.28 - 0.32) (-0.042 - 0.013) 

20:1 Eicosenoic 0.081 (0.016) 0.12 (0.016) -0.036 (0.020) -0.085, 0.012 0.118 

(0.080 - 0.083) (0.080 - 0.16) (-0.077 - 0.0032) 

22:0 Behenic 0.25 (0.023) 0.28 (0.023) -0.024 (0.032) -0.10, 0.055 0.485 

(0.21 - 0.30) (0.22 - 0.30) (-0.082 - 0.060) 

Vitamin (mg/100g dwt)Vitamin E 0.89 (0.11) 1.19 (0.11) -0.30 (0.15) -0.68, 0.069 0.093 

(0.81 - 0.94) (1.02 - 1.37) (-0.44 - -0.13) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-14. Summary of Site ILWY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contr

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 1.91 (0.23) 1.99 (0.23) -0.082 (0.33) -0.89, 0.72 0.812 

(1.37 - 2.23) (1.02 - 2.43) (-1.05 - 1.07) 

Phytic Acid (% dwt) 1.37 (0.072) 1.33 (0.072) 0.038 (0.10) -0.21, 0.29 0.719 

(1.33 - 1.48) (1.14 - 1.44) (-0.12 - 0.34) 

Raffinose (% dwt) 0.68 (0.023) 0.66 (0.023) 0.013 (0.032) -0.065, 0.091 0.689 

(0.63 - 0.69) (0.64 - 0.70) (-0.065 - 0.057) 

Stachyose (% dwt) 4.29 (0.055) 4.21 (0.055) 0.084 (0.078) -0.11, 0.27 0.322 

(4.14 - 4.37) (4.15 - 4.31) (-0.025 - 0.20) 

Trypsin Inhibitor (TIU/mg dwt) 35.39 (4.06) 37.57 (4.06) -2.18 (4.78) -13.87, 9.52 0.664 

(32.35 - 39.32) (27.58 - 53.09) (-20.74 - 8.70) 

Isoflavone (μg/g dwt)Daidzein 1397.02 (59.70) 1252.03 (59.70) 144.98 (71.95) -31.08, 321.05 0.090 

(1226.73 - 1609.37) (1126.30 - 1362.69) (-20.09 - 384.84) 
Monsanto Company 11-SY-217U 

Table F-14. Summary of Site ILWY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 1039.09 (47.68) 933.81 (47.68) 105.29 (61.87) -46.10, 256.67 0.139 

(929.19 - 1204.29) (810.28 - 1044.37) (-14.02 - 266.93) 

Glycitein 117.88 (4.74) 96.68 (4.74) 21.19 (5.09) 8.75, 33.64 0.005 

(112.20 - 122.62) (85.40 - 110.11) (9.72 - 33.87) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-15. Summary of Site ILWY Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 6.18 (0.18) 6.24 (0.18) -0.059 (0.26) -0.69, 0.57 0.825 

(5.82 - 6.53) (5.90 - 6.51) (-0.38 - 0.35) 

Carbohydrates 65.05 (0.44) 64.69 (0.44) 0.36 (0.47) -0.79, 1.51 0.473 

(63.87 - 66.05) (64.08 - 66.17) (-0.37 - 1.97) 

Moisture (% fwt) 72.18 (0.81) 70.08 (0.81) 2.10 (1.07) -0.52, 4.72 0.097 

(71.30 - 72.90) (66.60 - 71.60) (1.00 - 4.70) 

Protein 21.74 (0.46) 21.90 (0.46) -0.16 (0.49) -1.35, 1.03 0.751 

(20.70 - 22.67) (21.11 - 22.57) (-1.87 - 0.92) 

Total Fat 7.09 (0.30) 7.19 (0.30) -0.091 (0.35) -0.94, 0.76 0.802 

(6.94 - 7.35) (6.69 - 8.16) (-1.12 - 0.44) 

Fiber (% dwt)Acid Detergent Fiber 29.54 (1.33) 29.48 (1.33) 0.052 (0.99) -2.38, 2.49 0.960 

(26.09 - 32.03) (27.01 - 31.44) (-2.34 - 1.96) 
Monsanto Company 11-SY-217U 

Table F-15. Summary of Site ILWY Soybean Forage Nutrients for MON 87712 vs. Conventional Control (

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 30.73 (0.99) 32.78 (0.99) -2.05 (1.40) -5.49, 1.39 0.194 

(29.85 - 31.55) (29.47 - 35.33) (-4.21 - 2.08) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.69 (0.19) 4.92 (0.17) -0.23 (0.17) -0.68, 0.22 0.246 

(4.52 - 5.04) (4.58 - 5.46) (-0.68 - -0.028) 

Carbohydrates 38.77 (0.46) 39.38 (0.40) -0.61 (0.58) -2.09, 0.87 0.335 

(38.33 - 39.39) (38.13 - 40.43) (-1.87 - 0.72) 

Moisture (% fwt) 8.04 (0.14) 7.45 (0.12) 0.59 (0.18) 0.13, 1.04 0.021 

(7.91 - 8.11) (7.17 - 7.95) (-0.040 - 0.94) 

Protein 41.36 (0.31) 40.71 (0.28) 0.65 (0.33) -0.19, 1.50 0.103 

(40.37 - 42.01) (39.86 - 41.17) (0.42 - 1.29) 

Total Fat 15.11 (0.39) 14.99 (0.34) 0.12 (0.46) -1.07, 1.30 0.811 

(15.02 - 15.31) (13.91 - 16.42) (-1.19 - 0.58) 

Fiber (% dwt)Acid Detergent Fiber 14.25 (0.70) 14.96 (0.61) -0.71 (0.84) -2.87, 1.45 0.435 

(13.93 - 14.69) (13.25 - 16.31) (-2.38 - 1.19) 
Monsanto Company 11-SY-217U 

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 15.95 (0.64) 16.12 (0.55) -0.17 (0.84) -2.35, 2.00 0.844 

(15.53 - 16.43) (14.23 - 17.57) (-1.40 - 1.30) 

Amino Acid (% dwt)Alanine 1.76 (0.012) 1.76 (0.011) 0.00036 (0.016) -0.042, 0.042 0.983 

(1.75 - 1.77) (1.74 - 1.80) (-0.055 - 0.034) 

Arginine 3.09 (0.037) 3.10 (0.033) -0.011 (0.042) -0.12, 0.096 0.808 

(3.02 - 3.18) (3.02 - 3.14) (-0.12 - 0.086) 

Aspartic Acid 4.70 (0.031) 4.71 (0.027) -0.00071 (0.041) -0.11, 0.11 0.986 

(4.65 - 4.73) (4.66 - 4.75) (-0.031 - 0.069) 

Cystine 0.59 (0.011) 0.60 (0.0095) -0.010 (0.013) -0.045, 0.025 0.491 

(0.59 - 0.60) (0.56 - 0.63) (-0.034 - 0.030) 

Glutamic Acid 7.55 (0.063) 7.54 (0.054) 0.0074 (0.083) -0.21, 0.22 0.932 

(7.49 - 7.60) (7.43 - 7.62) (-0.058 - 0.16) 
Monsanto Company 11-SY-217U 

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.78 (0.010) 1.77 (0.0089) 0.0050 (0.014) -0.030, 0.040 0.728 

(1.77 - 1.78) (1.76 - 1.80) (-0.023 - 0.029) 

Histidine 1.06 (0.0089) 1.07 (0.0077) -0.014 (0.012) -0.044, 0.016 0.287 

(1.04 - 1.08) (1.05 - 1.09) (-0.046 - 0.022) 

Isoleucine 1.87 (0.016) 1.87 (0.014) -0.0016 (0.022) -0.057, 0.054 0.943 

(1.83 - 1.90) (1.83 - 1.90) (-0.073 - 0.051) 

Leucine 3.13 (0.018) 3.14 (0.016) -0.0098 (0.024) -0.072, 0.052 0.700 

(3.10 - 3.15) (3.10 - 3.16) (-0.053 - 0.043) 

Lysine 2.68 (0.017) 2.69 (0.015) -0.015 (0.023) -0.075, 0.044 0.533 

(2.67 - 2.69) (2.65 - 2.73) (-0.055 - 0.038) 

Methionine 0.57 (0.011) 0.56 (0.0095) 0.00094 (0.015) -0.036, 0.038 0.950 

(0.56 - 0.57) (0.53 - 0.59) (-0.033 - 0.036) 
Monsanto Company 11-SY-217U 

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.13 (0.019) 2.15 (0.016) -0.020 (0.025) -0.083, 0.044 0.460 

(2.08 - 2.17) (2.11 - 2.16) (-0.077 - 0.054) 

Proline 2.00 (0.025) 2.03 (0.022) -0.036 (0.032) -0.12, 0.046 0.309 

(1.94 - 2.06) (1.99 - 2.08) (-0.077 - 0.075) 

Serine 2.17 (0.027) 2.20 (0.024) -0.021 (0.036) -0.11, 0.071 0.579 

(2.13 - 2.21) (2.16 - 2.25) (-0.12 - 0.033) 

Threonine 1.59 (0.020) 1.58 (0.017) 0.0074 (0.026) -0.060, 0.075 0.788 

(1.55 - 1.63) (1.56 - 1.61) (-0.060 - 0.064) 

Tryptophan 0.44 (0.0096) 0.46 (0.0084) -0.023 (0.013) -0.055, 0.010 0.137 

(0.43 - 0.45) (0.45 - 0.47) (-0.040 - -0.0074) 

Tyrosine 1.48 (0.018) 1.50 (0.016) -0.020 (0.024) -0.082, 0.041 0.435 

(1.47 - 1.49) (1.49 - 1.52) (-0.054 - -0.0066) 
Monsanto Company 11-SY-217U 

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 1.97 (0.019) 1.95 (0.016) 0.015 (0.025) -0.049, 0.079 0.569 

(1.93 - 1.99) (1.91 - 1.97) (-0.040 - 0.085) 

Fatty Acid (% Total FA)16:0 Palmitic 11.23 (0.067) 11.24 (0.063) -0.0071 (0.049) -0.13, 0.12 0.889 

(11.10 - 11.35) (11.13 - 11.44) (-0.093 - 0.091) 

18:0 Stearic 3.67 (0.11) 3.96 (0.11) -0.29 (0.048) -0.41, -0.16 0.001 

(3.54 - 3.84) (3.78 - 4.21) (-0.37 - -0.24) 

18:1 Oleic 19.32 (0.43) 19.60 (0.41) -0.27 (0.27) -0.97, 0.43 0.361 

(18.72 - 20.01) (18.72 - 20.50) (-0.49 - -0.0016) 

18:2 Linoleic 55.27 (0.36) 54.55 (0.33) 0.72 (0.34) -0.15, 1.58 0.085 

(54.66 - 55.60) (53.75 - 55.02) (0.46 - 0.91) 

18:3 Linolenic 9.85 (0.17) 9.98 (0.16) -0.14 (0.072) -0.32, 0.049 0.116 

(9.49 - 10.22) (9.66 - 10.31) (-0.31 - 0.0045) 
Monsanto Company 11-SY-217U 

Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.27 (0.0097) 0.29 (0.0085) -0.020 (0.011) -0.049, 0.0085 0.129 

(0.25 - 0.29) (0.27 - 0.31) (-0.023 - -0.017) 

20:1 Eicosenoic 0.13 (0.019) 0.12 (0.017) 0.0068 (0.020) -0.045, 0.059 0.750 

(0.079 - 0.16) (0.079 - 0.16) (-0.0043 - 

0.00068)

22:0 Behenic 0.25 (0.026) 0.26 (0.023) -0.0089 (0.035) -0.098, 0.080 0.806 

(0.19 - 0.29) (0.18 - 0.29) (-0.014 - 0.0073) 

Vitamin (mg/100g dwt)Vitamin E 0.96 (0.050) 0.86 (0.043) 0.092 (0.066) -0.077, 0.26 0.219 

(0.90 - 1.03) (0.79 - 0.95) (0.077 - 0.13) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-17. Summary of Site INRC Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contr

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 2.03 (0.45) 2.35 (0.39) -0.32 (0.59) -1.85, 1.20 0.608 

(1.22 - 3.41) (1.43 - 3.05) (-1.84 - 1.21) 

Phytic Acid (% dwt) 1.08 (0.065) 1.17 (0.057) -0.089 (0.075) -0.28, 0.10 0.291 

(0.96 - 1.17) (1.03 - 1.24) (-0.12 - -0.066) 

Raffinose (% dwt) 0.63 (0.046) 0.59 (0.042) 0.035 (0.049) -0.090, 0.16 0.500 

(0.54 - 0.70) (0.52 - 0.66) (0.015 - 0.062) 

Stachyose (% dwt) 4.59 (0.12) 4.70 (0.11) -0.11 (0.13) -0.45, 0.23 0.437 

(4.46 - 4.76) (4.48 - 4.88) (-0.17 - -0.019) 

Trypsin Inhibitor (TIU/mg dwt) 31.45 (2.42) 32.94 (2.15) -1.49 (2.65) -8.31, 5.33 0.598 

(32.21 - 32.86) (24.37 - 38.89) (-6.64 - -1.61) 

Isoflavone (μg/g dwt)Daidzein 1036.00 (48.75) 878.45 (42.22) 157.54 (64.50) -8.25, 323.33 0.058 

(978.39 - 1120.78) (770.23 - 1050.52) (-72.13 - 350.56) 
Monsanto Company 11-SY-217U 

Table F-17. Summary of Site INRC Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 918.56 (57.59) 759.54 (49.88) 159.02 (76.19) -36.83, 354.87 0.091 

(852.43 - 974.97) (583.34 - 924.50) (-72.07 - 391.63) 

Glycitein 97.52 (5.54) 89.93 (4.80) 7.59 (7.17) -10.85, 26.02 0.338 

(90.56 - 104.90) (80.16 - 102.96) (-3.52 - 24.74) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-18. Summary of Site INRC Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 6.65 (0.19) 6.17 (0.17) 0.47 (0.25) -0.16, 1.10 0.113 

(6.38 - 6.93) (5.63 - 6.57) (-0.089 - 0.97) 

Carbohydrates 60.79 (0.98) 61.41 (0.93) -0.63 (0.70) -2.42, 1.16 0.408 

(59.41 - 62.73) (58.14 - 63.43) (-2.60 - 1.27) 

Moisture (% fwt) 73.13 (0.48) 73.35 (0.41) -0.22 (0.63) -1.84, 1.41 0.745 

(72.90 - 73.60) (72.90 - 74.20) (-1.30 - 0.50) 

Protein 24.85 (0.76) 23.91 (0.74) 0.94 (0.45) -0.23, 2.10 0.092 

(23.06 - 26.20) (21.87 - 26.12) (0.075 - 1.35) 

Total Fat 7.87 (0.37) 8.51 (0.32) -0.64 (0.45) -1.80, 0.51 0.212 

(7.60 - 8.18) (7.84 - 9.22) (-1.61 - 0.34) 

Fiber (% dwt)Acid Detergent Fiber 27.04 (1.79) 29.14 (1.57) -2.10 (2.11) -7.54, 3.33 0.365 

(25.61 - 29.37) (24.46 - 31.30) (-5.70 - -0.94) 
Monsanto Company 11-SY-217U 

Table F-18. Summary of Site INRC Soybean Forage Nutrients for MON 87712 vs. Conventional Control (

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 31.87 (1.00) 32.74 (0.86) -0.87 (1.32) -4.25, 2.52 0.539 

(29.59 - 34.10) (30.22 - 34.42) (-3.39 - 0.76) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.65 (0.081) 4.77 (0.081) -0.12 (0.11) -0.40, 0.16 0.348 

(4.56 - 4.73) (4.55 - 4.94) (-0.21 - 0.0049) 

Carbohydrates 38.06 (0.64) 38.18 (0.64) -0.12 (0.76) -1.97, 1.73 0.881 

(37.20 - 38.51) (37.34 - 39.61) (-1.29 - 1.16) 

Moisture (% fwt) 8.24 (0.43) 8.60 (0.43) -0.37 (0.61) -1.85, 1.12 0.570 

(7.60 - 8.93) (7.09 - 10.30) (-1.69 - 1.84) 

Protein 41.55 (0.50) 41.15 (0.50) 0.40 (0.71) -1.34, 2.15 0.594 

(41.02 - 42.28) (39.72 - 42.14) (-0.56 - 2.56) 

Total Fat 15.75 (0.39) 15.92 (0.39) -0.17 (0.47) -1.33, 0.99 0.729 

(14.71 - 16.63) (15.14 - 16.88) (-1.26 - 0.87) 

Fiber (% dwt)Acid Detergent Fiber 15.91 (0.71) 14.90 (0.71) 1.01 (0.72) -0.75, 2.78 0.209 

(14.97 - 16.96) (12.85 - 17.01) (-0.86 - 2.24) 
Monsanto Company 11-SY-217U 

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 16.38 (0.57) 16.78 (0.57) -0.41 (0.81) -2.38, 1.56 0.632 

(15.58 - 17.62) (15.38 - 18.40) (-2.59 - 2.23) 

Amino Acid (% dwt)Alanine 1.79 (0.020) 1.78 (0.020) 0.014 (0.019) -0.032, 0.061 0.476 

(1.78 - 1.82) (1.73 - 1.83) (-0.047 - 0.057) 

Arginine 3.19 (0.054) 3.21 (0.054) -0.021 (0.072) -0.20, 0.15 0.776 

(3.13 - 3.29) (3.11 - 3.31) (-0.16 - 0.18) 

Aspartic Acid 4.79 (0.056) 4.79 (0.056) -0.0033 (0.063) -0.16, 0.15 0.959 

(4.75 - 4.84) (4.67 - 4.89) (-0.14 - 0.14) 

Cystine 0.56 (0.0084) 0.58 (0.0084) -0.025 (0.012) -0.054, 0.0044 0.083 

(0.55 - 0.58) (0.57 - 0.62) (-0.072 - 0.010) 

Glutamic Acid 7.74 (0.12) 7.76 (0.12) -0.022 (0.13) -0.34, 0.30 0.874 

(7.69 - 7.80) (7.54 - 7.97) (-0.27 - 0.25) 
Monsanto Company 11-SY-217U 

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.81 (0.019) 1.80 (0.019) 0.014 (0.020) -0.035, 0.064 0.506 

(1.80 - 1.82) (1.74 - 1.84) (-0.036 - 0.079) 

Histidine 1.08 (0.013) 1.09 (0.013) -0.0072 (0.013) -0.040, 0.025 0.603 

(1.06 - 1.09) (1.06 - 1.12) (-0.038 - 0.038) 

Isoleucine 1.93 (0.025) 1.90 (0.025) 0.036 (0.028) -0.033, 0.10 0.247 

(1.92 - 1.95) (1.86 - 1.96) (-0.038 - 0.075) 

Leucine 3.19 (0.041) 3.19 (0.041) 0.0030 (0.045) -0.11, 0.11 0.948 

(3.17 - 3.21) (3.10 - 3.26) (-0.088 - 0.11) 

Lysine 2.69 (0.026) 2.69 (0.026) -0.0058 (0.025) -0.066, 0.055 0.823 

(2.67 - 2.70) (2.63 - 2.75) (-0.070 - 0.064) 

Methionine 0.55 (0.0069) 0.57 (0.0069) -0.016 (0.0097) -0.039, 0.0081 0.157 

(0.54 - 0.57) (0.55 - 0.59) (-0.054 - 0.0071) 
Monsanto Company 11-SY-217U 

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.18 (0.031) 2.18 (0.031) 0.0020 (0.034) -0.081, 0.085 0.956 

(2.13 - 2.22) (2.12 - 2.24) (-0.063 - 0.098) 

Proline 1.99 (0.038) 2.02 (0.038) -0.038 (0.053) -0.17, 0.092 0.499 

(1.87 - 2.11) (1.98 - 2.07) (-0.20 - 0.13) 

Serine 2.16 (0.037) 2.22 (0.037) -0.061 (0.035) -0.15, 0.024 0.129 

(2.13 - 2.18) (2.14 - 2.34) (-0.16 - 0.032) 

Threonine 1.62 (0.012) 1.59 (0.012) 0.032 (0.017) -0.0085, 0.072 0.101 

(1.60 - 1.64) (1.56 - 1.63) (-0.00038 - 0.064) 

Tryptophan 0.46 (0.014) 0.44 (0.014) 0.015 (0.020) -0.034, 0.064 0.478 

(0.44 - 0.48) (0.38 - 0.47) (-0.023 - 0.10) 

Tyrosine 1.50 (0.026) 1.51 (0.026) -0.015 (0.037) -0.11, 0.077 0.707 

(1.47 - 1.55) (1.42 - 1.56) (-0.095 - 0.13) 
Monsanto Company 11-SY-217U 

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 2.04 (0.025) 1.99 (0.025) 0.044 (0.028) -0.026, 0.11 0.174 

(2.02 - 2.06) (1.96 - 2.07) (-0.050 - 0.095) 

Fatty Acid (% Total FA)16:0 Palmitic 10.99 (0.075) 11.19 (0.075) -0.19 (0.11) -0.45, 0.067 0.120 

(10.78 - 11.21) (11.16 - 11.22) (-0.44 - 0.050) 

18:0 Stearic 4.62 (0.057) 4.57 (0.057) 0.045 (0.080) -0.15, 0.24 0.593 

(4.45 - 4.72) (4.45 - 4.65) (-0.20 - 0.25) 

18:1 Oleic 22.26 (0.13) 21.25 (0.13) 1.01 (0.18) 0.57, 1.45 0.001 

(22.07 - 22.50) (21.07 - 21.54) (0.90 - 1.19) 

18:2 Linoleic 52.72 (0.14) 53.25 (0.14) -0.52 (0.19) -0.99, -0.054 0.034 

(52.33 - 53.25) (53.21 - 53.27) (-0.92 - 0.037) 

18:3 Linolenic 8.70 (0.098) 8.98 (0.098) -0.28 (0.14) -0.62, 0.060 0.090 

(8.54 - 8.87) (8.83 - 9.18) (-0.64 - -0.0039) 
Monsanto Company 11-SY-217U 

Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (con

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.31 (0.0096) 0.33 (0.0096) -0.016 (0.013) -0.048, 0.016 0.269 

(0.27 - 0.33) (0.31 - 0.34) (-0.066 - 0.0050) 

20:1 Eicosenoic 0.14 (0.016) 0.15 (0.016) -0.018 (0.014) -0.051, 0.016 0.243 

(0.081 - 0.16) (0.15 - 0.16) (-0.066 - 0.0011) 

22:0 Behenic 0.26 (0.019) 0.29 (0.019) -0.029 (0.024) -0.088, 0.030 0.276 

(0.22 - 0.31) (0.21 - 0.32) (-0.091 - 0.022) 

Vitamin (mg/100g dwt)Vitamin E 1.32 (0.053) 1.35 (0.053) -0.024 (0.075) -0.21, 0.16 0.765 

(1.28 - 1.37) (1.20 - 1.47) (-0.19 - 0.15) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-20. Summary of Site INSH Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 1.58 (0.22) 2.17 (0.22) -0.60 (0.31) -1.35, 0.16 0.103 

(1.10 - 1.97) (1.63 - 3.05) (-1.40 - 0.30) 

Phytic Acid (% dwt) 0.99 (0.042) 0.94 (0.042) 0.048 (0.060) -0.098, 0.19 0.453 

(0.90 - 1.08) (0.87 - 1.03) (-0.13 - 0.21) 

Raffinose (% dwt) 0.66 (0.034) 0.68 (0.034) -0.024 (0.048) -0.14, 0.094 0.638 

(0.58 - 0.75) (0.62 - 0.74) (-0.12 - 0.13) 

Stachyose (% dwt) 4.12 (0.078) 4.05 (0.078) 0.069 (0.11) -0.20, 0.33 0.546 

(3.91 - 4.29) (3.78 - 4.15) (-0.21 - 0.28) 

Trypsin Inhibitor (TIU/mg dwt) 30.60 (4.05) 30.29 (4.05) 0.31 (5.69) -13.62, 14.23 0.958 

(28.25 - 32.72) (20.34 - 41.47) (-8.75 - 9.85) 

Isoflavone (μg/g dwt)Daidzein 981.19 (40.89) 1055.58 (40.89) -74.39 (45.24) -185.08, 36.30 0.151 

(926.76 - 1105.15) (910.56 - 1132.28) (-187.49 - 44.95) 
Monsanto Company 11-SY-217U 

Table F-20. Summary of Site INSH Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 728.13 (30.47) 773.66 (30.47) -45.53 (32.81) -125.82, 34.76 0.214 

(674.24 - 809.72) (675.92 - 836.15) (-125.71 - 37.81) 

Glycitein 106.91 (6.85) 108.31 (6.85) -1.40 (9.69) -25.11, 22.31 0.889 

(83.78 - 131.31) (102.05 - 116.49) (-22.23 - 22.61) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-21. Summary of Site INSH Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.95 (0.21) 5.50 (0.21) -0.55 (0.27) -1.20, 0.11 0.086 

(4.55 - 5.28) (4.98 - 5.93) (-1.04 - 0.11) 

Carbohydrates 63.40 (0.91) 63.49 (0.91) -0.086 (1.20) -3.02, 2.84 0.944 

(62.62 - 64.84) (62.18 - 64.82) (-1.80 - 2.66) 

Moisture (% fwt) 68.85 (0.66) 68.38 (0.66) 0.48 (0.92) -1.78, 2.73 0.624 

(68.00 - 69.50) (67.30 - 69.30) (-0.40 - 1.40) 

Protein 23.23 (0.65) 22.59 (0.65) 0.64 (0.83) -1.40, 2.67 0.472 

(22.52 - 23.57) (20.98 - 24.01) (-1.49 - 2.53) 

Total Fat 8.45 (0.52) 8.41 (0.52) 0.039 (0.63) -1.50, 1.58 0.952 

(8.16 - 8.59) (7.59 - 9.33) (-0.83 - 1.00) 

Fiber (% dwt)Acid Detergent Fiber 25.96 (1.24) 27.36 (1.24) -1.40 (1.76) -5.70, 2.90 0.455 

(24.43 - 27.31) (24.68 - 29.02) (-4.54 - 0.90) 
Monsanto Company 11-SY-217U 

Table F-21. Summary of Site INSH Soybean Forage Nutrients for MON 87712 vs. Conventional Control (c

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 28.21 (1.41) 30.37 (1.41) -2.16 (1.78) -6.51, 2.19 0.270 

(26.56 - 30.13) (26.89 - 32.60) (-4.37 - 1.04) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 5.63 (0.092) 5.55 (0.092) 0.082 (0.13) -0.24, 0.40 0.553 

(5.25 - 5.85) (5.33 - 5.71) (-0.41 - 0.38) 

Carbohydrates 37.84 (0.65) 38.65 (0.65) -0.81 (0.90) -3.01, 1.40 0.406 

(36.54 - 38.71) (37.51 - 40.40) (-2.05 - 0.88) 

Moisture (% fwt) 6.80 (0.35) 6.86 (0.35) -0.055 (0.50) -1.28, 1.17 0.916 

(5.72 - 8.32) (6.44 - 7.33) (-0.72 - 1.62) 

Protein 39.25 (0.33) 38.62 (0.33) 0.62 (0.35) -0.23, 1.48 0.124 

(38.90 - 39.92) (37.73 - 39.23) (0.013 - 1.20) 

Total Fat 17.31 (0.41) 17.18 (0.41) 0.13 (0.58) -1.28, 1.53 0.834 

(16.57 - 17.89) (16.25 - 17.81) (-1.16 - 0.95) 

Fiber (% dwt)Acid Detergent Fiber 14.62 (0.84) 14.30 (0.84) 0.32 (1.19) -2.59, 3.23 0.796 

(12.94 - 15.27) (12.41 - 17.85) (-2.58 - 2.81) 
Monsanto Company 11-SY-217U 

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 15.22 (0.58) 16.08 (0.58) -0.86 (0.68) -2.53, 0.82 0.256 

(13.68 - 17.02) (15.28 - 16.62) (-1.60 - 0.62) 

Amino Acid (% dwt)Alanine 1.70 (0.010) 1.67 (0.010) 0.037 (0.013) 0.0045, 0.069 0.031 

(1.69 - 1.72) (1.65 - 1.68) (0.021 - 0.051) 

Arginine 3.05 (0.025) 2.90 (0.025) 0.15 (0.028) 0.083, 0.22 0.001 

(3.03 - 3.08) (2.85 - 2.98) (0.074 - 0.23) 

Aspartic Acid 4.56 (0.031) 4.45 (0.031) 0.10 (0.039) 0.0079, 0.20 0.037 

(4.51 - 4.62) (4.36 - 4.49) (0.030 - 0.18) 

Cystine 0.62 (0.0072) 0.63 (0.0072) -0.016 (0.010) -0.041, 0.0090 0.169 

(0.60 - 0.63) (0.61 - 0.65) (-0.053 - 0.014) 

Glutamic Acid 7.24 (0.057) 7.03 (0.057) 0.21 (0.066) 0.046, 0.37 0.020 

(7.19 - 7.36) (6.86 - 7.10) (0.10 - 0.34) 
Monsanto Company 11-SY-217U 

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.72 (0.010) 1.69 (0.010) 0.031 (0.011) 0.0032, 0.059 0.034 

(1.70 - 1.75) (1.66 - 1.70) (0.013 - 0.052) 

Histidine 1.04 (0.0082) 1.01 (0.0082) 0.031 (0.010) 0.0064, 0.055 0.021 

(1.03 - 1.06) (1.00 - 1.03) (0.013 - 0.044) 

Isoleucine 1.80 (0.019) 1.77 (0.019) 0.029 (0.026) -0.035, 0.092 0.316 

(1.78 - 1.84) (1.70 - 1.81) (-0.023 - 0.10) 

Leucine 3.02 (0.020) 2.96 (0.020) 0.063 (0.025) 0.0023, 0.12 0.043 

(3.00 - 3.05) (2.90 - 2.99) (0.017 - 0.12) 

Lysine 2.57 (0.016) 2.53 (0.016) 0.036 (0.016) -0.0029, 0.075 0.063 

(2.55 - 2.61) (2.49 - 2.56) (0.015 - 0.055) 

Methionine 0.57 (0.0058) 0.57 (0.0058) 0.0048 (0.0081) -0.015, 0.025 0.573 

(0.55 - 0.59) (0.56 - 0.57) (-0.012 - 0.031) 
Monsanto Company 11-SY-217U 

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.04 (0.018) 1.98 (0.018) 0.055 (0.023) 0.00024, 0.11 0.049 

(1.98 - 2.07) (1.95 - 2.02) (0.027 - 0.097) 

Proline 1.91 (0.028) 1.93 (0.028) -0.017 (0.038) -0.11, 0.076 0.666 

(1.90 - 1.93) (1.89 - 1.97) (-0.074 - 0.039) 

Serine 2.10 (0.019) 2.04 (0.019) 0.053 (0.025) -0.0086, 0.11 0.080 

(2.06 - 2.13) (2.01 - 2.08) (-0.027 - 0.091) 

Threonine 1.56 (0.012) 1.55 (0.012) 0.015 (0.017) -0.027, 0.057 0.410 

(1.55 - 1.57) (1.51 - 1.58) (-0.029 - 0.063) 

Tryptophan 0.44 (0.0095) 0.42 (0.0095) 0.019 (0.012) -0.0096, 0.048 0.154 

(0.43 - 0.45) (0.40 - 0.44) (-0.0065 - 0.038) 

Tyrosine 1.45 (0.016) 1.40 (0.016) 0.048 (0.018) 0.0044, 0.091 0.035 

(1.41 - 1.48) (1.37 - 1.44) (0.023 - 0.11) 
Monsanto Company 11-SY-217U 

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 1.91 (0.020) 1.87 (0.020) 0.045 (0.025) -0.018, 0.11 0.129 

(1.88 - 1.96) (1.78 - 1.91) (-0.013 - 0.11) 

Fatty Acid (% Total FA)16:0 Palmitic 11.89 (0.033) 12.12 (0.033) -0.23 (0.046) -0.34, -0.11 0.002 

(11.84 - 11.94) (12.04 - 12.23) (-0.39 - -0.13) 

18:0 Stearic 3.97 (0.023) 3.92 (0.023) 0.053 (0.028) -0.014, 0.12 0.102 

(3.92 - 4.03) (3.87 - 4.01) (0.022 - 0.091) 

18:1 Oleic 19.70 (0.11) 19.71 (0.11) -0.0098 (0.10) -0.27, 0.25 0.928 

(19.60 - 19.81) (19.45 - 19.99) (-0.20 - 0.15) 

18:2 Linoleic 54.62 (0.14) 54.37 (0.14) 0.25 (0.16) -0.16, 0.65 0.186 

(54.38 - 54.90) (53.90 - 54.70) (-0.038 - 0.48) 

18:3 Linolenic 9.08 (0.058) 9.23 (0.058) -0.15 (0.082) -0.35, 0.049 0.113 

(8.96 - 9.19) (9.03 - 9.41) (-0.45 - 0.033) 
Monsanto Company 11-SY-217U 

Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.30 (0.0084) 0.26 (0.0084) 0.032 (0.0078) 0.013, 0.051 0.006 

(0.29 - 0.30) (0.25 - 0.30) (0.0020 - 0.049) 

20:1 Eicosenoic 0.16 (0.0030) 0.15 (0.0030) 0.0054 (0.0042) -0.0047, 0.016 0.239 

(0.15 - 0.16) (0.15 - 0.16) (-0.0062 - 0.011) 

22:0 Behenic 0.29 (0.018) 0.23 (0.018) 0.052 (0.016) 0.013, 0.092 0.017 

(0.28 - 0.30) (0.20 - 0.30) (-0.0048 - 0.079) 

Vitamin (mg/100g dwt)Vitamin E 1.33 (0.092) 1.23 (0.092) 0.094 (0.10) -0.16, 0.34 0.392 

(1.17 - 1.45) (1.01 - 1.37) (-0.084 - 0.23) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-23. Summary of Site KSLA Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contr

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 2.17 (0.32) 1.28 (0.32) 0.88 (0.46) -0.23, 2.00 0.101 

(1.22 - 2.81) (0.58 - 2.39) (-1.17 - 1.63) 

Phytic Acid (% dwt) 1.72 (0.069) 1.61 (0.069) 0.11 (0.097) -0.12, 0.35 0.280 

(1.63 - 1.81) (1.39 - 1.77) (-0.034 - 0.42) 

Raffinose (% dwt) 0.87 (0.011) 0.82 (0.011) 0.043 (0.012) 0.013, 0.073 0.011 

(0.86 - 0.88) (0.80 - 0.85) (0.020 - 0.063) 

Stachyose (% dwt) 4.33 (0.065) 4.21 (0.065) 0.12 (0.092) -0.10, 0.35 0.236 

(4.27 - 4.42) (4.16 - 4.25) (0.064 - 0.26) 

Trypsin Inhibitor (TIU/mg dwt) 34.65 (4.00) 36.99 (4.00) -2.34 (5.66) -16.20, 11.52 0.693 

(31.85 - 39.35) (26.29 - 54.88) (-23.03 - 6.80) 

Isoflavone (μg/g dwt)Daidzein 1311.42 (35.73) 1341.83 (35.73) -30.41 (48.92) -150.10, 89.29 0.557 

(1232.55 - 1393.21) (1268.27 - 1442.92) (-148.91 - 98.29) 
Monsanto Company 11-SY-217U 

Table F-23. Summary of Site KSLA Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contro

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 1017.66 (44.06) 1064.05 (44.06) -46.39 (59.96) -193.11, 100.33 0.468 

(887.87 - 1093.13) (1002.79 - 1175.72) (-159.29 - 66.50) 

Glycitein 98.83 (10.16) 101.86 (10.16) -3.03 (12.66) -34.01, 27.95 0.818 

(75.71 - 125.39) (92.22 - 117.90) (-28.68 - 23.31) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-24. Summary of Site ILCY Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 7.90 (0.30) 7.32 (0.30) 0.58 (0.42) -0.45, 1.62 0.216 

(6.93 - 8.75) (7.07 - 8.04) (-0.13 - 1.34) 

Carbohydrates 68.63 (1.06) 67.21 (1.06) 1.42 (1.49) -2.23, 5.08 0.377 

(66.67 - 70.88) (65.25 - 70.63) (-3.97 - 4.90) 

Moisture (% fwt) 75.45 (0.59) 73.90 (0.59) 1.55 (0.73) -0.24, 3.34 0.078 

(73.90 - 76.90) (73.10 - 75.00) (0.10 - 3.80) 

Protein 19.75 (0.61) 19.41 (0.61) 0.33 (0.86) -1.78, 2.44 0.713 

(18.45 - 21.17) (17.88 - 20.50) (-2.05 - 3.29) 

Total Fat 3.74 (0.42) 6.14 (0.42) -2.40 (0.42) -3.42, -1.38 0.001 

(3.41 - 4.03) (4.57 - 7.18) (-3.15 - -0.86) 

Fiber (% dwt)Acid Detergent Fiber 33.37 (1.43) 29.10 (1.43) 4.26 (2.03) -0.70, 9.22 0.080 

(30.74 - 38.10) (26.28 - 32.84) (-0.081 - 11.81) 
Monsanto Company 11-SY-217U 

Table F-24. Summary of Site ILCY Soybean Forage Nutrients for MON 87712 vs. Conventional Control (

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 38.88 (1.58) 34.35 (1.58) 4.53 (2.18) -0.80, 9.87 0.082 

(33.84 - 41.65) (32.51 - 38.64) (1.33 - 7.93) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 4.98 (0.16) 4.94 (0.16) 0.045 (0.19) -0.42, 0.51 0.818 

(4.54 - 5.30) (4.69 - 5.21) (-0.27 - 0.25) 

Carbohydrates 38.54 (0.60) 39.13 (0.60) -0.60 (0.85) -2.68, 1.49 0.509 

(37.56 - 40.03) (37.69 - 40.86) (-1.37 - 1.05) 

Moisture (% fwt) 8.46 (0.20) 7.88 (0.20) 0.58 (0.25) -0.039, 1.20 0.061 

(7.98 - 8.94) (7.74 - 8.07) (-0.090 - 1.17) 

Protein 41.05 (0.14) 40.14 (0.14) 0.91 (0.15) 0.54, 1.28 <0.00

(40.85 - 41.36) (40.03 - 40.21) (0.74 - 1.17) 

Total Fat 15.46 (0.55) 15.80 (0.55) -0.34 (0.77) -2.22, 1.55 0.677 

(14.44 - 16.47) (14.09 - 16.95) (-2.32 - 0.53) 

Fiber (% dwt)Acid Detergent Fiber 14.94 (0.47) 15.41 (0.47) -0.48 (0.67) -2.11, 1.16 0.503 

(14.11 - 15.87) (14.25 - 16.05) (-1.56 - 1.62) 
Monsanto Company 11-SY-217U 

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 16.28 (0.54) 17.20 (0.54) -0.93 (0.76) -2.79, 0.94 0.269 

(14.95 - 17.17) (15.45 - 18.10) (-2.65 - 1.72) 

Amino Acid (% dwt)Alanine 1.78 (0.013) 1.73 (0.013) 0.052 (0.013) 0.020, 0.084 0.007 

(1.76 - 1.80) (1.70 - 1.74) (0.042 - 0.063) 

Arginine 3.19 (0.035) 3.02 (0.035) 0.17 (0.026) 0.10, 0.23 <0.00

(3.16 - 3.24) (2.93 - 3.12) (0.095 - 0.23) 

Aspartic Acid 4.77 (0.025) 4.64 (0.025) 0.13 (0.031) 0.053, 0.20 0.005 

(4.74 - 4.81) (4.57 - 4.67) (0.082 - 0.17) 

Cystine 0.60 (0.0069) 0.62 (0.0069) -0.023 (0.0097) -0.046, 0.0013 0.059 

(0.58 - 0.62) (0.61 - 0.63) (-0.051 - 0.0037) 

Glutamic Acid 7.68 (0.056) 7.41 (0.056) 0.27 (0.068) 0.11, 0.44 0.007 

(7.60 - 7.76) (7.28 - 7.48) (0.22 - 0.32) 
Monsanto Company 11-SY-217U 

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Glycine 1.79 (0.0080) 1.74 (0.0080) 0.052 (0.0096) 0.028, 0.076 0.001 

(1.78 - 1.80) (1.71 - 1.76) (0.033 - 0.081) 

Histidine 1.09 (0.0059) 1.06 (0.0059) 0.025 (0.0043) 0.014, 0.035 0.001 

(1.08 - 1.09) (1.04 - 1.07) (0.017 - 0.035) 

Isoleucine 1.92 (0.013) 1.85 (0.013) 0.066 (0.018) 0.023, 0.11 0.009 

(1.90 - 1.93) (1.82 - 1.88) (0.020 - 0.096) 

Leucine 3.18 (0.016) 3.09 (0.016) 0.099 (0.020) 0.050, 0.15 0.002 

(3.17 - 3.21) (3.05 - 3.11) (0.084 - 0.13) 

Lysine 2.68 (0.011) 2.62 (0.011) 0.058 (0.013) 0.025, 0.090 0.004 

(2.66 - 2.68) (2.59 - 2.65) (0.034 - 0.074) 

Methionine 0.57 (0.0048) 0.57 (0.0048) 0.0020 (0.0068) -0.015, 0.019 0.783 

(0.55 - 0.58) (0.56 - 0.58) (-0.018 - 0.018) 
Monsanto Company 11-SY-217U 

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Phenylalanine 2.17 (0.018) 2.08 (0.018) 0.095 (0.020) 0.046, 0.14 0.003 

(2.15 - 2.19) (2.03 - 2.13) (0.064 - 0.15) 

Proline 2.02 (0.014) 1.97 (0.014) 0.051 (0.020) 0.0011, 0.10 0.046 

(2.00 - 2.04) (1.95 - 1.99) (0.029 - 0.091) 

Serine 2.19 (0.027) 2.12 (0.027) 0.074 (0.029) 0.0023, 0.14 0.044 

(2.17 - 2.24) (2.10 - 2.13) (0.052 - 0.11) 

Threonine 1.55 (0.016) 1.58 (0.016) -0.028 (0.023) -0.084, 0.028 0.261 

(1.52 - 1.59) (1.55 - 1.63) (-0.089 - 0.040) 

Tryptophan 0.44 (0.0084) 0.45 (0.0084) -0.012 (0.012) -0.041, 0.016 0.324 

(0.41 - 0.47) (0.44 - 0.47) (-0.030 - 0.0028) 

Tyrosine 1.47 (0.032) 1.45 (0.032) 0.028 (0.017) -0.013, 0.070 0.148 

(1.41 - 1.54) (1.33 - 1.51) (-0.0033 - 0.078) 
Monsanto Company 11-SY-217U 

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Amino Acid (% dwt)Valine 2.03 (0.017) 1.95 (0.017) 0.083 (0.024) 0.025, 0.14 0.013 

(2.00 - 2.05) (1.91 - 1.99) (0.0091 - 0.13) 

Fatty Acid (% Total FA)16:0 Palmitic 11.63 (0.052) 11.89 (0.052) -0.26 (0.073) -0.44, -0.079 0.012 

(11.59 - 11.71) (11.83 - 12.00) (-0.41 - -0.11) 

18:0 Stearic 4.00 (0.033) 4.02 (0.033) -0.021 (0.033) -0.10, 0.060 0.549 

(3.92 - 4.10) (3.94 - 4.07) (-0.12 - 0.032) 

18:1 Oleic 19.45 (0.11) 19.13 (0.11) 0.32 (0.15) -0.056, 0.70 0.082 

(19.38 - 19.60) (18.73 - 19.38) (0.0019 - 0.68) 

18:2 Linoleic 54.44 (0.14) 54.47 (0.14) -0.029 (0.20) -0.52, 0.46 0.890 

(54.10 - 54.61) (54.29 - 54.81) (-0.35 - 0.27) 

18:3 Linolenic 9.80 (0.091) 9.82 (0.091) -0.017 (0.12) -0.30, 0.27 0.889 

(9.76 - 9.85) (9.59 - 9.99) (-0.14 - 0.20) 
Monsanto Company 11-SY-217U 

Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (co

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fatty Acid (% Total FA)20:0 Arachidic 0.30 (0.0029) 0.30 (0.0029) 0.00053 (0.0039) -0.0089, 0.010 0.894 

(0.29 - 0.31) (0.29 - 0.30) (-0.0097 - 0.0091) 

20:1 Eicosenoic 0.095 (0.016) 0.095 (0.016) 0.00010 (0.023) -0.057, 0.057 0.996 

(0.075 - 0.14) (0.073 - 0.15) (-0.066 - 0.071) 

22:0 Behenic 0.29 (0.0028) 0.29 (0.0028) 0.0028 (0.0040) -0.0069, 0.013 0.500 

(0.29 - 0.30) (0.28 - 0.30) (-0.00065 - 0.011) 

Vitamin (mg/100g dwt)Vitamin E 0.96 (0.045) 1.13 (0.045) -0.17 (0.059) -0.31, -0.027 0.027 

(0.94 - 0.99) (1.02 - 1.30) (-0.31 - -0.065) 

1dwt = dry weight; fwt = fresh weight; FA = fatty acid. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-26. Summary of Site NEYO Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contr

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

AntinutrientLectin (H.U./mg dwt) 2.18 (0.27) 1.72 (0.27) 0.45 (0.24) -0.12, 1.03 0.103 

(1.31 - 2.59) (1.25 - 2.27) (0.066 - 0.97) 

Phytic Acid (% dwt) 0.99 (0.064) 0.97 (0.064) 0.013 (0.073) -0.17, 0.19 0.863 

(0.88 - 1.12) (0.86 - 1.08) (-0.027 - 0.043) 

Raffinose (% dwt) 0.66 (0.011) 0.71 (0.011) -0.046 (0.015) -0.084, -0.0087 0.023 

(0.65 - 0.66) (0.69 - 0.74) (-0.072 - -0.028) 

Stachyose (% dwt) 4.27 (0.030) 4.11 (0.030) 0.16 (0.042) 0.057, 0.26 0.009 

(4.24 - 4.29) (4.05 - 4.16) (0.11 - 0.21) 

Trypsin Inhibitor (TIU/mg dwt) 33.52 (2.48) 33.08 (2.48) 0.44 (3.44) -7.98, 8.87 0.901 

(29.21 - 40.86) (29.37 - 36.43) (-7.22 - 11.49) 

Isoflavone (μg/g dwt)Daidzein 1425.70 (61.46) 1262.35 (61.46) 163.36 (86.92) -49.34, 376.05 0.109 

(1378.10 - 1471.56) (1076.47 - 1533.78) (-121.04 - 363.95) 
Monsanto Company 11-SY-217U 

Table F-26. Summary of Site NEYO Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Contr

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Isoflavone (μg/g dwt)Genistein 1050.47 (50.27) 898.64 (50.27) 151.83 (71.10) -22.14, 325.79 0.076 

(1006.30 - 1085.77) (761.46 - 1087.78) (-81.48 - 324.31) 

Glycitein 99.73 (9.66) 99.40 (9.66) 0.33 (13.66) -33.10, 33.75 0.981 

(89.15 - 107.95) (77.28 - 129.45) (-31.42 - 30.67) 

1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 

Table F-27. Summary of Site NEYO Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Proximate (% dwt)Ash 6.76 (0.35) 7.83 (0.35) -1.07 (0.50) -2.29, 0.15 0.074 

(6.14 - 7.33) (6.56 - 8.74) (-2.60 - 0.77) 

Carbohydrates 66.98 (0.83) 65.75 (0.83) 1.22 (1.17) -1.65, 4.10 0.337 

(64.83 - 68.67) (63.01 - 67.97) (-2.36 - 5.67) 

Moisture (% fwt) 75.38 (0.33) 74.73 (0.33) 0.65 (0.37) -0.26, 1.56 0.132 

(74.80 - 76.40) (74.40 - 75.40) (-0.30 - 1.70) 

Protein 21.01 (0.48) 20.57 (0.48) 0.44 (0.68) -1.23, 2.11 0.542 

(20.44 - 21.53) (18.71 - 22.24) (-1.79 - 2.40) 

Total Fat 5.37 (0.29) 5.84 (0.29) -0.47 (0.41) -1.48, 0.54 0.298 

(4.84 - 6.53) (5.57 - 6.21) (-1.37 - 0.95) 

Fiber (% dwt)Acid Detergent Fiber 33.04 (1.31) 31.24 (1.31) 1.80 (1.85) -2.72, 6.32 0.367 

(30.84 - 35.04) (27.71 - 33.66) (-2.82 - 7.33) 
Monsanto Company 11-SY-217U 

Table F-27. Summary of Site NEYO Soybean Forage Nutrients for MON 87712 vs. Conventional Control 

 Difference (Test minus Control)

Analytical Component (Units)1

Test2 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significa

(p-Valu

Fiber (% dwt)Neutral Detergent Fiber 36.33 (1.71) 35.65 (1.71) 0.67 (2.42) -5.26, 6.60 0.790 

(34.14 - 40.76) (32.73 - 41.41) (-6.68 - 8.04) 

1dwt = dry weight; fwt = fresh weight. 2Test refers to MON 87712. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control, A3525. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 11-SY-217U 337 of 467 

References for Appendix F 

AACC. 1998. Approved methods of the American Association of Cereal Chemists. 

American Association of Cereal Chemists, Inc., St. Paul, Minnesota. 

AOAC. 2005a. AOAC official method 982.30: Protein efficiency ratio. Official Methods 

of Analysis of AOAC International, Association of Analytical Communities 

International, Gaithersburg, Maryland. 

AOAC. 2005b. AOAC official method 923.03: Ash of flour. Official Methods of 

Analysis of AOAC International, Association of Analytical Communities International, 

Gaithersburg, Maryland. 

AOAC. 2005c. AOAC official method 983.23: Fat in foods. Official Methods of Analysis 

of AOAC International, Association of Analytical Communities International, 

Gaithersburg, Maryland. 

AOAC. 2005d. AOAC official method 955.04: Nitrogen (total) in fertilizers. Official 

Methods of Analysis of AOAC International, Association of Analytical Communities 

International, Gaithersburg, Maryland. 

AOAC. 2007a. AOAC official method 960.39 and 948.22. Official Methods of Analysis 

of AOAC International, Association of Analytical Communities International, 

Gaithersburg, Maryland. 

AOAC. 2007b. AOAC official method 926.08 and 925.09. Official Methods of Analysis 

of AOAC International, Association of Analytical Communities International, 

Gaithersburg, Maryland. 

AOCS. 1997a. Official Methods and Recommended Practices of the American Oil 

Chemists' Society, 5th Edition. Method Ce 2-66, American Oil Chemists' Society, 

Champaign, Illinois. 

Brobst, K.H. 1972. Gas-liquid chromatography of trimethylsilyl derivatives. Pages 3-8 in 

Methods in Carbohydrate Chemistry. Volume 6. Academic Press, New York, New York. 

Cort, W., T.S. Vicente, E.H. Waysek and B.D. Williams. 1983. Vitamin E content of 

feedstuffs determined by high-performance liquid chromatographic fluorescence. Journal 

of Agriculture and Food Chemistry 31:1330-1333. 

Goering, H.K. and P.J. Van Soest. 1970. Forage and fiber analyses (apparatus, reagents, 

procedures, and some applications). Agriculture Handbook No. 379. U.S. Department of 

Agriculture, Agricultural Research Service, Washington, D.C. 
Monsanto Company 11-SY-217U 338 of 467 

Kakade, M.L., J.J. Rackis, J.E. McGhee and G. Puski. 1974. Determination of trypsin 

inhibitor activity of soy products: A collaborative analysis of an improved procedure. 

Pages 376-382 in Cereal Chemistry. Volume 51. American Association of Cereal 

Chemists, St. Paul, Minnesota. 

Klurfeld, D.M. and D. Kritchevsky. 1987. Isolation and quantitation of lectins from 

vegetable oils. Lipids 22:667-668. 

Lehrfeld, J. 1989. High-performance liquid chromatography analysis of phytic acid on a 

pH-stable macroporous polymer column. Cereal Chemistry 66:510-515. 

Lehrfeld, J. 1994. HPLC separation and quantitation of phytic acid and some inositol 

phosphates in foods: Problems and solutions. Journal of Agricultural and Food Chemistry

42:2726-2731. 

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. 

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 19:551-554. 

McMurray, C.H., W.J. Blanchflower and D.A. Rice. 1980. Influence of extraction 

techniques on determination of Tocopherol in animal feedstuffs. Journal of the 

Association of Official Analytical Chemists 63:1258-1261. 

Merrill, A.L. and B.K. Watt. 1973. Energy value of foods... Basis and derivation. 

Agricultural Handbook No. 74. US Department of Agriculture, Agricultural Research 

Service, Washington, D.C. 

OECD. 2001. Consensus document on compositional considerations for new varieties of 

soybean: Key food and feed nutrients and anti-nutrients. ENV/JM/MONO(2001)15. 

Series on the Safety of Novel Foods and Feeds No.2. Organisation for Economic Cooperation and Development, Paris, France. 

Pettersson, H. and K.-H. Kiessling. 1984. Liquid chromatographic determination of the 

plant estrogens coumestrol and isoflavones in animal feed. Journal of Association of 

Official Analytical Chemists 67:503-506. 

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 32:530-533. 

Speek, A.J., J. de Schrijver and W.H.P. Schreurs. 1985. Vitamin E composition of some 

seed oils as determined by high-performance liquid chromatography with fluorometric 

detection. Journal of Food Science 50:121-124. 
Monsanto Company 11-SY-217U 339 of 467 

Appendix G: Materials, Methods, and Individual-Site Results for Seed 

Dormancy and Germination Assessment of MON 87712 

G.1. Materials 

Seed dormancy and germination characteristics were assessed on seed from MON 87712, 

a conventional control A3525, and commercial reference varieties produced at the Stark 

County, Illinois; Macon County, Missouri; and Butler County, Missouri sites in 2009 

field trials (Appendix G). The seed from MON 87712, the conventional control A3525, 

and the commercial reference varieties were harvested from four replications at each of 

the three field sites and pooled to produce one seed lot of MON 87712, the conventional 

control A3525, and each commercial reference variety from each field site for dormancy 

and germination testing (Table G-1). 

G.2. Characterization of the Materials 

For the MON 87712, conventional control A3525, and commercial reference variety 

starting seed, the presence or absence of MON 87712 was verified by event-specific 

polymerase chain reaction analyses. 

G.3. Germination Testing Facility and Experimental Methods 

Seed 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; 2006; 2007). 

Seed lots of MON 87712, the conventional control A3525, and four commercial 

reference varieties were produced from each of three sites and tested under six different 

temperature regimes. Six germination chambers were maintained under dark conditions 

with one the of the following temperature regimes: constant temperature of 

approximately 10, 20, or 30°C or alternating temperatures of approximately 10/20, 10/30, 

or 20/30°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 assessment. For each seed lot, four replicated paper 

germination towels were prepared per facility SOPs for each temperature regime. Wax 

coated paper was placed on a large tray followed by a water-moistened germination 

towel. A target of 100 seeds per seed lot were placed on the germination towel (i.e., one 

seed lot per towel) using a vacuum planting system. A second water-moistened 

germination towel was placed on top of the seed. The towels were then rolled up and 

secured with a rubber band. All rolled germination towels were placed into appropriately 

labeled buckets that were then covered with ventilated plastic bags attached with rubber 

bands. The buckets were arranged in the germination chambers in a split-plot design. 

For each split-plot design, the whole-plot was the seed production location and the subplot was the seed substance (i.e., test, control, or reference substance). 
Monsanto Company 11-SY-217U 340 of 467 

A description of each germination characteristic evaluated and the timing of evaluations 

are presented in Table VII-1. The types of data collected depended on the temperature 

regime. Each rolled germination towel in the AOSA-recommended temperature regime 

(i.e., 20/30°C) was evaluated periodically during the study for normal germinated, 

abnormal germinated, hard, dead, and firm-swollen seed as defined by AOSA guidelines 

(AOSA, 2006; 2007). AOSA only provides guidelines (AOSA, 2007) for testing seed 

under optimal temperatures (20/30°C); however, additional temperature regimes were 

included to test a range of temperature conditions. Each rolled germination towel in the 

additional temperature regimes (i.e., 10, 20, 30, 10/20, and 10/30°C) was evaluated 

periodically for germinated, viable hard, dead, and viable firm-swollen seed. Emergence 

and/or development of essential structures of seedlings that otherwise would be 

categorized as “normal germinated” under optimal temperature conditions may not be so 

at non-optimal temperatures. Therefore, for the additional temperature regimes, no 

distinction was made between normal and abnormal germinated seed. 

G.4. Statistical Analysis 

An analysis of variance was conducted using SAS (2008) according to a split-plot 

design with four replications. MON 87712 was compared to the conventional soybean 

control for germination characteristics of seed produced within each site (i.e., individualsite analyses) and a combined-site analysis in which the data were pooled across seed 

production sites. The seed germination characteristics analyzed included 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 

5% (α=0.05). MON 87712 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 across the 

seed production sites. The following is a summary of the results from the individual-site 

analysis. Results from the combined-site analysis are presented in Table VII-2. 

G.5. Individual-Site Seed Dormancy and Germination Analysis 

In the individual-site analysis, four statistically significant differences were detected 

between MON 87712 and the conventional control A3525 (Table G-2). Four statistically 

significant differences were detected between MON 87712 and the conventional control 

A3525 for seed produced at the ILWY and MOAN site. At 10/20°C, MON 87712 had 

higher percent germinated seed than the conventional control A3525 at 10/20°C (100.0% 

vs. 99.3%) and lower percent dead seed than the conventional control A3525 (0.0% vs 

0.8%) at the ILWY site. At 20/30°C, MON 87712 had higher percent normal germinated 

seed than the conventional control A3525 (99.8% vs. 97.8%) and lower percent abnormal 

germinated seed than the conventional control A3525 (0.0% vs. 2.0%) at the MOAN site. 

 

 SAS is a registered trademark of SAS Institute, Inc. 
Monsanto Company 11-SY-217U 341 of 467 

Statistical differences detected between MON 87712 and the conventional control A3525 

for germination characteristics in the individual-site analysis were not consistently 

detected across temperature regimes or seed production sites. Furthermore, the 

differences were not detected in the combined site analysis indicating that there is no 

increased weed potential for MON 87712 compared to conventional soybean. 
Monsanto Company 11-SY-217U 342 of 467 

Table G-1. Starting Seed of MON 87712, Conventional Control and Commercial 

Reference Varieties Used in Dormancy Assessment 

Site1 

Material 

Type Material Name2 Phenotype Sample ID 

MOAN Control A3525 Conventional 11262199 

MOAN Test MON 87712 Soybean Intrinsic Yield 11262197 

MOAN Reference SB3369R Glyphosate-Tolerant3 11262200 

MOAN Reference Pioneer 93M14 Conventional 11262201 

MOAN Reference NutriPride 8339 Glyphosate-Tolerant3 11262211 

MOAN Reference Pioneer 93M62 Conventional 11262212 

MOFI Control A3525 Conventional 11262057 

MOFI Test MON 87712 Soybean Intrinsic Yield 11262055 

MOFI Reference Stine 3300-0 Conventional 11262058 

MOFI Reference SB3888R Glyphosate-Tolerant3 11262059 

MOFI Reference NK S30-D4 Glyphosate-Tolerant3 11262061 

MOFI Reference LG-C3540 Conventional 11262063 

ILWY Control A3525 Conventional 11262070 

ILWY Test MON 87712 Soybean Intrinsic Yield 11262065 

ILWY Reference SB3369R Glyphosate-Tolerant3 11262072 

ILWY Reference Pioneer 93M11 Glyphosate-Tolerant3 11262075 

ILWY Reference Stine 3300-0 Conventional 11262077 

ILWY Reference SB3819 Conventional 11262081 

 

1

 Site = Site where seed lot was produced; ILWY = Stark County, IL; MOAN = Macon 

County, MO; and MOFI = Butler County, MO 

2

 Material Name = Test material name is a Monsanto Regulatory ID. Control and 

reference material names are commercial names. 

3

 Glyphosate - tolerant = Commercially-available Roundup Ready soybean. 
Monsanto Company 11-SY-217U 

Table G-2. Dormancy and Germination Characteristics of MON 87712 and the Conventional Control Aat each of the Three Field Sites 

ILWY2 MOAN2 MOFMean % (S.E.) 3 Mean % (S.E.) 3 Mean 

Temperature Regime Germination Category1 MON 87712 Control MON 87712 Control MON 10 °C Germinated 99.8 (0.3) 99.5 (0.3) 100.0 (0.0) 99.8 (0.3) 99.5 

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0Dead 0.3 (0.3) 0.5 (0.3) 0.0 (0.0) 0.3 (0.3) 0.5 (0Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

20 °C Germinated 99.8 (0.3) 100.0 (0.0) 99.8 (0.3) 99.3 (0.5) 98.8 

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0Dead 0.3 (0.3) 0.0 (0.0) 0.3 (0.3) 0.8 (0.5) 1.3 (0Viable Firm Swollen † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

30 °C Germinated 100.0 (0.0) 100.0 (0.0) 99.0 (0.4) 99.5 (0.3) 99.0 

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0Dead 0.0 (0.0) 0.0 (0.0) 1.0 (0.4) 0.5 (0.3) 1.0 (0Viable Firm Swollen † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0
Monsanto Company 11-SY-217U 

Table G-2. Dormancy and Germination Characteristics of MON 87712 and the Conventional Control Aat each of the Three Field Sites (continued) 

ILWY2 MOAN2 MOFMean % (S.E.)3 Mean % (S.E.)3 Mean 

Temperature Regime Germination Category1 MON 87712 Control MON 87712 Control MON 10/20 °C Germinated 100.0 (0.0)* 99.3 (0.3) 99.8 (0.3) 100.0 (0.0) 99.8 

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0Dead 0.0 (0.0)* 0.8 (0.3) 0.3 (0.3) 0.0 (0.0) 0.3 (0Viable Firm Swollen † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

10/30 °C Germinated 99.8 (0.3) 100.0 (0.0) 100.0 (0.0) 99.5 (0.3) 98.8 

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0Dead 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.5 (0.3) 1.3 (0Viable Firm Swollen † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

20/30 °C Normal Germinated 96.5 (1.0) 97.8 (1.0) 99.8 (0.3)* 97.8 (1.1) 94.0 (AOSA) Abnormal Germinated 2.5 (0.9) 2.0 (0.8) 0.0 (0.0)* 2.0 (0.9) 5.3 (0

Viable Hard † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0Dead 1.0 (0.4) 0.3 (0.3) 0.3 (0.3) 0.3 (0.3) 0.8 (0Viable Firm Swollen † 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

Note: The experimental design was a split-plot with four replications. * Indicates a statistically significant difference (

α=0.05) between MON 87712 and the conventional control A352

† No statistical comparisons could be made due to lack of variability in the data. 1 Dormancy and germination characteristics were analyzed using an analysis of variance (ANOVA) model. 2 Site codes are as follows: ILWY = Stark County, IL; MOAN = Macon County, MO; and MOFI = Butler Count3 Mean based on n = 4. S.E. = Standard Error. In some instances, the total percentage of both MON 87712 

control A3525 did not equal exactly 100% due to numerical rounding of the means. 
Monsanto Company 11-SY-217U 345 of 467 

References for Appendix G 

AOSA. 2000. Tetrazolium testing handbook. Contribution no. 29. Association of Official 

Seed Analysts, Lincoln, Nebraska. 

AOSA. 2006. Seedling evaluation handbook. Contribution no. 35. Association of Official 

Seed Analysts, Lincoln, Nebraska. 

AOSA. 2007. Tetrazolium testing handbook - Family: Brassicaceae I. Association of 

Official Seed Analysts, Lincoln, Nebraska. 
Monsanto Company 11-SY-217U 346 of 467 

Appendix H: Materials, Methods, and Individual-Site Results from Phenotypic, 

Agronomic, and Environmental Interaction Assessment of MON 87712 under 

Field Conditions 

H.1. Materials 

The soybean materials for the phenotypic and environmental assessment in the field 

included MON 87712, the conventional soybean control A3525, and 18 commercial 

reference varieties. The list of the soybean materials planted at each of 19 field sites is 

presented in Table H-1. 

H.2. Characterization of the Materials 

The identities of MON 87712 and the conventional control A3525 seed were verified by 

event-specific polymerase chain reaction analyses. 

H.3. Field Sites and Plot Design 

Data were collected from field sites conducted in 2009 at 19 sites within the U.S. soybean 

production regions (Table VII-3). These 19 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 19 sites in a randomized complete block 

design with four replications. Each plot at the ARNE, ILSE, MOCB, and MOSL sites 

consisted of twelve rows spaced approximately 30 inches apart and approximately 30 feet 

in length. Rows # 2 and 3 were designated for the collection of phenotypic data. 

Rows # 4 and 5 were designated for the collection of abiotic stress response, disease 

damage, and arthropod-related damage data. Rows # 7 and 9 were designated for the 

collection of arthropod samples. Rows # 1, 6, 8, and 10-12 were used as buffer rows. 

Each plot was surrounded by approximately 5-15 feet of a commercial soybean variety 

by planting border rows in the alleyways between the blocks and around the entire 

perimeter of the plot area. The purpose of the planted borders was 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 IAJA, IARL, ILCY, ILMS, ILWY, INRC, INSH, KSLA, MOAN, MOFI, 

MOKI, MOWR, NEYO, PAGR, and PAHM sites consisted of four 20 feet long rows 

spaced approximately 30 inches apart. Rows # 2 and 3 were designated for the collection 

of phenotypic, abiotic stress response, disease damage, and arthropod-related damage 

data. Rows # 1 and 4 were used as buffer rows. The entire plot area was surrounded by a 

border of a commercial soybean variety approximately 10 feet (four rows) in width. 

H.4. Planting and Field Operations 

Field and planting information are listed in Table H-2. Agronomic practices used to 

prepare and maintain each study site were characteristic of those used in each respective 
Monsanto Company 11-SY-217U 347 of 467 

geographic region. All maintenance operations were performed uniformly over the entire 

trial area. 
Monsanto Company 11-SY-217U 

Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interaction Assessment Note: The study also included two additional experimental materials that were outside of the scope of the objectives of this evaluation of MON 877121 T = Test, C = Control, and R = reference 2 Phenotypic abbreviations: SIY = soybean intrinsic yield, GT = glyphosate-tolerant, Conventional = conventional commercial 3 Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL; IL

ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = Pawnee County, KS; M

MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County, MO; MOSL = St. Louis County, MO; MOWR = Lincoln 

County, NE; PAGR = Berks County, PA, site 1; PAHM = Berks County, PA, site 2. 

Material 

Material Type1 Phenotype2

Monsanto Lot Number Sites3

MON 87712 T SIY 11223539 All A3525 C Conventional 11223542 All SB3369R R GT 11226924 ILWY, MOAN, MOSL, NEYO SB3579R R GT 11226925 ILCY, MOCB, MOKI SB3888R R GT 11226926 ARNE, INRC, KSLA NuPride 8339 R GT 11226939 ILCY, ILMS, MOAN, MOKI NC+ 2A95 R GT 11226838 IARL, ILMS, KSLA, MOCB, PAHM Pioneer 93M43 R GT 11226839 IAJA, ILSE, INSH, MOWR, PAHM Pioneer 93M11 R GT 11226840 ARNE, ILWY, MOWR, NEYO NK S28-B4 R GT 11226842 IARL, INRC, INSH, MOSL, PAGR NK S30-D4 R GT 11226843 IAJA, ILSE, MOLP, MOFI, PAGR Midland 363 R Conventional 11226698 IAJA, INRC, KSLA, MOCB Stine 3300-0 R Conventional 10001134 ILCY, ILWY, MOCB, MOFI, NEYO Pioneer 9382 R Conventional 11226581 IAJA, ILSE, MOWR, PAHM Pioneer 93M62 R Conventional 11226582 ILMS, MOAN, MOWR, PAGR SB3819 R Conventional 11226928 ILWY, INSH, MOKI, NEYO LG 3211 R Conventional 11226860 IARL, ILCY, INSH, MOSL, PAHM LG C3540 R Conventional 11226858 IARL, INRC, MOFI, PAGR Pioneer 93M14 R Conventional 11226720 ARNE, ILMS, MOAN, MOSL NuPride 3202 R Conventional 11226938 ARNE, ILSE, KSLA, MOKI 
Monsanto Company 11-SY-217U 

Table H-2. Field and Planting Information 1 Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL; IL

ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = Pawnee County, KS; M

MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County, MO; MOSL = St. Louis County, MO; MOWR = Lincoln 

County, NE; PAGR = Berks County, PA, site 1; PAHM = Berks County, PA, site 2. 2 Month-day-year. 

3 Width × length. Sites with arthropod collection (ARNE, ILSE, MOCB, and MOSL) had larger plot areas. 

Site1

Planting Date2 

Planting rate (seeds/ft)

Planting depth (in) 

Plot size (ft)3 

Rows/ plot Soil series, organic matter, pH 

Cropping History 

2007 

ARNE 6-9-09 9 1.0 30 × 30 12 Bosket sandy loam; 1.0%; 6.0 Soybean IAJA 6-5-09 9 1.5 10 × 20 4 Clarion loam; 2.5%; 5.6 Soybean IARL 6-5-09 9 1.0 10 × 20 4 Mahaska silty clay loam; 3.54%; 7.01 Corn / Wheat ILCY 6-30-09 9 1.4 10 × 20 4 Hoyleton-Darmstadt complex / silt loam; 

2.6%; 7.3 

Soybean 

ILMS 6-30-09 9 1.0 10 × 20 4 Bluford silt loam; 2.0%; 6.3 Corn ILSE 6-24-09 9 1.5 30 × 30 12 Flaragan silt loam; 5.0%; 6.5 Sweet corn ILWY 6-6-09 9 2.0 10 × 20 4 Plano silt loam; 3.4%; 6.3 Soybean INRC 6-25-09 9 1.0 10 × 20 4 Reesville silt clay loam; 1.4%; 7.0 Soybean INSH 6-8-09 9 1.5 10 × 20 4 Crosby silt loam; 2.1%; 5.8 Corn KSLA 6-8-09 9 2.0 10 × 20 4 Silt loam; 2.6%; 7.6 Sunflower 

Sorghum / Fallow Corn 

MOAN 6-29-09 9 1.0 10 × 20 4 Mexico silt loam; 2.1%; 7.2 Soybean MOCB 6-25-09 9 1.0 30 × 30 12 Putnam silt loam; 2.5%; 6.8 Soybean MOFI 6-12-09 9 0.5 10 × 20 4 Amagon silt loam; 1.8%; 5.0 Soybean MOKI 6-25-09 9 1.25 10 × 20 4 Mexico silt loam; 3.1%; 6.4 Soybean MOSL 6-5-09 9 1.0 30 × 30 12 Peers silty clay; 2.9%; 7.5 Soybean MOWR 

6-6-09 9 1.0 10 × 20 4 Hatton silt loam; 2.9%; 6.5 Soybean 

NEYO 6-5-09 9 1.0 10 × 20 4 Hastings silt loam; 3.0%; 6.2 Soybean PAGR 6-8-09 9 1.5 10 × 20 4 Bedington-Berks complex clay loam; 1.4%; 

5.2 

Corn 

PAHM 6-25-09 9 1.5 10 × 20 4 Philo / Atkins loam; 1.2%; 6.4 Soybean 
Monsanto Company 11-SY-217U 350 of 467 

H.5. Phenotypic Observations 

The description of characteristics measured and the designated developmental stages 

when observations occurred are listed in Table VII-1. 

H.6. Environmental Observations 

Environmental interactions (i.e., interactions between the crop plants and their receiving 

environment) were used to characterize MON 87712 by evaluating plant response to 

abiotic stressors, disease damage, arthropod-related damage, and pest and beneficial 

arthropod abundance in the plots using the methods described in G.7 ad G.8. 

H.7. Abiotic Stress Response, Disease Damage, and Arthropod-Related Damage 

MON 87712 and the conventional control A3525 were evaluated at all 19 sites for 

differences in plant response to abiotic stressors, disease damage, and arthropod-related 

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 – V5 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, diseased, and arthropod 

pests that were either actively causing plant injury in the study area or were likely to 

occur in soybean during the 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 observations were collected from each plot using a 

continuous 0 – 9 scale of increasing severity. 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) 

Arthropod-related damage was assessed from each plot on the upper four nodes of 10 

non-systematically selected plants using the arthropod-specific 0 – 5 rating scales of 

increasing severity listed below. 
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Defoliating arthropods (e.g., corn earworm, bean leaf beetle, 

Japanese beetle, soybean looper)

Rating Severity of plant damage 

0 None 

1 1 – 20 % defoliation 

2 21 – 40% defoliation 

3 41 – 60% defoliation 

4 61 – 80% defoliation 

5 > 80% defoliation 

Pod feeding arthropods (e.g., corn earworm, bean leaf beetle, 

stink bug, Lygus bug on reproductive plant parts)

Rating Severity of plant damage 

0 None 

1 1 – 20 % damaged pods 

2 21 – 40% damaged pods 

3 41 – 60% damaged pods 

4 61 – 80% damaged pods 

5 > 80% damaged pods 

Leafhoppers (e.g., potato leafhopper)

Rating Severity of plant damage 

0 None 

1 1 – 50% of foliage with leaf yellowing; no leaf puckering 

or leaf margin necrosis 

2 1 – 50% of foliage with leaf yellowing, leaf puckering 

and/or leaf margin necrosis 

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 

5 > 50% of foliage with necrotic leaves (leaves dead due to 

leafhopper damage) 

Aphids (e.g., soybean aphid)

Rating Severity of plant damage 

0 None 

1 1 – 100 aphids per plant; no leaf puckering 

2 101 – 250 aphids per plant; no leaf puckering 

3 ≥ 250 aphids per plant with leaf puckering 

4 ≥ 250 aphids per plant with leaf puckering and leaf 

yellowing and/or necrosis 

5 ≥ 250 aphids per plant with plant stunting 
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H.8. Arthropod Abundance 

Pest and beneficial arthropods were collected at the ARNE, ILSE, and MOCB sites four 

times during the growing season at the following intervals: 

Collection 1: R1 – R2 growth stage 

Collection 2: Approximately two weeks after collection 1 

Collection 3: Approximately two weeks after collection 2 

Collection 4: Approximately two weeks after collection 3 

Arthropods were collected using a vertical beat sheet sampling method (Drees and Rice, 

1985). The beat sheet was approximately 36×36 inch sheet constructed of a stiff material 

with a collecting trough at the bottom. The sheet was placed between row 7 and row 8 

and the collecting trough was positioned near the base of the plants in row 7. Plants were 

shaken vigorously along the length of the beat sheet to dislodge arthropods from the 

plants. Another sub-sample was collected from the same row, approximately 8 ft from 

the first sub-sample. Two sub-samples were collected from row 9 in the same manner. A 

total of four sub-samples were collected in this way from each plot. The four subsamples were combined into one pre-labeled container and placed on freezer packs. The 

samples were then sent to Monsanto Company, St. Louis, MO for arthropod 

identification and enumeration. 

A maximum of six pest and six beneficial arthropods were enumerated for each 

collection. Three preselected pest and beneficial arthropods (or arthropod groups), 

namely bean leaf beetle, green clover worm, and stink bugs for the pests, and Araneae 

(spiders), Nabis sp., and Orius sp. for the beneficial arthropods, were enumerated at all 

sites for each collection time. Additionally, for each individual collection (e.g.,

Collection 1, ARNE site), four non-systematically selected samples were examined to 

determine presence and relative abundance of up to three additional pest and beneficial 

arthropods to be enumerated for that particular collection and site. Thus, the suite of pest 

and beneficial arthropods assessed often varied between collections from a site and 

between sites due to differences in temporal activity and geographical distribution of 

arthropod taxa. 

 
Monsanto Company 11-SY-217U 353 of 467 

H.9. Environmental Interactions Evaluation Criteria 

For the assessments of abiotic stress response and disease damage, MON 87712 and the 

conventional control A3525 were considered different in susceptibility or tolerance to an 

abiotic stressor or disease on a particular observation date at a site if the range of injury 

severity to MON 87712 did not overlap with the range of injury severity to the 

conventional control A3525 across all four replications. These data are categorical and 

were not subjected to statistical analysis. For each observation at a site, the range of 

injury severity across the commercial reference varieties provided data that are 

representative of commercial soybean varieties. Arthropod-related damage and arthropod 

abundance were quantitatively evaluated and subjected to statistical analysis, as 

appropriate. 

H.10. Data Assessment 

Experienced scientists familiar with the 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. 

Any unexpected observations or issues that would impact the study objectives were 

noted. Data were then subjected to statistical analysis as indicated below. 

H.11. Statistical Analysis 

The data from each experiment were analyzed separately according to a randomized 

complete block design using SAS® (2008). MON 87712 was compared to the 

conventional control A3525 within each site (individual-site analyses) and in a 

combined-site analysis, in which the data were pooled across sites, for early stand count, 

seedling vigor, days to 50% flowering, days to 50% end of flowering, days to 50% 

senescence, days to physiological maturity, plant height, lodging, pod shattering, final 

stand count, seed moisture, 100 seed weight, and yield. Growth stage, flower color, 

abiotic stress response, and disease damage data were categorical and not statistically 

analyzed. Arthropod-related damage and pest and beneficial arthropod abundance data 

were statistically analyzed only within individual observations/collections and sites due 

to the variation in temporal activity and geographical distribution of the taxa. 

No statistical comparisons were made between MON 87712 and commercial reference 

varieties. The reference range for each measured phenotypic characteristic was 

determined from the minimum and maximum mean values from the 18 commercial 

reference varieties planted among the sites. The reference range for the abundance and 

damage of each arthropod evaluated from a given collection/observation and site was 

determined from the minimum and maximum mean abundance or damage values 

collected from the reference varieties at the site. Data excluded from the study and the 

reasons for their exclusions are listed in Table H-3. 
Monsanto Company 11-SY-217U 

Table H-3. Data Missing or Excluded from Analysis Site1 Material name Material type Plots Characteristics Reason for exclusionAll All All All Animal damage Animal damage i

environmental stressor.

All All All All Herbicide injury Herbicide injury i

environmental stressor.

ILCY All All All Brown stem rot, 

observation 4

Assessment of brown performed at all plots.

ILCY All All All Plant height Data were not collected.IAJA All All All Growth stage 

Monitoring

Growth stage monitorisenescence indicated tmade after harvest which Therefore, growth stacollected at senescence w

IAJA All All All Arthropod damage Data were not collected.

IAJA All All All Disease and 

abiotic stressors, observations 2 & 3

Data were not collected.

IAJA MON 87712

A3525Pioneer 93B82

TestControlReference

209, 305;303;208

Final stand count Data were not collected.
Monsanto Company 11-SY-217U 

Table H-3. Data Missing or Excluded from Analysis (continued) Site1 Material name Material type Plots Characteristics Reason for exclusionIAJE All All All All Site was dropped due 

frost.

IARL MON 87712 Test 405 Flower color Data was not collected.

IARL All All All Disease and 

abiotic stressors, observation 2

Inconsistent use of stress

IARL NC+ 2A95 Reference 408 Drought, 

observation 4

Data were not collected.

ILMS Pioneer 93M14 Reference 103, 207, 301, 404 All The wrong number of 

placed into the planting excessive number of see

ILSE All All All Plant height Data were not collected.

ILSE MON 87712 Test 408 Days to 50% end 

of flowering

Original data in paper out but no explanation result, the electronic data 

ILSE MON 87712 Test 408 Days to 50% end 

of senescence

Original data in paper out but no explanation result, the electronic data 
Monsanto Company 11-SY-217U 

Table H-3. Data Missing or Excluded from Analysis (continued) Site1 Material name Material type Plots Characteristics Reason for exclusionILSE All All All Disease and abiotic 

stressors, observation 2, 3, and 4

Data were not collected.

ILSE All All All Growth stage 

Monitoring after R4

Data were not collected.

ILSE All All All Arthropod damage Incorrect rating scale waINSH LG 3211 Reference 206 Yield Only one row of the plo

equipment malfunction.

INSH All All All Disease and abiotic 

stressors, observation 2

Data were not collected.

INRC All All All Arthropod damage Data were not collected.INRC All All All Seedling vigor Data were not collected.

INRC All All All Disease and abiotic 

stressors, observation 2

Data were not collected.

KSLA All All All Days to 50% end of 

flowering

Data were not collected.
Monsanto Company 11-SY-217U 

Table H-3. Data Missing or Excluded from Analysis (continued) Site1 Material name Material type Plots Characteristics Reason for exclusionKSLA All All All Eyespot Eyespot is a corn diseas

be evaluated as a soybea

KSLA All All All Frost damage Assessment of frost dam

on all plots.

MOAN Pioneer 93M14 Reference 110, 205, 309, 402 All The wrong number of 

placed into the planting excessive number of see

MOAN MON 87712 Test 407 Days to 

physiological maturity

Data was not collected.

MOCB Stine 3300-0 Reference 401 Days to 50% end 

of flowering

Data was not collected.

MOCB SB3579R Reference 110 Days to 

physiological maturity

Data was not collected.

MOLP All All All All Site was dropped due to 

flood.
Monsanto Company 11-SY-217U 

Table H-3. Data Missing or Excluded from Analysis (continued) Site1 Material name Material type Plots Characteristics Reason for exclusionMOKI A3525 Control 108 Early stand count Data was recorded on 

majority of the seedlings 

MOKI All All All Seedling vigor Data were not collected.

MOKI All All All Disease and 

abiotic stressors, observations 1 & 2

Data were not collected.

MOSL SB3369R Reference 304 Sudden death, 

observation 4

Data were not collected.

MOSL All All All Arthropod 

abundance

Data were not collected

MOSL All All All Biomass and 

photosynthetic data

Data were of poor quaavailable sites.

MOWR Pioneer 93M62 Reference 406 Arthropod 

stressors, observation 2

Data from two of the thwere not collected.

MOWR Pioneer 93M43 Reference 110 Disease and 

abiotic stressors, observation 3

Data were not collected.
Monsanto Company 11-SY-217U 

Table H-3. Data Missing or Excluded from Analysis (continued) Site1 Material name Material type Plots Characteristics Reason for exclusionPAGR LG 3211 Reference 101 Alternaria, 

observation 1

Data were not collected.

PAGR MON 87712

MON 87712(-)A3525A3525(-)Pioneer 93M62

TestControlControlControlReference

102, 203, 304, 404; 302, 401; 204, 306; 205, 305, 405; 101, 206, 308, 409

Plant height Data were not collected.

PAHM LG C3540 Reference 409 Bacterial blight, 

observation 2

Data were not collected

1 Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IAJE = Greene County, IA; IARL = Jefferson County, IA; 

ILMS = Effingham County, IL; ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; K

MOAN = Macon County, MO; MOCB = Shelby County, MO; MOFI = Butler County, MO; MOLP = Adair County, MO; MOKI = Callaway Cou

County, MO; MOWR = Lincoln County, MO; NEYO = York County, NE; PAHM = Berks County, PA, site 1; PAGR = B
Monsanto Company 11-SY-217U 360 of 467 

H.12. Individual Field Site Plant Growth and Development Results and Discussion 

In the individual-site analysis, a total of 67 statistically significant differences (α = 0.05) 

were detected out of a total of 211 comparisons made between MON 87712 and the 

conventional control A3525 (Table H-4). These differences were distributed among 

eleven of the 14 phenotypic characteristics. No statistical comparisons could be made in 

30 instances where p-values could not be generated due to lack of variability. The 

nineteen flower color comparisons were categorical and were not statistically analyzed; 

however, at each site, all plants of MON 87712 and the conventional control A3525 had 

purple flowers as expected (Table H-4). MON 87712 and the conventional control A3525 

were within the same range of plant growth stage observations among the sites (Table H5). 

Differences in stand count were observed between MON 87712 and the conventional 

control A3525 at the individual sites. Early stand count was higher for MON 87712 than 

the conventional control A3525 at the IAJA site (321.3 vs. 303.3 plants/plot), the KSLA 

site (312.8 vs. 293.8 plants/plot), the MOFI site (289.3 vs. 266.5 plants/plot), and the 

MOWR site (354.0 vs. 343.3 plants/plot), and lower than the control at the IARL site 

(333.3 vs. 349.0 plants/plot). Final stand count was higher for MON 87712 than the 

conventional control A3525 at the IAJA site (310.0 vs. 249.0 plants/plot), the ILSE site 

(260.3 vs. 232.3 plants/plot), the INSH site (327.5 vs. 305.8 plants/plot), the KSLA site 

(300.5 vs. 277.8 plants/plot), the MOWR site (347.8 vs. 338.3 plants/plot), and the 

NEYO site (343.5 vs. 307.8 plants/plot). 

Statistically significant differences in flowering time were observed between 

MON 87712 and the conventional control A3525 at individual sites. Plants of 

MON 87712 flowered earlier than the conventional control A3525 at the IAJA site (204.3 

vs. 207.3 days after 1 Jan. 2009) and the PAGR site (209.3 vs. 210.5 days after 1 Jan. 

2009), and later than the conventional control A3525 at the ARNE site (193.0 vs. 190.8 

days after 1 Jan. 2009), the ILWY site (206.3 vs. 205.3 days after 1 Jan. 2009), the INRC 

site (218.0 vs. 216.5 days after 1 Jan. 2009), and the NEYO site (206.3 vs. 205.0 days 

after 1 Jan. 2009). Plants of MON 87712 ended flowering earlier than the conventional 

control A3525 at the INRC site (254.0 vs. 255.0 days after 1 Jan. 2009) and the MOFI 

site (221.5 vs. 223.3 days after 1 Jan. 2009), and later than the conventional control 

A3525 at the ILCY site (234.0 vs. 232.8 days after 1 Jan. 2009), the MOKI site (243.0 vs. 

239.3 days after 1 Jan. 2009), and the PAGR site (226.8 vs. 225.5 days after 1 Jan. 2009). 

Plants of MON 87712 reached 50% senescence significantly later than the conventional 

control A3525 at all but three sites (ILWY, INRC, and INSH). Plants of MON 87712 

reached physiological maturity significantly later than the conventional control A3525 at 

16 sites. MON 87712 had significantly more lodging than the conventional control 

A3525 at the KSLA site (3.0 vs. 1.8 ranting). 

Pod shattering was significantly lower for MON 87712 than the conventional control 

A3525 at the NEYO site (1.0 vs. 1.8 rating). 
Monsanto Company 11-SY-217U 361 of 467 

Grain moisture was significantly higher for MON 87712 than the conventional control 

A3525 at the IARL site (13.0 vs. 12.0%) and the PAGR site (16.0 vs. 15.7%), and 

significantly lower than the conventional control A3525 at the MOFI site (11.6 vs. 

11.9%). 

The weight of 100 seeds was significantly higher for MON 87712 than the conventional 

control A3525 at the INRC site (14.7 vs. 13.7 g) and the MOKI site (20.5 vs. 17.5 g). 

Statistically significant differences in yield were observed between MON 87712 and the 

conventional control A3525 at the individual sites. Yield was higher for MON 87712 

than the conventional control A3525 at the INSH site (54.5 vs. 47.9 bu/ac), the KSLA 

site (68.9 vs. 60.0 bu/ac), the MOWR site (65.1 vs. 58.3 bu/ac), the NEYO site (51.7 vs. 

46.9 bu/ac), the PAGR site (69.3 vs. 61.4 bu/ac), and the PAHM site (52.4 vs. 48.2 

bu/ac). 

Considering that the statistical differences detected in the individual-site analyses for 

days to 50% flowering, days to 50% end of flowering, lodging, pod shattering, grain 

moisture, and 100 seed weight 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 

weediness potential of MON 87712 compared to the conventional control A3525. While 

statistical differences were detected for early stand count at five sites, days to 50% 

senescence at sixteen sites, days to physiological maturity at sixteen sites, final stand 

count at six sites, and yield at six sites, and statistical differences were detected for these 

phenotypic characteristics in the combined-site analysis, the assessed phenotypic values 

of MON 87712 were within their respective variation of the study references. 

H.13 Individual Field Site Environmental Interaction Results and Discussion 

In an individual site assessment, no statistically significant differences (α = 0.05) were 

observed between MON 87712 and the conventional control A3525 for any of the 186 

comparisons for the assessed abiotic stressors (Table H-6) or for any of the 198 

comparisons for the assessed diseases (Table H-7). 

In an assessment of arthropod-related damage, no statistically significant differences 

were detected (α = 0.05) between MON 87712 and the conventional control A3525 for 

129 out of 137 comparisons for the assessed arthropods (Table H-8). Statistical 

comparisons could not be made between MON 87712 and the conventional control 

A3525 for 55 additional arthropod-related damage comparisons for which p-values could 

not be generated due to lack of variability in the data. A total of eight statistically 

significant differences involving three taxa were detected between MON 87712 and the 

conventional control A3525 out of the 137 comparisons. The bean leaf beetle damage 

was higher in MON 87712 than the conventional control A3525 in Observation 1 at the 

ILCY site (0.53 vs. 0.23 rating) but lower in Observation 2 at the MOAN site (0.65 vs. 

0.93 rating). MON 87712 had more damage than the conventional control A3525 from 

green cloverworms in Observation 4 from the MOWR site (0.60 vs. 0.48 rating). 

MON 87712 had more damage than the conventional control A3525 from Japanese 
Monsanto Company 11-SY-217U 362 of 467 

beetles in Observation 1 at the ILMS site (0.50 vs. 0.30 rating). MON 87712 had more 

damage than the conventional control A3525 from leafhoppers in Observation 1at the 

PAHM site (0.55 vs. 0.15 rating). MON 87712 had less damage than the conventional 

control A3525 from soybean loopers in Observation 2 at the MOAN site (0.55 vs. 0.90 

rating) and Observation 3 at the MOFI site (0.45 vs. 0.85 rating). MON 87712 had less 

damage than the conventional control A3525 from velvetbean caterpillars in 

Observation 3 at the MOFT site (0.45 vs. 0.85 rating). The mean damage ratings for 

these detected differences were outside their respective reference range. However, the 

differences detected were small in magnitude and these differences were not consistent 

across observations or sites. Thus, these detected differences in arthropod-related 

damage were not indicative of a consistent response associated with the trait and are not 

considered biologically meaningful in terms of an adverse environmental impact of 

MON 87712 compared to the conventional control A3525. 

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

differences were detected (α = 0.05) between MON 87712 and the conventional control 

A3525 for 106 out of 115 comparisons, including 59 pest arthropod comparisons 

(Table H-9) and 56 beneficial arthropod comparisons (Table H-10). Statistical 

comparisons could not be made between MON 87712 and the conventional control 

A3525 for eight additional comparisons, including six pest arthropod comparisons and 

two beneficial arthropod comparisons, for which p-values could not be generated due to 

lack of variability in the data. A total of nine statistically significant differences were 

detected between MON 87712 and the conventional control A3525 for arthropod 

abundance, including five differences for pest arthropods and four differences for 

beneficial arthropods. 

The five differences detected out of the 59 pest arthropod comparisons involved aphids, 

bean leaf beetles, green cloverworms, and stink bugs (Table H-8). MON 87712 had 

higher aphid abundance than the conventional control A3525 in Collection 4 at the 

MOCB site (37.5 vs. 19.3 per plot). MON 87712 had higher bean leaf beetle abundance 

than the conventional control A3525 in Collection 2 at the ILSE site (1.3 vs. 0.3 per plot). 

MON 87712 had lower green cloverworm abundance than the conventional control 

A3525 in Collection 3 at the MOCB site (0.8 vs. 5.5 per plot). The abundance of stink 

bug was lower in MON 87712 then the conventional control A3525 in Collection 3 at the 

ARNE site (0.3 vs. 2.0 per plot) but higher in Collection 3 at the MOCB site (0.8 vs. 0.0 

per plot). The mean abundance values for aphids in Collection 4 at the MOCB site, green 

cloverworms in Collection 3 at the MOCB, and stink bugs in Collection 3 at the ARNE 

site were within their respective reference range. The mean abundance values for bean 

leaf beetle in Collection 2 at the ILSE site and stink bug in Collection 3 at the MOCB site 

were outside their respective reference range. However, the differences detected for 

these taxa were not consistently detected across collections or sites. Thus, the detected 

differences in pest arthropod abundance were not indicative of a consistent response 

associated with the trait and are not considered biologically meaningful in terms of an 

adverse environmental impact of MON 87712 compared to conventional soybean. 

The four differences detected out of the 56 beneficial arthropod comparisons involved 

big-eyed bugs, ladybird beetles, micro-parasitic hymenoptera, and Orius spp. (Table H-
Monsanto Company 11-SY-217U 363 of 467 

9). MON 87712 had higher big-eyed bug abundance than the conventional control 

A3525 in Collection 1 at the ARNE site (4.3 vs. 1.8 per plot). MON 87712 had higher 

ladybird beetle abundance than the conventional control A3525 in Collection 4 at the 

ILSE site (8.3 vs. 3.5 per plot). MON 87712 had higher micro-parasitic hymenoptera 

abundance than the conventional control A3525 in Collection 4 at the MOCB site (2.3 vs. 

0.8 per plot). MON 87712 had lower Orius spp. abundance than the conventional control 

A3525 in Collection 1 at the ILSE site (0.0 vs. 1.0 per plot). The mean abundance values 

for micro-parasitic hymenoptera in Collection 4 at the MOCB site and Orius spp. in 

Collection 1 at the ILSE site were within their respective reference range. The mean 

abundance value for big-eyed bugs in Collection 1 at the ARNE site and ladybird beetles 

in Collection 4 at the ILSE site were outside their respective reference range. However, 

the differences detected for these taxa were not consistently detected across collections or 

sites. Thus, the detected differences in beneficial arthropod abundance were not 

indicative of a consistent response associated with the trait and are not considered 

biologically meaningful in terms of adverse environmental impact of MON 87712 

compared to conventional soybean. 
Monsanto Company 11-SY-217U 

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control A3525 

Phenotypic Characteristic (units)

Early stand count (# of plants/plot) Seedling vigor (1-9 scale)

Days after 1 Jan 2009 t

50% flowering3

Site1 MON 87712 (SE)2 Control (SE) MON 87712 (SE) Control (SE) MON 87712 (SE) ControARNE 301.5 (2.7) 299.0 (7.4) 4.8 (0.6) 4.5 (0.7) 193.0 (0.0)* 190.8 (IAJA 321.3 (4.4)* 303.3 (16.4) 2.0 (0.0) 2.3 (0.3) 204.3 (0.5)* 207.3 (

IARL 333.3 (4.9)* 349.0 (2.5) 1.3 (0.3) 1.3 (0.3) 205.0 (0.0) 204.5 (ILCY 325.5 (9.6) 319.5 (6.9) 1.0 (0.0) 1.3 (0.3) 214.8 (0.3) 215.8 (ILMS 310.3 (2.1) 320.3 (6.9) 2.5 (0.3) 2.5 (0.3) 218.5 (0.9) 219.3 (ILSE 234.8 (17.7) 224.3 (9.6) 7.3 (0.5) 7.3 (0.3) 218.3 (0.3) 218.0 (ILWY 336.8 (9.6) 347.3 (3.1) 2.0 (0.0)† 2.0 (0.0) 206.3 (0.3)* 205.3 (INRC 273.3 (11.4) 269.5 (10.9)

—

— 218.0 (0.0)* 216.5 (

INSH 321.5 (9.1) 315.0 (6.8) 5.3 (0.3) 5.5 (0.5) 205.8 (0.6) 205.8 (KSLA 312.8 (3.8)* 293.8 (10.8) 3.0 (0.0) 3.3 (0.3) 201.0 (0.0) 200.8 (MOAN 330.8 (1.1) 322.3 (5.5) 3.0 (0.0) 3.0 (0.0) 219.3 (0.3) 219.5 (MOCB 308.0 (6.7) 278.2 (33.7) 2.5 (0.3) 3.5 (0.5) 216.8 (0.3) 217.5 (MOFI 289.3 (2.8)* 266.5 (7.6) 3.0 (0.0) 3.0 (0.4) 201.0 (0.4) 200.3 (MOKI 153.5 (12.2) 139.3 (28.6)

—

— 217.5 (0.5) 217.0 (

MOSL 349.5 (2.7) 351.0 (3.2) 1.0 (0.0)† 1.0 (0.0) 196.0 (0.4) 195.8 (MOWR 354.0 (0.9)* 343.3 (1.4) 1.0 (0.0)† 1.0 (0.0) 199.3 (0.3) 198.5 (NEYO 338.3 (4.6) 314.5 (8.4) 3.0 (0.0)† 3.0 (0.0) 206.3 (0.3)* 205.0 (PAGR 244.5 (5.7) 238.8 (14.2) 4.0 (0.6) 4.3 (0.5) 209.3 (0.3)* 210.5 (PAHM 312.7 (7.4) 309.5 (4.4) 2.3 (0.5) 2.8 (0.3) 220.0 (0.4) 220.5 (
Monsanto Company 11-SY-217U 

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control A3525 (contin

Phenotypic Characteristic (units)

Days after 1 Jan 2009 to 

50% end of flowering4

Days after 1 Jan 2009 to 

50% senescence5

Days after 1 Jan 2009 t

physiological maturity

Site1 MON 87712 (SE) Control (SE) MON 87712 (SE) Control (SE) MON 87712 (SE) ControARNE 222.0 (0.0) 220.3 (0.5) 256.3 (0.3)* 253.0 (0.0) 268.0 (0.0)* 263.8 IAJA 230.8 (0.3) 231.5 (0.5) 266.8 (0.3)* 264.8 (0.3) 287.3 (0.5) 285.8 

IARL 225.8 (0.5) 226.0 (0.0) 268.3 (0.3)* 264.8 (0.3) 290.5 (0.3)* 281.5 ILCY 234.0 (0.6)* 232.8 (0.3) 267.0 (0.0)* 264.5 (0.7) 285.0 (0.4)* 280.5 ILMS 237.0 (0.4) 237.5 (0.5) 269.3 (0.3)* 268.0 (0.4) 282.8 (0.5)* 277.5 ILSE 241.0 (1.0) 239.8 (0.9) 280.3 (0.7)* 275.0 (0.7) 284.8 (0.3)* 280.8 ILWY 225.5 (0.5) 226.5 (0.5) 269.8 (1.0) 268.5 (0.9) 279.0 (0.0)† 279.0 INRC 254.0 (0.0)* 255.0 (0.0) 272.5 (0.3) 272.0 (0.7) 283.0 (0.0)† 283.0 INSH 228.0 (0.0) 228.5 (0.3) 265.3 (0.9) 264.0 (1.0) 274.3 (1.0)* 271.0 KSLA

—

— 265.0 (0.0)* 259.5 (0.3) 273.0 (0.0)* 266.5 

MOAN 244.3 (0.3) 243.3 (1.1) 275.0 (0.7)* 271.3 (0.8) 290.7 (0.3)* 287.0 MOCB 240.5 (1.2) 240.0 (1.4) 277.0 (0.0)* 271.8 (1.4) 291.8 (0.3)* 287.0 MOFI 221.5 (0.3)* 223.3 (0.3) 255.8 (0.3)* 252.0 (0.4) 283.8 (0.3)* 282.0 MOKI 243.0 (0.0)* 239.3 (0.6) 276.8 (0.3)* 273.3 (0.3) 276.8 (0.3)* 273.0 MOSL 219.0 (0.0) 218.0 (0.0) 259.5 (0.5)* 255.8 (0.5) 267.3 (0.5)* 263.0 MOWR 219.5 (0.3) 219.8 (0.3) 260.0 (0.0)* 257.0 (1.0) 268.0 (0.0)* 262.8 NEYO 224.5 (0.3) 224.8 (0.3) 267.0 (0.0)* 264.8 (0.3) 278.0 (0.0)* 277.3 PAGR 226.8 (0.5)* 225.5(0.3) 266.0 (0.0)* 264.8(0.3) 280.5 (0.3)* 278.5 PAHM 235.8 (0.3) 235.5 (0.3) 272.8 (0.3)* 270.5 (0.7) 294.0 (0.6)* 291.5 
Monsanto Company 11-SY-217U 

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control A3525 (contin

Phenotypic Characteristic (units)

Flower color7 Plant height (in) Lodging (1-9 scale)

Site1 MON 87712 Control MON 87712 (SE) Control (SE) MON 87712 (SE) Control ARNE purple purple 38.3 (0.7) 37.1 (0.7) 4.8 (0.3) 4.0 (0.0IAJA purple purple 41.9 (0.5) 40.9 (0.2) 1.0 (0.0)† 1.0 (0.0

IARL purple purple 36.0 (0.6) 35.9 (1.1) 4.5 (0.3) 3.8 (0.ILCY purple purple 

—

— 1.0 (0.0)† 1.0 (0.0

ILMS purple purple 21.0 (0.7) 21.0 (0.5) 1.0 (0.0)† 1.0 (0.0ILSE purple purple 

—

— 3.8 (0.3) 3.8 (0.3

ILWY purple purple 35.7 (0.5) 35.1 (1.8) 3.3 (0.3) 3.3 (0.3INRC purple purple 24.3 (0.6) 24.1 (0.7) 1.0 (0.0)† 1.0 (0.0INSH purple purple 27.1 (0.8) 26.4 (0.4) 1.0 (0.0)† 1.0 (0.0KSLA purple purple 41.2 (1.2) 38.9 (1.1) 3.0 (0.0)* 1.8 (0.3MOAN purple purple 26.7 (0.5) 26.5 (0.4) 1.0 (0.0) 1.0 (0.0MOCB purple purple 26.0 (0.4) 26.0 (0.9) 4.0 (0.4) 3.3 (0.3MOFI purple purple 25.8 (0.5) 22.8 (1.7) 1.0 (0.0)† 1.0 (0.0MOKI purple purple 25.2 (0.6) 26.7 (0.7) 1.0 (0.0)† 1.0 (0.0MOSL purple purple 34.3 (0.8) 32.8 (0.7) 1.0 (0.0)† 1.0 (0.0MOWR purple purple 31.8 (0.6) 30.1 (0.9) 1.0 (0.0)† 1.0 (0.0NEYO purple purple 36.0 (0.5) 35.8 (0.4) 1.0 (0.0)† 1.0 (0.0PAGR purple purple 

— 36.7 (1.9) 4.3 (0.5) 4.0 (0.4

PAHM purple purple 35.1 (0.5) 34.5 (0.1) 3.3 (1.0) 4.0 (0.7
Monsanto Company 11-SY-217U 

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control A3525 (contin

Phenotypic Characteristic (units)

Pod Shattering (1-9 scale)

Final stand count (# of 

plants/plot) Grain moisture (%)

Site1 MON 87712 (SE) Control (SE) MON 87712 (SE) Control (SE) MON 87712 (SE) ControARNE 1.0 (0.0)† 1.0 (0.0) 293.5 (4.4) 284.3 (5.0) 12.3 (0.1) 12.6 (0IAJA 1.0 (0.0)† 1.0 (0.0) 310.0 (7.0)* 249.0 (10.1) 13.1 (0.4) 12.6 (0

IARL 1.0 (0.0)† 1.0 (0.0) 316.0 (7.3) 318.5 (8.1) 13.0 (0.5)* 12.0 (0

ILCY 1.0 (0.0)† 1.0 (0.0) 287.0 (22.8) 294.5 (8.7) 10.1 (0.1) 10.2 (0ILMS 1.0 (0.0)† 1.0 (0.0) 284.3 (7.1) 276.8 (7.6) 13.4 (0.1) 13.4 (0ILSE 1.0 (0.0) 1.3 (0.3) 260.3 (9.9)* 232.3 (9.1) 13.7 (0.1) 13.8 (0ILWY 1.0 (0.0)† 1.0 (0.0) 317.0 (3.9) 320.5 (5.6) 12.8 (0.0) 12.7 (0INRC 1.0 (0.0)† 1.0 (0.0) 273.3 (11.4) 269.5 (10.9) 11.7 (0.2) 12.1 (0

INSH 1.0 (0.0)† 1.0 (0.0) 327.5 (3.6)* 305.8 (5.8) 12.8 (0.1) 13.1 (0

KSLA 2.5 (0.3) 4.0 (0.8) 300.5 (3.3)* 277.8 (12.4) 11.7 (0.2) 12.3 (0

MOAN 1.0 (0.0)† 1.0 (0.0) 314.5 (4.7) 314.0 (9.0) 11.2 (0.1) 16.8 (0

MOCB 1.0 (0.0)† 1.0 (0.0) 278.7 (4.7) 260.0 (28.7) 14.2 (0.3) 14.0 (0

MOFI 1.0 (0.0)† 1.0 (0.0) 274.5 (2.0) 259.3 (7.6) 11.6 (0.1)* 11.9 (0

MOKI 1.0 (0.0)† 1.0 (0.0) 188.8 (12.1) 206.8 (29.9) 11.9 (0.3) 12.5 (0

MOSL 1.0 (0.0)† 1.0 (0.0) 337.5 (2.7) 336.8 (2.5) 16.7 (0.4) 17.1 (0

MOWR 1.0 (0.0)† 1.0 (0.0) 347.8 (1.4)* 338.3(0.8) 13.1 (0.5) 12.7 (0NEYO 1.0 (0.0)* 1.8 (0.3) 343.5 (1.4)* 307.8 (6.4) 11.1 (0.0) 11.1 (0PAGR 1.0 (0.0) 1.5 (0.5) 266.3 (10.2) 261.8 (6.6) 16.0 (0.3)* 15.7 (0PAHM 2.5 (0.3) 2.8 (0.5) 325.3(2.3) 319.3 (0.0) 14.9 (0.5) 14.8 (0
Monsanto Company 11-SY-217U 

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control A3525 (contin

Phenotypic Characteristic (units)

100 seed weight (g) Yield (bu/ac)

Site1 MON 87712 (SE) Control (SE) MON 87712 (SE) Control (SE)ARNE 16.4 (0.2) 16.1 (0.3) 65.0 (1.1) 62.6 (1.5)IAJA 15.9 (0.2) 16.0 (0.2) 64.1 (3.2) 61.7 (0.3)

IARL 16.0 (0.4) 15.0 (0.0) 48.3 (2.1) 45.8 (2.2)ILCY 15.8 (0.2) 15.5 (0.5) 66.0 (1.4) 58.4 (4.0)ILMS 14.0 (0.1) 13.2 (0.3) 45.6 (2.2) 40.9 (3.6)ILSE 15.3 (0.1) 15.6 (0.5) 33.7 (2.1) 29.9 (2.9)ILWY 15.8 (0.3) 15.8 (0.2) 33.0 (1.9) 34.6 (2.0)INRC 14.7 (0.3)* 13.7 (0.5) 37.2 (2.9) 33.1 (1.3)INSH 15.7 (0.2) 15.8 (0.2) 54.5 (1.5)* 47.9 (1.7)KSLA 17.5 (0.3) 18.3 (0.3) 68.9 (2.9)* 60.0 (2.3)MOAN 14.0 (0.7) 13.5 (0.3) 48.3 (0.9) 47.7(1.5)MOCB 14.8 (0.2) 14.5 (0.1) 54.3 (1.4) 50.7 (3.5)MOFI 17.0 (0.2) 16.6 (0.3) 44.1(1.6) 43.7 (2.9)MOKI 20.5 (1.0)* 17.5 (0.7) 42.3 (1.5) 46.9 (2.6)MOSL 19.5 (0.3) 19.0 (0.0) 55.2 (1.8) 52.5 (1.3)MOWR 17.9 (0.5) 17.3 (0.1) 65.1 (1.7)* 58.3 (0.9)NEYO 15.3 (0.3) 14.8 (0.3) 51.7 (3.3)* 46.9 (0.2)PAGR 17.7 (0.1) 16.8 (0.3) 69.3 (1.6)* 61.4 (0.8)PAHM 16.3 (0.1) 17.0 (0.2) 52.4 (2.2)* 48.2 (1.6)

Note: The experimental design was a randomized complete block with four replications. * Statistically significant differences were detected (

α=0.05) between MON 87712 and the conventional control A3525. † No statistical comparisons were made due to lack of variability in the data. 

1 Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IARL = Jefferson County, IA; ILCY = Cl

Effingham County, IL; ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone CouCounty, KS; MOAN = Macon County, MO; MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County

County, MO; MOWR = Lincoln County, MO; NEYO = York County, NE; PAGR = Berks County, PA, site 1; PAHM = Berks County
Monsanto Company 11-SY-217U 

Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control (continued)2 Mean based on n = 4 for MON 87712 and the conventional control A3525 for all data characteristics except as follows. For Early S

conventional control A3525 at the MOKI site. For Days to 50% end of flowering n = 3 for MON 87712 at the ILSE site. For Days to the conventional control A3525 at the ILSE site. For Days to physiological maturity n = 3 for MON 87712 at the MOAN site. For 

conventional control A3525 at the PAGR site. For Final stand count n = 3 for the conventional control A3525 at the IAJA site. SE = S

3 Calendar day number (days after 1 Jan 2009) when 50% of the plants in each plot were flowering. 4 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot have stopped flowering. 5 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot reached 50% senescence. 6 Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plot reached physiological maturity. 7 Flower color data were categorical and were not statistically analyzed. 

— Dashes indicate information not collected.
Monsanto Company 11-SY-217U 

Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercial R

 Assessment Date and Range of Growth Stages Observed1

Site2 Substance Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 ARNE 6/29/2009 7/13/2009 7/21/2009 8/3/2009 8/26/2009 9/11/2009 — —  MON 87712 V2 R2 R2 R4 R6 R6 — —  Control V2 R2 R2 R4 R6 R6 — —  References V2 R2 — — — — — — IAJA 7/9/2009 7/16/2009 8/19/2009 8/27/2009 — — — —  MON 87712 V2 V5 R2 R3 — — — —  Control V2 V5 R2 R3 — — — —  References V2 V5 R2 R3 — — — — IARL 6/30/2009 7/2/2009 7/14/2009 7/27/2009 8/4/2009 8/17/2009 9/7/2009 9/14/2009  MON 87712 V2 V2 V6-V7 R2 R3 R5 R6 R6  Control V2 V2 V5-V6 R2 R3-R4 R5 R6 R6  References V2 V2 V5-V8 R2 R3-R4 R5 — — ILCY 7/22/2009 7/29/2009 8/10/2009 8/14/2009 8/22/2009 — — —  MON 87712 V2 V4 V5-V6 V9 R3 — — —  Control V2 V4 V5-V6 V9 R3 — — —  References V2 — V5-V6 — R3 — — — ILMS 7/24/2009 7/27/2009 8/20/2009 9/8/2009 9/29/2009 — — —  MON 87712 V2 V3 R2 R4-R5 R6 — — —  Control V2 V3 R2 R4-R5 R6 — — —  References V2 — R2 R4-R5 — — — — 
Monsanto Company 11-SY-217U 

Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercia(continued) 

 Assessment Date and Range of Growth Stages Observed1

Site2 Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 ILSE 7/17/2009 8/3/2009 8/12/2009 9/25/2009 — — —  MON 87712 V2 V5 R2 R4 — — —  Control V2 V5 R2 R4 — — —  References V2 — R2 R4 — — — ILWY 7/10/2009 7/27/2009 8/17/2009 9/7/2009 9/28/2009 — —  MON 87712 V2 R1 R4 R6 R7 — —  Control V2 R1 R4 R6 R7 — —  References V2 — — — — — — INRC 7/18/2009 7/28/2009 8/22/2009 10/2/2009 — — —  MON 87712 V1 V2 R2 R6 — — —  Control V1 V2 R2 R6 — — —  References V1 V2 R2 R6 — — — INSH 7/1/2009 7/7/2009 7/15/2009 7/29/2009 8/12/2009 8/28/2009 9/28/2009  MON 87712 V2 V2 V5 R1-R2 R3 R4 R7  Control V2 V2 V5 R1-R2 R3 R4 R7  References V2 V2 V5 R1-R2 R3 R4 R7 KSLA 7/2/2009 7/17/2009 7/22/2009 7/31/2009 8/14/2009 8/31/2009 9/11/2009  MON 87712 V3 R1 R2 R2 R4 R5 R6  Control V3 R1 R2 R2 R4 R5-R6 R6  References V3 R1 R2 R2 R4 R5-R6 R6 
Monsanto Company 11-SY-217U 

Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercia(continued) 

Assessment Date and Range of Growth Stages Observed1

Site2 Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 MOAN 7/22/2009 8/10/2009 8/27/2009 9/25/2009 10/13/2009 — — —  MON 87712 V2 R1 R5 R6 R6-R7 — — —  Control V2 R1 R5 R6 R7 — — —  References V2 R1 — — — — — — MOCB 7/20/2009 8/4/2009 8/25/2009 9/24/2009 10/13/2009 — — —  MON 87712 V2-V3 V6-V7 R4-R5 R6 R7 — — —  Control V2-V3 V6 R4-R5 R6 R7-8 — — —  References V2-V3 — — — — — — — MOFI 7/1/2009 7/7/2009 7/22/2009 8/12/2009 9/2/2009 — — —  MON 87712 V2 V3 V6-V7 R3 R6 — — —  Control V2 V3 V6 R3 R6 — — —  References V2 V3 V5-V7 R3 R6 — — — MOKI 7/20/2009 8/1/2009 8/10/2009 8/20/2009 9/24/2009 — — —  MON 87712 V1 V4-V5 V5 R4 R6 — — —  Control V1 V4-V5 V5 R4 R6 — — —  References V1 V4 V5 R4 R6 — — — MOSL 6/28/2009 7/15/2009 8/1/2009 8/21/2009 9/13/2009 — — —  MON 87712 V2 V7-V8 R3 R5 R6 — — —  Control V2 V7-V8 R3 R5 R6 — — —  References V2 V7-V8 — — — — — — 
Monsanto Company 11-SY-217U 

Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercia(continued) 

Assessment Date and Range of Growth Stages Observed1

Site2 Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 MOWR 6/28/2009 7/13/2009 7/23/2009 8/1/2009 8/20/2009 9/13/2009 — —  MON 87712 V2 V5-V6 R2 R3 R5 R6 — —  Control V2 V5 R2 R3 R5 R6 — —  References V2 V5 R2 — — R6 — — NEYO 7/2/2009 7/10/2009 7/22/2009 8/3/2009 8/11/2009 9/1/2009 9/28/2009 —  MON 87712 V3 V5 V7 R2 R3-R4 R5 R7 —  Control V3 V5 V7 R2 R3-R4 R5 R7 —  References V3 V5 — R2 — R5 — — PAGR 7/2/2009 7/22/2009 8/11/2009 8/31/2009 9/21/2009 10/6/2009 — —  MON 87712 V2 V6-V7 R3-R4 R6 R6 R7 — —  Control V2 V6 R3-R4 R6 R6 R7-R8 — —  References V2 V6 R4 R6 R6 R7-R8 — — PAHM 7/22/2009 8/12/2009 9/3/2009 9/23/2009 — — — —  MON 87712 V2-V3 R1 R5 R6 — — — —  Control V2 R1 R5 R6 — — — —  References — R2 R5 R6 — — — — Obs. = Observation number 1 Month-day-year. 2 Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IA; IARL = Jefferson County, IA; ILCY = Cl

Effingham County, IL; ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone CouCounty, KS; MOAN = Macon County, MO; MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County

County, MO; MOWR = Lincoln County, MO; NEYO = York County, NE; PAGR = Berks County, PA, site 1; PAHM = Berks County

— Dashes indicate information not provided. 
Monsanto Company 11-SY-217U 374 of 467 

Table H-6. Abiotic Stress Response Evaluations of MON 87712 and Conventional 

Control A3525 Using an Observational Severity Scale 

Abiotic Stressor 

Number of 

observations 

across all sites

Number of observations where no 

differences were observed between 

MON 87712 and the Conventional 

Control A3525 

Total 186 186 

Cold 6 6 

Compaction1 12 12 

Drought2 45 45 

Flood3 41 41 

Frost damage 5 5 

Hail damage 14 14 

Mineral toxicity 2 2 

Nutrient deficiency 29 29 

Wind damage 32 32 

 

No differences were observed between MON 87712 and the conventional control A3525 during any 

observation for damage caused by any of the assessed abiotic stressors. Data were not subjected to 

statistical analysis. 

Note: The experimental design was a randomized complete block with four replications. All observations 

were made during the following four crop developmental stages: V2 –V5; R1 - R2; R3 - R5; and R6 - R8. 

1

Includes soil compaction. 

2

Includes heat. 

3

Includes wet soil, excess moisture. 
Monsanto Company 11-SY-217U 375 of 467 

Table H-7. Disease Damage Evaluations of MON 87712 and Conventional Control 

A3525 Using an Observational Severity Scale 

Disease stressor 

Number of 

observations 

across all 

sites

Number observations where no 

differences were observed between 

MON 87712 and the Conventional 

Control A3525 

Total 198 198 

Anthracnose 6 6 

Aster yellow phytoplasma 1 1 

Bacterial blight1 19 19 

Bacterial leaf spot 3 3 

Brown stem rot 6 6 

Cercospora2

 7 7 

Charcoal rot 3 3 

Downy mildew 25 25 

Frogeye leaf spot 17 17 

Fusarium 1 1 

Leaf spots (Septoria and 

Alternaria)

3

18 18 

Phytophtora4 13 13 

Powdery mildew 16 16 

Pythium 3 3 

Rhizoctonia 1 1 

Seedling blight 1 1 

Septoria brown spot5 24 24 

Southern blight 1 1 

Soybean mosaic virus 3 3 

Soybean rust 6 6 

Stem canker 3 3 

Sudden death 15 15 

White mold 6 6 

 

No differences were observed between MON 87712 and the conventional control A3525 

during any observation for damage caused by any of the assessed disease stressors. Data 

were not subjected to statistical analysis. 

Note: The experimental design was a randomized complete block with four replications. 

All observations were made during the following four crop developmental stages: V3 –

V5; R1 - R2; R3 - R5; and R6 - R8. 

1 Includes Pseudomonas. 2 Includes Cercospora leaf blight and Cercospora leaf spot. 

3 Includes Alternaria black spot and Alternaria leaf spot. 

4 Includes root rot. 

5 Includes brown spot. 
Monsanto Company 11-SY-217U 

Table H-8. Arthropod-Related Damage Evaluations of MON 87712 and Conventional Control A3525 UsSeverity Scale Arthropod 

Number of observations across sites1

Number of observations where no differences were detected between MON 87712 and the conventional control A35252 

Statistically Significant Differences4Site3 

Observation Number 

Arthropod Damage Ra

MON 87712 Mean (S.E.) 

ContrMean 

Aphids5 (Aphididae) 20 20 

−

−

−

−

Armyworms6

(Spodoptera spp.) 10 10 

−

−

−

−

Bean leaf beetles (Cerotoma trifurcata) 35 33 ILCY 1 0.53(0.05) 0.23(

 MOAN 2 0.65(0.12) 0.93(

Blister beetles (Meloidae) 5 5 

−

−

−

−

Cabbage loopers (Trichoplusia ni) 1 1 

−

−

−

−

Grasshoppers (Acrididae) 29 29 

−

−

−

−

Green cloverworms (Plathypena scabra) 16 15 MOW R 4 0.60(0.04) 0.48 Japanese beetles (Popillia japonica) 21 20 ILMS 1 0.50(0.04) 0.30(

Leafhoppers7 

(Cicadellidae) 6 5 PAHM 1 0.55(0.12) 0.15(Silver spotted skippers (Epargyreus clarus) 1 1 

−

−

−

−
Monsanto Company 11-SY-217U 

Table H-8. Arthropod-Related Damage Evaluations of MON 87712 and Conventional Control A3525 UsSeverity Scale (continued) Arthropod 

Number of observations across sites1 

Number of observations where no differences were detected between MON 87712 and the conventional control A35252

Statistically Significant Differences4

Site3 

Observation Number 

Arthropod Damage Ra

MON 87712 Mean (S.E.) 

ContrMean 

Soybean loopers (Pseudoplusia includens) 11 9 MOAN 2 0.55(0.10) 0.90(

 MOFI 3 0.45(0.05) 0.85(

Stink bugs (Pentatomidae) 22 22 

−

−

−

−

Three-cornered alfalfa hoppers (Spissistilus 

f i )

4 4 

−

−

−

−

Thrips (Thripidae) 2 2 

−

−

−

−

Velvetbean caterpillars 

(Anticarsia 

gemmatalis) 4 3 MOFI 3 0.45(0.05) 0.85(

Note: The experimental design was a randomized complete block with four replications. Observations were conducted at four crop developmental staObservation 2 = R1-R2, Observation 3 = R3-R5, and Observation 4 = R6-R8. 1 A total of 192 arthropod damage observations were made across sites. Lack of variability in the data precluded statistical comparisons for 55 of 

comparisons could be made between MON 87712 and conventional control A3525 for 137 of the observations. 

2 No statistically significant differences were detected (α=0.05) or numerical differences between MON 87712 and the conventional control A3525 

generated due to lack of variability in the data. 

3 Site codes are as follows: ILCY = Clinton County, IL; ILMS = Effingham County, IL; MOAN = Macon County, MO; MOFI = Butler County, MO

MO; PAHM = Berks County, PA site 2. 

4 Means, standard errors (S.E.), and reference ranges are reported for a statistically significant difference that was detected (α=0.05) between MON 

control A3525. Reference range = minimum and maximum mean values among the commercial reference varieties. 5 Aphid includes soybean aphid. 

6 Armyworm includes fall armyworm and beet armyworm. 7 Leafhopper includes potato leafhopper. 

Dash (

−) indicates that there were no statistically significant differences were detected (α=0.05) between MON 87712 and the conventional control A3
Monsanto Company 11-SY-217U 

Table H-9. Abundance of Pest Arthropods in Samples Collected from MON 87712, Conventional ConCommercial Reference Varieties 

 Pest Arthropod1  Aphids (Aphididae) Bean leaf beetles (Cerotoma trifurcata) Corn earworms (HeColl. Site3 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

ConMea

1 ARNE 

−

−

− 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0 

−

−

 ILSE 0.8 (0.5) 0.0 (0.0) 0.5 - 2.0 1.5 (1.0) 1.8 (0.3) 0.8 - 2.5 

−

−

 MOCB 

−

−

− 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0 

−

−

2 ARNE 

−

−

− 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0 0.8 (0.3) 0.5 

 ILSE 2.3 (0.9) 2.8 (2.1) 1.3 - 4.0 1.3 (0.3)* 0.3 (0.3) 0.5 - 1.0 

−

−

 MOCB 

−

−

− 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3 

−

−

3 ARNE 

−

−

− 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0 

−

−

 ILSE 0.5 (0.5) 1.0 (0.6) 0.5 - 2.0 10.3 (1.9) 12.8 (3.6) 5.8 - 10.0 

−

−

 MOCB 61.5 (56.2) 31.8 (27.9) 16.3 - 61.8 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3 

−

−

4 ARNE 

−

−

− 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3 

−

−

 ILSE 1.5 (0.6) 1.0 (0.6) 0.0 - 1.8 35.8 (5.1) 25.5 (4.3) 17.0 - 30.8 

−

−

 MOCB 37.5 (12.0)* 19.3 (7.1) 18.3 - 42.5 0.8 (0.3) 0.5 (0.3) 0.5 - 1.3 

−

−
Monsanto Company 11-SY-217U 

Table H-9. Abundance of Pest Arthropods in Samples Collected from MON 87712, Conventional ConCommercial Reference Varieties (continued) 

 Pest Arthropod1  Green cloverworms 

(Plathypena scabra) 

Garden flea hoppers (Halticus bractacus) 

Leafhoppers2

(Cicadellidae) 

Coll. Site3 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

ConMe

1 ARNE 1.8 (0.5) 4.3 (2.3) 1.5 - 2.0 

−

−

−

−

−

 ILSE 7.3 (2.8) 13.5 (3.7) 9.0 - 12.8 

−

−

−

−

−

 MOCB 4.0 (1.7) 3.0 (1.2) 1.3 - 3.0 1.0 (0.7) 1.0 (0.7) 0.8 - 1.8 1.0 (0.7) 0.0 2 ARNE 1.5 (0.9) 4.5 (2.1) 2.3 - 3.0 

−

−

−

−

−

 ILSE 10.0 (2.0) 11.0 (2.9) 9.0 - 15.5 

−

−

−

−

−

 MOCB 3.8 (1.1) 2.0 (0.6) 1.3 - 3.0 0.3 (0.3) 0.3 (0.3) 0.0 - 1.3 

−

−

3 ARNE 2.8 (0.5) 4.3 (1.9) 2.0 - 3.5 

−

−

−

−

−

 ILSE 0.0 (0.0) 0.5 (0.5) 0.0 - 0.3 0.0 (0.0) 0.3 (0.3) 0.0 - 0.5 

−

−

 MOCB 0.8 (0.5)* 5.5 (2.6) 0.8 - 4.8 2.8 (1.3) 1.5 (1.2) 0.8 - 2.8 

−

−

4 ARNE 3.5 (1.6) 4.8 (1.8) 0.8 - 4.0 

−

−

−

−

−

 ILSE 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0 

−

−

−

−

−

 MOCB 0.3 (0.3) 0.0 (0.0) 0.0 - 0.0 8.0 (4.3) 4.3 (2.3) 1.5 - 4.5 

−

−
Monsanto Company 11-SY-217U 

Table H-9. Abundance of Pest Arthropods in Samples Collected from MON 87712, Conventional ConCommercial Reference Varieties (continued) 

 Pest Arthropod1  Soybean loopers 

(Pseudoplusia includens) 

Stink bugs (Pentatomidae) 

Three-cornered (Spissistilus festinu

Coll. Site3 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

ConMea

1 ARNE 

−

−

− 0.8 (0.5) 0.5 (0.5) 0.0 - 2.5 3.5 (0.9) 6.3 

 ILSE 0.0 (0.0) 0.0 (0.0) 0.0 - 0.5 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3 

−

−

 MOCB 

−

−

− 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0 

−

−

2 ARNE 

−

−

− 1.8 (0.8) 1.8 (0.6) 0.5 - 3.5 0.3 (0.3) 1.8 

 ILSE 

−

−

− 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3 

−

−

 MOCB 

−

−

− 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0 

−

−

3 ARNE 

−

−

− 0.3 (0.3)* 2.0 (0.4) 0.3 - 2.3 0.3 (0.3) 0.8 

 ILSE 

−

−

− 1.3 (0.3) 0.5 (0.3) 0.0 - 1.5 

−

−

 MOCB 

−

−

− 0.8 (0.5)* 0.0 (0.0) 0.0 - 0.5 

−

−

4 ARNE 

−

−

− 1.0 (1.0) 2.5 (1.0) 1.0 - 5.5 3.5 (1.2) 4.8 

 ILSE 

−

−

− 0.8 (0.5) 1.3 (0.8) 0.3 - 0.8 

−

−

 MOCB 

−

−

− 0.3 (0.3) 0.0 (0.0) 0.0 - 1.0 

−

−
Monsanto Company 11-SY-217U 

Table H-9. Abundance of Pest Arthropods in Samples Collected from MON 87712, Conventional ConCommercial Reference Varieties (continued) 

 Pest Arthropod1  Tarnished plant bugs 

(Lygus lineolaris) 

Yellow-striped armyworms (Spodoptera ornithogalli) 

Yellow woollybear ca(Spilosoma virginica) 

Coll. Site3 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

ContrMean 

1 ARNE 0.3 (0.3) 0.0 (0.0) 0.3 - 0.5 

−

−

−

−

−

 ILSE 0.0 (0.0) 0.5 (0.5) 0.0 - 0.8 

−

−

−

−

−

 MOCB 

−

−

−

−

−

−

−

−

2 ARNE 

−

−

− 1.0 (0.4) 0.3 (0.3) 0.0 - 1.8 

−

−

 ILSE 0.3 (0.3) 0.5 (0.3) 0.3 - 0.3 

−

−

− 0.3 (0.3) 0.0 (0

 MOCB 

−

−

−

−

−

−

−

−

3 ARNE 

−

−

− 0.3 (0.3) 0.5 (0.5) 0.0 - 0.8 

−

−

 ILSE 1.0 (0.4) 2.3 (1.9) 0.3 - 1.0 

−

−

−

−

−

 MOCB 

−

−

−

−

−

−

−

−

4 ARNE 

−

−

− 0.5 (0.5) 0.5 (0.5) 0.0 - 0.3 0.3 (0.3) 0.0 (0

 ILSE 2.3 (0.5) 7.8 (2.8) 1.5 - 3.5 

−

−

−

−

−

 MOCB 1.0 (0.7) 0.3 (0.3) 0.0 - 0.8 

−

−

−

−

−

Note: The experimental design was a randomized complete block with four replications. Arthropod collection 1 was made at R1 - R2 growth st

collections at approximately two week intervals thereafter. Numbers represent sample means with standard error (SE) in parentheses. 

* Indicates a statistically significant difference (α=0.05) between MON 87712 and the conventional control A3525.

† No p-values were generated due to lack of variability in the data.

1 MON 87712 and conventional control A3525 values represent mean number of arthropods from four replications. A dash (

−) indicates arthropod no

2 Leafhopper includes potato leafhopper. 3 Site codes are as follows: ARNE = Jackson County, AR; ILSE = Champaign County, IL; MOCB = Shelby County, MO. 
Monsanto Company 11-SY-217U 

Table H-10. Abundance of Beneficial Arthropods in Samples Collected from MON 87712, Conventional the Commercial Reference Varieties 

 Beneficial Arthropod1 Spiders 

(Araneae) 

Big-eyed bugs (Geocoris spp.) 

Carabid beetles (Carabidae) 

Coll. Site2 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

CM(

1 ARNE 1.5 (0.6) 2.8 (1.3) 0.5 - 3.0 4.3 (0.9)* 1.8 (1.0) 0.8 - 3.8 

−

−

 ILSE 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3 

−

−

−

−

−

 MOCB 0.8 (0.5) 0.3 (0.3) 0.0 - 0.3 

−

−

−

−

−

2 ARNE 2.8 (1.0) 2.5 (0.6) 1.8 - 5.8 4.0 (1.5) 2.5 (1.0) 0.8 - 7.0 

−

−

 ILSE 0.5 (0.5) 1.3 (0.5) 0.3 - 1.5 

−

−

−

−

−

 MOCB 1.3 (0.5) 0.3 (0.3) 0.3 - 1.0 

−

−

−

−

−

3 ARNE 2.8 (0.9) 1.0 (0.4) 0.5 - 2.0 1.0 (0.6) 1.0 (0.6) 0.3 - 1.0 

−

−

 ILSE 1.8 (0.3) 0.5 (0.3) 0.5 - 1.3 

−

−

− 0.5 (0.3) 0

 MOCB 1.3 (1.3) 0.8 (0.3) 1.8 - 2.8 

−

−

−

−

−

4 ARNE 1.8 (0.9) 1.3 (0.3) 0.8 - 2.5 0.3 (0.3) 1.8 (1.4) 0.3 - 1.8 

−

−

 ILSE 2.0 (0.7) 0.8 (0.5) 0.8 - 1.5 

−

−

−

−

−

 MOCB 2.8 (0.8) 1.3 (0.6) 0.5 - 2.0 

−

−

−

−

−
Monsanto Company 11-SY-217U 

Table H-10. Abundance of Beneficial Arthropods in Samples Collected from MON 87712, Conventional the Commercial Reference Varieties (continued) 

 Beneficial Arthropod1 Lacewings 

(Chrysopidae) 

Ladybird beetles (Coccinellidae) 

 Parasitic Wasps (Micro-parasitic 

Coll. Site2 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

CM(S

1 ARNE 

−

−

−

−

−

−

−

−

 ILSE 0.0 (0.0) 0.0 (0.0) 0.0 - 0.5 0.0 (0.0) 0.0 (0.0) 0.0 - 0.5 

−

−

 MOCB 

−

−

− 1.0 (0.4) 0.5 (0.3) 0.3 - 1.3 

−

−

2 ARNE 

−

−

−

−

−

− 0.3 (0.3) 0.

 ILSE 1.0 (0.4) 0.3 (0.3) 0.0 - 1.3 0.8 (0.3) 0.0 (0.0) 0.5 - 1.0 

−

−

 MOCB 

−

−

− 1.3 (0.5) 0.5 (0.3) 0.0 - 2.0 

−

−

3 ARNE 

−

−

−

−

−

−

−

−

 ILSE 0.3 (0.3) 0.3 (0.3) 0.0 - 0.8 4.0 (1.2) 3.5 (1.0) 3.8 - 8.8 

−

−

 MOCB 0.3 (0.3) 0.8 (0.5) 0.5 - 1.0 1.0 (0.6) 0.5 (0.3) 0.5 - 3.0 

−

−

4 ARNE 

−

−

−

−

−

−

−

−

 ILSE 0.5 (0.3) 0.0 (0.0) 0.0 - 0.3 8.3 (1.7)* 3.5 (1.0) 1.0 - 2.8 4.3 (1.4) 3 MOCB 2.8 (0.8) 1.0 (0.7) 1.5 - 3.3 3.8 (0.6) 2.8 (0.9) 2.5 - 5.3 2.3 (0.5)* 0
Monsanto Company 11-SY-217U 

Table H-10. Abundance of Beneficial Arthropods in Samples Collected from MON 87712, Conventional the Commercial Reference Varieties (continued) 

 Beneficial Arthropod1 Damsel bugs 

(Nabis spp.) Orius spp. 

Coll. Site2 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

MON 87712 Mean (SE) 

Control Mean (SE) 

Reference range 

1 ARNE 0.3 (0.3) 0.3 (0.3) 0.0 - 1.3 0.0 (0.0) 0.8 (0.5) 0.0 - 1.0  ILSE 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3 0.0 (0.0)* 1.0 (0.4) 0.0 - 0.8  MOCB 0.3 (0.3) 0.0 (0.0) 0.0 - 0.5 5.0 (2.6) 2.8 (1.0) 1.3 - 4.5 2 ARNE 2.3 (1.3) 2.5 (1.0) 0.5 - 4.3 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0  ILSE 0.0 (0.0) 0.0 (0.0) 0.0 - 1.3 2.8 (0.5) 1.3 (0.8) 1.0 - 2.5  MOCB 0.5 (0.5) 0.0 (0.0) 0.0 - 0.5 0.3 (0.3) 0.0 (0.0) 0.5 - 2.3 3 ARNE 1.5 (0.6) 1.0 (1.0) 0.3 - 2.0 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0  ILSE 0.3 (0.3) 0.8 (0.5) 0.5 - 1.5 17.3 (6.3) 6.3 (1.3) 4.3 - 11.5  MOCB 0.8 (0.3) 0.5 (0.5) 0.0 - 0.5 0.8 (0.8) 3.5 (2.4) 6.0 - 11.5 4 ARNE 1.0 (0.6) 0.3 (0.3) 0.8 - 2.0 0.0 (0.0)† 0.0 (0.0) 0.0 - 0.0  ILSE 0.8 (0.5) 0.5 (0.5) 0.5 - 2.0 19.5 (4.9) 16.3 (8.1) 4.5 - 11.0  MOCB 1.0 (0.4) 0.0 (0.0) 0.5 - 1.3 14.5 (4.2) 16.3 (4.7) 7.5 - 13.0 

* Indicates a statistically significant difference (

α=0.05) between MON 87712 and the conventional control A3525.

Note: The experimental design was a randomized complete block with four replications. Arthropod collection 1 was made at R1 - R2 

subsequent collections at approximately two week intervals thereafter. Numbers represent sample means with standard error (SE) in pa

† No p-values were generated due to lack of variability in the data.

1 MON 87712 and conventional control A3525 values represent mean number of arthropods from four replications. A dash (

−) indicate

2 Site codes are as follows: ARNE = Jackson County, AR; ILSE = Champaign County, IL; MOCB = Shelby County, MO. 
Monsanto Company 11-SY-217U 385 of 467 

References for Appendix H

Drees, B.M. and M.E. Rice. 1985. The vertical beat sheet: A new device for sampling 

soybeans insects. Journal of Economic Entomology 78:1507-1510. 
Monsanto Company 11-SY-217U 386 of 467 

Appendix I: Materials and Methods for Pollen Morphology and Viability 

Assessment 

I.1. Plant Production 

MON 87712, a conventional soybean control A3525, and four commercial reference 

varieties were grown under similar agronomic conditions in a field trial in St. Louis 

County, MO (Table H-1; MOSL site). The trial was arranged in a randomized completed 

block design with four replications. Each plot consisted of twelve rows approximately 30 

feet in length. 

I.2. Flower Collection 

When the soybean plants were flowering, approximately twenty whole flowers were 

collected from the first and fourth row of each plot. One flower from the bottom of the 

plant, two flowers from the middle, and one flower from the top were collected from each 

of five representative plants per plot. All flowers from a plot were placed in a single, 

clean container. The container was labeled with the plot number from which the sample 

originated, entry number, and entry name. The containers were kept on wet ice from 

collection until the pollen was prepared and stained. 

I.3. Pollen Sample Preparation 

Pollen samples were prepared in a laboratory. Clean microscope slides were labeled with 

the plot number. A circle of approximately one centimeter in diameter was drawn in the 

center of the slide with a pap hydrophobic barrier pen. Tweezers and a dissecting needle 

were used to open flowers from each plot and to 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 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. 

I.4. Data Collection 

Pollen characteristics were assessed by viewing samples with an Olympus BX51 

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 and installed with associated DP Controller c 1.2.1.108 and DP 

Manager V1.2.1.107 camera software and Image-Pro Plus v6.2.1.491 imaging software. 

I.4.1. 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 collapsed 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 
Monsanto Company 11-SY-217U 387 of 467 

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. 

I.4.2. Pollen Diameter 

Micrographs (400× resolution) of 10 selected pollen grains 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 from each plot 

was calculated from 20 total measurements. 

I.4.3. General Pollen Morphology 

General pollen morphology was observed from digital images of MON 87712, the 

conventional control A3525, and commercial reference varieties that also were used for 

pollen diameter measurements. 

I.5. Statistical Analysis 

An analysis of variance was conducted according to a randomized complete block design 

using SAS® (2008). The level of statistical significance was predetermined to be 5% 

(α = 0.05). MON 87712 was compared to the conventional control for percent viable 

pollen and pollen diameter. No statistical comparisons were made between MON 87712 

and the commercial reference varieties. A reference range for each measured 

characteristic was determined from the minimum and maximum mean values from 

among the references. General pollen morphology was qualitative; therefore, no 

statistical analysis was conducted on these observations. 
Monsanto Company 11-SY-217U 388 of 467 

References for Appendix I 

Alexander, M.P. 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain 

Technology 55:13-18. 
Monsanto Company 11-SY-217U 389 of 467 

Appendix J: Materials and Methods for Symbiont Assessment 

J.1. Materials 

The soybean materials for the symbiont interaction assessment included MON 87712, the 

conventional control A3525, and six commercial references varieties (Table J-1). 

Nodule, root tissue, and shoot tissue collected from MON 87712, the conventional 

control A3525, and the commercial reference varieties were evaluated. 

Table J-1. Starting Seed of MON 87712, Conventional Control, and Commercial 

Reference Varieties Used in the Symbiont Assessment 

T/C/R1 Starting Seed Name2 Phenotype Orion Number 

Test MON 87712 High yield 11223539 

Control A3525 Conventional 11223542 

Reference A2553 Conventional 10000961 

Reference Midland 363 Conventional 11243106 

Reference Stewart SB3454 Conventional 11242910 

Reference Gateway 427 Conventional 11225759 

Reference Pioneer 93B15 Conventional 11242904 

Reference Hoegemeyer 333 Conventional 11242905 

 

1

 T/C/R = test, control, and reference 

2

 The test starting seed name is a Monsanto Regulatory identification; the conventional control A3525 and 

commercial reference varieties starting seed names are variety names. 
Monsanto Company 11-SY-217U 390 of 467 

J.2. Characterization of the Methods 

The identities of MON 87712 and the conventional control A3525 seed were verified by 

event-specific polymerase chain reaction analyses. 

J.3. Greenhouse Phase and Experimental Design 

MON 87712, the conventional control A3525, and the commercial reference varieties 

starting seed were planted in 6-inch pots containing nitrogen-deficient potting medium 

(Sunshine® Mix #3 Basic/LB2) composed of primarily peat, vermiculite, and perlite. 

Plants from MON 87712, the conventional control A3525, and commercial reference 

varieties starting seed were grown in a greenhouse where actual temperatures ranged 

from approximately 18 to 35°C. Eight replicate pots were planted with three seeds per 

pot for each of MON 87712, the conventional control A3525, and commercial reference 

varieties. At planting, each seed was inoculated with approximately 1 × 107 cells of 

Bradyrhizobium japonicum (VAULT® NP, Becker, Underwood, Ames, IA) in phosphatebuffered saline. Pots were arranged in eight replicated blocks for the 6-week sampling 

period using a randomized complete block design. 

The starting seeds for replicates 1, 2, and 3 were planted on July 27, 2010, replicates 4, 5, 

and 6 were planted on July 28, 2010, and replicates 7, 8, and 9 were planted on July 29, 

2010. In all cases, replicate pots had a minimum of one plant emerge within one week. 

A solution of nitrogen-free nutrient solution (approximately 250 ml) was added weekly 

after plant emergence. 

J.4. 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 pots. The shoot material was cut 

into smaller pieces and placed in labeled 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 weighed to determine the fresh weight (fwt) of the 

nodules. 

The remaining root and shoot fresh weight were determined for each plant. Nodules as 

well as root and shoot material were placed in a drying oven on the same day as 

collected. The plant material was dried for at least 72 hours at approximately 65°C to 

determine dry weight (dwt). The shoot tissue was ground using 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). 

 
Monsanto Company 11-SY-217U 391 of 467 

J.5. Statistical Analysis 

The data consisted of six measurements taken at the six week sampling period: nodule 

number, nodule dwt (g), shoot dwt (g), root dwt (g), shoot percent total nitrogen, and 

shoot total nitrogen (g). An analysis of variance was conducted using a randomized 

complete block design with eight replications. Data were analyzed using SAS® (2008). 

The level of statistical significance was predetermined to be 5% (α = 0.05). No statistical 

comparisons were made between MON 87712 and the commercial reference varieties. 

Instead, a reference range for each measured characteristic was determined from the 

minimum and maximum mean values from among the six commercial reference varieties. 
Monsanto Company 11-SY-217U 392 of 467 

References for Appendix J 

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina.. 
Monsanto Company 11-SY-217U 393 of 467 

Appendix K: Materials and Methods for Volunteer Potential Assessment 

K.1. Materials 

The soybean materials for the volunteer potential assessment in the field included 

MON 87712, the conventional control A3525, and commercial reference varieties. The 

list of the soybean materials planted at each of four field sites is presented in Table K-1. 

The viability of the starting seed material was determined in the laboratory 

(BioDiagnostics Inc.) by conducting warm germination testing of each seed lot. The 

viability of MON 87712 and the conventional control A3525 from all sites was ≥90%. 

The viability of the commercial reference varieties from among the sites was ≥80% with 

two exceptions: two commercial reference varieties harvested at the Jackson County, MO 

(ARNE) site had germination rates of 42 and 31% respectively. Based upon the viability 

data, it was determined that the seed were acceptable for evaluating volunteer potential. 
Monsanto Company 11-SY-217U 

Table K-1. Starting Seed for Volunteer Potential Assessment Substance Substance Type Phenotype/Genotype Site1 

Orion ID # 

Rep 1 Rep 2 Rep 3 

MON 87712 Test Soybean Intrinsic Yield ARAU 11259962-002 11259970-002 11259979-002 MON 87712 Test Soybean Intrinsic Yield ARNE 11260951-002 11260927-002 11260953-002 MON 87712 Test Soybean Intrinsic Yield ILWY 11259999-002 11260022-002 11260014-002 MON 87712 Test Soybean Intrinsic Yield MOAN 11259953-002 11259931-002 11259939-002 A3525 Control Conventional ARAU 11259967-002 11259974-002 11259990-002 A3525 Control Conventional ARNE 11260932-002 11260949-002 11260947-002 A3525 Control Conventional ILWY 11260003-002 11260010-002 11260018-002 A3525 Control Conventional MOAN 11259958-002 11259943-002 11259934-002 SB3888R Reference Conventional ARAU 11259986-002 11259978-002 11259971-002 NK S30-D4 Reference Conventional ARAU 11259984-002 11259977-002 11259968-002 LG-C3540 Reference Conventional ARAU 11259982-002 11259966-002 11259975-002 Stine 3300-0 Reference Conventional ARAU 11259989-002 11259980-002 11259973-002 NuPride 3202 Reference Conventional ARNE 11260946-002 11260956-002 11260931-002 Pioneer 93M14 Reference Conventional ARNE 11260944-002 11260954-002 11260929-002 Pioneer 93M11 Reference Conventional ARNE 11260933-002 11260938-002 11260948-002 SB3888R Reference Conventional ARNE 11260935-002 11260930-002 11260945-002 Stine 3300-0 Reference Conventional ILWY 11260011-002 11260002-002 11259994-002 Pioneer 93M11 Reference Conventional ILWY 11260006-002 11260015-002 11259998-002 SB3819 Reference Conventional ILWY 11260009-002 11260000-002 11259992-002 SB3369R Reference Conventional ILWY 11260005-002 11260012-002 11260021-002 SB3369R Reference Conventional MOAN 11259936-002 11259944-002 11259952-002 NutriPride 8339 Reference Conventional MOAN 11259937-002 11259946-002 11259955-002 Pioneer 93M62 Reference Conventional MOAN 11259942-002 11259951-002 11259935-002 Pioneer 93M14 Reference Conventional MOAN 11259948-002 11259940-002 11259933-002 1 ARAU = Woodruff County, AR; ARNE = Jackson County, AR; ILWY = Stark County, IL; MOAN = Shelby County, MO.
Monsanto Company 11-SY-217U 395 of 467 

K.2. Characterization of the Materials 

The identities of MON 87712 and the conventional control A3525 were verified by 

event-specific polymerase chain reaction analyses. 

K.3. Field Sites and Plot Designs

Data were collected from field sites conducted in 2009-2010 growing season at four sites 

within the U.S. soybean production regions. These four sites provided a range of 

environmental and agronomic conditions representative of major U.S. soybean-growing 

regions. The Principle Investigator at each site was familiar with soybean production and 

the identification of soybean volunteers. 

At each site, the study area used for volunteer potential assessment had not been used for 

soybean production the previous growing season. Planting, soil, and cropping history 

information for each site are presented in Table K-2. The experiment was established at 

each of four locations in a randomized complete block design with four replications. 

Each plot was 5-8 ft wide by 20 ft long. To avoid mixing of the materials between 

adjacent plots during seed incorporation, a confined seeding area was established within 

each plot that consisted of the center area of the plot located approximately 1 ft from each 

plot edge. Consequently, the actual seeded area of each plot was 3-6 ft wide by 18 ft 

long. Alleyways between experimental blocks were 5-10 ft wide. 
Monsanto Company 11-SY-217U 

Table K-2. Seeding Information, Soil Description, and Cropping History Information for Volunteer PotenSite1 

Seeding Date2 

Target Seeding Rate (seed/plot) 

Approximate Seed Incorporation Depth (in) 

Plot Size (ft) 

Actual Seeding Area (ft) Reps 

Soil Series Description; Organic Matter (%); and pH 

Pr

ARAU 11/23/2009 400 1-3 8 x 20 6 x 18 4 Wiville fine sandy loam; 1.25 %; 6.2 ARNE 11/23/2009 400 1-3 8 x 20 6 x 18 4 Bosket fine sandy loam; 2.0 %; 5.8 SILWY 11/23/2009 400 1-3 5 x 20 3 x 18 4 Plano silt loam; 3.6 %; 6.1 MOAN 12/02/2009 400 0.5-2 5 x 20 3 x 18 4 Mexico silt loam; 2.7 %; 6.0 1ARAU = Woodruff County, AR; ARNE = Jackson County, AR; ILWY = Stark County, IL; MOAN = Shelby County, MO. 2 month/day/year. 
Monsanto Company 11-SY-217U 397 of 467 

K.4. Planting and Field Operations 

Each plot was hand-seeded by uniformly scattering approximately 400 seed on the soil 

surface within the confined seeding area. Seed were then mechanically incorporated to a 

maximum depth of approximately depth of approximately 0.5-3 inches to avoid surface 

predation. 

Agronomic practices used to prepare and maintain each study site were characteristic of 

each respective region. No irrigation was applied to the study areas, and no plot 

management was required after the seed were scattered in the plots. 

K.5. Data Collection 

The plots at each site were monitored for volunteer plants starting approximately two 

weeks after planting. Counts occurred approximately every two weeks thereafter until 

environmental conditions were no longer conducive for germination and emergence. 

Monitoring resumed in the spring of 2010, when environmental conditions became 

favorable for soybean germination and emergence. Counts continued approximately 

every two weeks until mid-June 2010. A total of seven germination and emergence 

observations were conducted at each site throughout the study. 

K.6. Data Assessment 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all stages of data collection, summarization, and analysis. Study personnel 

ensured 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 by the Lead Scientist for evidence of 

biologically relevant changes and for possible evidence of an unexpected plant response. 

Any unexpected observations or issues during the study that would impact the study 

objectives were noted by the Lead Scientist. 

K.7. Statistical Analysis 

There were few volunteers observed in the study. Due to insufficient number of 

volunteer plants and lack of variability, statistical analysis of the data could not be 

performed. 

K.8. Individual-Site Volunteer Potential Results and Discussion 

The environmental conditions after planting were not favorable for germination and 

emergence at any of the four sites, therefore no volunteers were observed in fall of 2009. 

Monitoring resumed in the spring of 2010, when environmental conditions became 

favorable for germination and emergence of soybean volunteers. No volunteer plants 

were observed at the ARAU and ARNE sites for MON 87712, the conventional control 

A3525 or commercial reference varieties. A small number of volunteer plants were 

observed at the ILWY and MOAN sites (Table VII-9). The small number of volunteer 

observed for MON 87712 and the conventional control A3525 where within the range of 
Monsanto Company 11-SY-217U 398 of 467 

the number of volunteers observed for the commercial reference varieties. However, no 

statistical analysis was performed due to insufficient number of volunteers observed and 

lack of variability 
Monsanto Company 11-SY-217U 399 of 467 

Appendix L: Materials and Methods for Persistence Outside of Cultivation 

Assessment 

L.1. Materials 

The soybean materials for the persistence outside of cultivation assessment in the field 

included MON 87712, the conventional control A3525, and commercial reference 

varieties. The list of the soybean materials planted at each of four field sites is presented 

in Table L-1. 

Table L-1. Starting Seed for Persistence Outside of Cultivation Assessment 

Material

Name1

Material

Type Phenotype

Monsanto

Lot Number Sites2

MON 87712 Test High yield 11223539 All

A3525 Control Conventional 11223542 All

Garst 3585N Reference Conventional 10001119 INSH, NEYO

Pioneer 93M62 Reference Conventional 11226582 INSH, NEYO

Stewart SB3579R Reference Glyphosate-tolerant 11226925 INSH, NEYO

Pioneer 93M11 Reference Glyphosate-tolerant 11226840 INSH, NEYO

Pioneer 93B82 Reference Conventional 11226581 MOCB

Trisoy 3535CN Reference Conventional 10001811 MOCB

NuPride 8339 Reference Glyphosate-tolerant 11226939 MOCB

NK S30-D4 Reference Glyphosate-tolerant 11226843 MOCB

Midland/Phillips 363 Reference Conventional 11226698 MOFI

Stewart SB3454 Reference Conventional 10001130 MOFI

Stewart SB3993R Reference Glyphosate-tolerant 11226927 MOFI

Pioneer 93M43 Reference Glyphosate-tolerant 11226839 MOFI

 

1 The use of the word “material” is synonymous with the use of the word “substance” in the 

statistical report (Appendix A). 

2

 Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; 

and NEYO = York County, NE 
Monsanto Company 11-SY-217U 400 of 467 

L.2. Characterization of the Materials 

Identities of MON 87712 and the conventional control A3525 seed were verified by 

event-specific polymerase chain reaction analyses. 

L.3. Field Sites and Plot Design 

Data were collected from field sites conducted in 2009 at four sites within the U.S. 

soybean production regions. The study sites were located within major soybean growing 

regions of the U.S. where seed or grain may be incidentally returned to the environment 

during harvest, handling, or transport. 

The experiment was established in 2009 at four sites in a randomized complete block 

design with three replications. Each plot was approximately 100 ft2

 in size, consisting of 

four rows, approximately 10 ft in length, with an inter-row spacing of approximately 30 

inches. 

Due to strong soybean plant stands in all plots at the MOCB site, the experiment was 

continued for a second season. The site was established in January, 2010 and continued 

through July, 2010. Each entry of every replication was established in exactly the same 

plot area in which that entry had previously been located during 2009. An exception was 

that the three plots which were planted with a material dropped from development were 

left unused after the plant and seed material from these plots was removed and destroyed. 

The seed for each plot was hand scattered over the existing vegetation in three ten foot 

rows. Each row was marked with a colored flag to facilitate locating new plants in the 

spring of 2010. Each plot was then covered with a wire mesh with 0.5 inch openings in 

order to prevent surface predation of the seeds by animals. The mesh was secured on the 

edges of all plots. An animal exclusion electric fence was constructed around the 

perimeter of the study area. A 15 foot isolation border was established around the outside 

of the fenced study area. This isolation border was the same area as the original 15 foot 

isolation border. 

L.4. Planting and Field Operations 

One hundred seeds were planted in each row and the planting density at all sites was 10 

seeds/ft resulting in 400 seeds/plot being planted at all sites. 

Within each study area, the study materials were grown under similar conditions. The 

study areas consisted of unmanaged areas and no plot preparation was performed other 

than mowing the area at approximately 3-6 inches. No fertilizers, tillage, or herbicides 

were applied to the study area prior to or after planting. Planting information and a soil 

description of each study site are provided in Tables L-2 and L-3. 

The INSH study area was well-drained unused pasture. The ground cover was mostly 

perennial grass with some broadleaf species and was estimated to be approximately 100% 

cover. The MOCB study area was on the perimeter of a cultivated field and had not been 

in agricultural use during the previous two years. The ground cover was estimated to be 

approximately 90% and consisted of fescue grass and clover. The MOFI study area was 
Monsanto Company 11-SY-217U 401 of 467 

a frequently mowed area near the farm house that was 100% covered in annual grasses 

and broadleaf annuals. The area had not been in agricultural production for at least ten 

years. The NEYO study area was a well drained level field margin adjacent to a field 

under cultivation and contained mostly perennial brome grass. The ground cover was 

estimated to be approximately 100%. None of the study sites had been used for crop 

production for at least the previous two years. Weather conditions at the sites during 

2009 and 2010 were typical for their respective regions and there were no unusual 

weather events.
Monsanto Company 11-SY-217U 

Table L-2. Seeding Information, Soil Description, and Cropping History Information for Persistence O

Assessment Site1

Planting Date 

(mm/dd/yyyy) 

Planting Rate 

(seeds/plot) 

Planting 

Depth (in) Plot Size (ft) Reps Soil Series; Org

INSH 06/05/2009 400 1.5 10 × 10 3 Crosby silt loam; 2.5MOCB 06/23/2009 400 1.0 10 × 10 3 Mexico silt loam; 2.MOFI 06/12/2009 400 0.5 10 × 10 3 Wiville loamy fine sNEYO 06/03/2009 400 0.75 10 × 10 3 Hastings silt loam; 3MOCB 01/25/2010 varies by plot surface 10 × 10 3 Mexico silt loam; 2.1 Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; and NEYO = York CounTable L-3. Study Area Planting Information and Soil Description for 2010 Extended Season at MOCB SitSite1

Planting Date 

(mm/dd/yyyy) 

Planting Rate 

(seeds/plot) 

Planting 

Depth (in) Plot Size (ft) Reps Soil Series; Org

MOCB 01/25/2010 varies by plot surface 10 × 10 3 Mexico silt loam; 2.1 Sites: MOCB = Shelby County, MO
Monsanto Company 11-SY-217U 403 of 467 

L.5. Data Collection 

The characteristics evaluated and the timing of their evaluation are presented in Table L4. Replacement values were calculated and used to evaluate the ability of the study 

materials to persist outside of cultivation. The replacement value is the ratio of the 

number of seeds produced to the number of seeds sown. A replacement value greater 

than one means that more seeds were produced than were sown and indicates a 

population that is increasing. A replacement value of less than one means that fewer 

seeds were produced than sown. This is indicative of a population that will not replace 

itself and will not persist. 

Table L-4. Data Characteristics Evaluated at Each Site for Persistence Outside of 

Cultivation Assessment 

Characteristic Timing of Evaluation Evaluation Description 

Stand count Approximately every 14 days Number of emerged plants per plot 

Growth stage 

monitoring 

Approximately every 14 days Average plant growth stage of emerged 

soybean plants using guidelines outlined 

in Soybean Growth and Development

(Pedersen, 2004). 

Vigor monitoring Approximately every 14 days 

up to R1 growth stage 

Vigor rating on a 1 – 9, where 1 = 

excellent vigor and 9 = poor vigor 

Number of plants 

producing pods 

R8 growth stage Total number of plants in a plot which 

produced pods 

Number of seed 

produced 

R8 growth stage Total number of seeds produced in a plot 

Seed weight R8 growth stage Total weight (g) of all seeds produced in 

a plot 

 
Monsanto Company 11-SY-217U 404 of 467 

L.6. Data Assessment 

Experienced scientists familiar with the 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 by the Study Director for 

evidence of biologically relevant changes and for possible evidence of an unexpected 

plant response. Any unexpected observations or issues during the study that would 

impact the study objectives were noted by the Study Director. 

L.7. Statistical Analysis 

Analysis of variance was conducted according to a randomized complete block design 

using SAS (2008). The level of significance was α = 0.05. For each statistically 

analyzed characteristic, MON 87712 was compared to the conventional control A3525 at 

each site. There was no intention to analyze the data across all sites since the sites were 

selected for their varying attributes. The reference range was determined for each 

characteristic from the minimum and maximum mean values of the references. 

L.8. Individual-Site Persistence Outside of Cultivation Results and Discussion 

Plant growth stage data were categorical and not statistically analyzed. However, there 

were no instances at any of the sites where the range of growth stages of MON 87712 and 

the conventional control A3525 did not overlap (Table L-4). 

No statistically significant differences were observed between MON 87712 and the 

conventional control A3525 at the MOFI, MOCB, and NEYO sites for stand count at any 

data collection time during the 2009 growing season. At the INSH site, MON 87712 had 

a greater stand count than the conventional control A3525 at the second observation (82.0 

vs. 28.3 plants/plot). Although the mean values for stand count for MON 87712 and the 

conventional control A3525 at the INSH site were outside the reference range (29-81 

plants/plot), the difference was not observed in the subsequent observation (1.3 vs. 3.3 

plants/plot) at the INSH site, at any of the six other observation times at the INSH site, or 

at any of the observations at the other sites (Table L-5). 

No statistically significant differences were observed between MON 87712 and the 

conventional control A3525 at the INSH, MOFI, and NEYO sites for plant vigor at any 

data collection time during the 2009 growing season (Table L-6). At the MOCB site, 

MON 87712 had slightly decreased plant vigor than the conventional control A3525 at 

the sixth observation (5.0 vs. 4.7 rating). The mean value for plant vigor for MON 87712 

at the MOCB site was within the range of the references (4.0-5.7 rating) and the 

difference was not observed at the other observation times at the MOCB site or at any 

observations at the other sites (Table VII-8). 

 

 SAS is a registered trademark of the SAS Institute. 
Monsanto Company 11-SY-217U 405 of 467 

No statistically significant differences were observed between MON 87712 and the 

conventional control A3525 at the end of the 2009 season for number of plants producing 

pods per plot, number of seeds produced per plot, or weight of seeds produced per plot 

(Table VII-8). 

At the INSH, MOFI, and NEYO sites, MON 87712 and the conventional control A3525 

produced no seed per plot (Table VII-8). This resulted in replacement values of zero at 

these sites. Therefore, the experiment was completed at these sites upon harvest in 2009. 

The replacement values of 2.72 for MON 87712 and 2.63 for the conventional control 

A3525 at the MOCB site in 2009 indicated that the plots produced more seed than was 

planted. However, no plants emerged in any of the plots during the second season at the 

MOCB site and thus, the replacement value was zero. This indicates that the populations 

were all in decline, and did not increase over time in the unmanaged conditions. 
Monsanto Company 11-SY-217U 

Table L-5. Growth Stage Monitoring of MON 87712, the Conventional Control A3525, and the CoVarieties in 2009 

Assessment Date and Range of Growth Stages Observed2

Site1 Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 INSH 6-24-09 7-8-09 7-21-09 8-12-09 8-24-09 9-11-09 9-28-09 10-14-09

−

MON 87712 V1 V2 V3 V2 V1-R3 R3 R5-R6 R7

−

A3525 V1 V2 V3 V2 R1-R3 R3-R4 R6 R7

−

References V1 V2 V3 V2 V2-R3 V1-R3 R6 R7

−

MOFI 6-26-09 7-10-09 7-27-09 8-8-09 8-21-09 9-4-09 9-19-09 10-5-09 10-21-0

MON 87712 V1 V2-V3 V2-V4 R1 R3-R4 R5 R7 R7-R8 R8 A3525 V1 V2 V2-V3 R1 R3 R5 R7 R8 R8 References V1 V2-V3 V2-V4 R1-R2 R3-R4 R5-R6 R7-R8 R7-R8 R8 

MOCB 7-7-09 7-21-09 8-4-09 8-19-09 8-31-09 9-16-09 9-28-09 10-13-09 10-29-0

MON 87712 VC V2 V4 R3-R4 R5 R5 R6 R7 R8A3525 VC V2 V4 R4 R5 R5 R6 R7 R8References VC V2 V4-R1 R4 R5 R5 R6-R7 R7 R8

NEYO 6-18-09 7-1-09 7-17-09

−

−

−

−

−

−

MON 87712 VC V2 V3

−

−

−

−

−

−

A3525 VC V2 V3

−

−

−

−

−

−

References VC V2 V3

−

−

−

−

−

−

Obs. = Observation number — Dashes indicate information not provided due to no plants remaining in the field. 1 Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; and NEYO = York Coun2 Month-day-year. 
Monsanto Company 11-SY-217U 

Table L-6. Stand Counts of MON 87712, the Conventional Control A3525, and the Commercial Reference 

Means and Ranges of Stand Counts Observed

Site1 Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 INSH

MON 87712 124.0 82.0* 1.3 4.7 0.3 0.7 0.7 0.7A3525 99.0 28.3 3.3 3.7 1.0 0.7 0.3 0.3References 89-140 29-81 2-9 2-9 0-2 0-1 0-1 0-1

MOFI

MON 87712 187.3 121.0 92.7 67.3 62.7 62.3 14.0 12.7 A3525 170.3 97.0 45.3 36.0 27.3 22.7 7.0 6.0 References 151-243 93-207 50-179 45-118 36-84 32-70 10-23 10-20 

MOCB

MON 87712 330.0 342.3 328.3 321.3 327.0 312.3 296.3 278.3 A3525 341.3 345.7 340.3 335.3 332.7 321.0 295.7 285.3 References 221-331 250-344 241-345 261-323 253-342 249-339 219-339 211-330 

NEYO

MON 87712 193.7 110.3 11.0

−

−

−

−

−

A3525 188.0 103.3 51.3

−

−

−

−

−

References 140-200 44-139 3-77

−

−

−

−

−

Note: The experimental design at each site was a randomized complete block with three replications. Values fo

conventional control A3525 are means. References are a range of the minimum and maximum mean values of the fouvarieties at each site. — Dashes indicate information not provided due to no plants remaining in the field. * Indicates a statistically significant difference between MON 87712 and the control (

α = 0.05). 1 Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; and NEYO = York Coun
Monsanto Company 11-SY-217U 

Table L-7. Plant Vigor Monitoring of MON 87712, the Conventional Control A3525, and the Commerciain 2009 

Means and Ranges of Vigor Ratings Observed2

Site1 Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 INSH

MON 87712 2.0 2.0 9.0 9.0† 3.0

−

−

−

A3525 2.0 2.0 8.0 9.0 3.0

−

−

−

References 1.3-2.0 2.0-2.7 7.3-8.7 9.0-9.0 0.0-6.0

−

−

−

MOFI

MON 87712 5.0 6.7 7.7 8.7

−

−

−

−

A3525 5.3 7.3 8.3 9.0

−

−

−

−

References 3.7-5.7 4.7-7.0 5.7-7.7 8.0-9.0

−

−

−

−

MOCB

MON 87712 2.0 3.0 NA 2.0 4.7 5.0* 5.0 3.3 A3525 2.7 3.0 NA 1.7 3.7 4.7 5.7 3.0 References 2.0-4.0 3.0-4.0 NA 1.3-2.7 4.3-5.7 4.0-5.7 5.3-7.0 2.7-3.3 

NEYO

MON 87712 5.0 8.0 8.7

−

−

−

−

−

A3525 5.3 7.7 8.3

−

−

−

−

−

References 5.0-5.7 7.0-9.0 7.7-9.0

−

−

−

−

−

Note: The experimental design at each site was a randomized complete block with three replications. Values for MON 87712 anA3525 are means. References are a range of the minimum and maximum mean values of the four commercial reference varieties at eac1 Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; and NEYO = York County, NE 2 Vigor was rated on a 1-9 scale, where 1 = excellent vigor and 9 = poor vigor. Plant vigor was not required after the plants reached the 

— Dashes indicate information not provided due to no plants remaining in the field or data was not required. NA = not available. * Indicates a statistically significant difference between MON 87712 and the control (

α = 0.05). 

† No statistical comparisons were made due to lack of variability in the data. 
Monsanto Company 11-SY-217U 409 of 467 

Table L-8. Phenotypic Comparison of MON 87712 to the Control within Each Site 

in 2009 

Number of plants producing pods per plot

Means (SE) Reference Range2

Site1 MON 87712 Control Minimum Maximum

INSH 0.7 (0.7) 0.3 (0.3) 0.0 1.0

MOFI 0.0 (0.0) 0.3 (0.3) 0.3 1.0

MOCB 136.0 (60.0) 140.7 (40.2) 124.3 201.7

NEYO 0.0 (0.0)† 0.0 (0.0) 0.0 0.0

 

Note: The experimental design at each site was a randomized complete block with three replications. The 

number of values used in the calculation of the means (n)=3. SE = standard error. 

† No statistical comparisons were made due to lack of variability in the data. 

1

 Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; and 

NEYO = York County, NE 

2

 Reference range = Minimum and maximum mean values among the commercial reference varieties. 

 
Monsanto Company 11-SY-217U 410 of 467 

References for Appendix L 

Pedersen, P. 2004. Soybean growth and development. Iowa State University, Ames, 

Iowa. 

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina. 
Monsanto Company 11-SY-217U 411 of 467 

Appendix M: Petitioner’s Environmental Report 

Summary

Monsanto has developed the biotechnology derived soybean line MON 87712 that will be 

used in traditional breeding programs to produce varieties with increased yield 

opportunity compared to conventional, non-biotechnology-derived soybeans of the same 

genetic background. Crop yield is a variable and complex parameter that depends on 

genetic as well as environmental factors. In field trials using MON 87712, yield increases 

of 7 to 11 percent were measured compared to control soybean grown in the same trials. 

The yield increases are achieved using a gene from the plant Arabadopsis thaliana that 

produces a protein that impacts the plant’s day/night processes. Research data indicate 

this change in day/night processes results in increased availability of carbon and nitrogen 

assimilates in the plant leading to higher yield. 

This environmental report (ER), which has been prepared to support APHIS’ obligations 

under the National Environmental Policy Act (NEPA), evaluates two alternatives: the 

“deregulation in whole” alternative and the no action alternative. Under the deregulation 

in whole alternative, MON 87712 would no longer be a regulated article under 7 CFR 

Part 340 and would be widely available for planting. Under the no action alternative 

MON 87712 would remain a regulated article. 

Affected Environment 

U.S. Soybean Production: 

In 2009, soybean represented 59% of world oilseed production, and approximately 35% 

of those soybeans (valued at $33.6 billion) were produced in the U.S. on 77.5 million 

acres throughout the eastern half of the U.S., with a concentration in the Midwest. 

During 2009, the U.S. exported 40.9 million metric tons (1.50 billion bushels) of 

harvested soybean seed, which accounted for 44% of the world's soybean exports. Most 

of the soybean supply (85%) is crushed to produce meal and oil, which is used to supply 

either the feed industry for livestock use or the food industry for edible vegetable oil and 

soybean protein isolates. Soybean is also used on a limited basis as forage for animal 

feed. 

Soybean Growth, Varieties, Yield and Land Use: 

Every year growers can choose from among hundreds of soybean varieties with different 

characteristics such as maturity group and subgroup, disease resistance, insecticide seed 

treatment, and herbicide tolerance. 

Soybean growth stages are important to growers because different growth stages are 

critical for certain agronomic practices (e.g., weed control, application of 

fungicide/insecticide), and certain other stages are critical for yield determination. The 

time of onset and the duration of the various growth stages in soybean are highly 

dependent on day length, which has led to different varieties being developed for 

different latitudes. In the U.S., soybean varieties are classified into one of ten major 
Monsanto Company 11-SY-217U 412 of 467 

“maturity groups”, which are designated by 10 north-south bands from North Dakota to 

Florida. 

Soybean varieties are tested in performance trials by public and private institutions to 

assess yield and other desirable characteristics. Breeders have historically used desirable 

traits such as yield for crop domestication and improvement by retaining plants that 

exhibit desirable traits for future breeding. In recent years molecular breeding techniques 

have been used to accelerate this process. As a result of conventional breeding combined 

with improved agronomic practices, soybean yields have steadily increased over the 

years, at a rate of approximately 0.35 bushels/acre since 1924, to the current U.S. average 

yield of 44 bushels per acre. While acres planted to some crops such as soybeans have 

increased over the years, the total acre of crops planted to cropland is the same as it was 

100 years ago. Some crops such as soybean have increased in acreage, and others, such 

as wheat, barley and sorghum have decreased in acreage. 

Seed Production: 

Because seeds from a given maturity group are produced within the general geographic 

boundaries of that maturity group, seed production occurs throughout the soybean 

growing area. While field operations and management practices for seed production are 

similar to those for commercial soybeans, extra care must be taken to ensure high purity 

and practices that ensure high germination rates of the harvested seed. Not all 

commercial seed is certified, but most commercial seed meets certification standards. 

Organic Soybean Production: 

In the 2008 Census of Agriculture, the most recent data source available, USDA reported 

that organic soybeans were harvested on 98,199 acres in 28 states, with a total production 

of 2.58 million bushels, representing 0.09% of the total U.S. soybean production for 

2008. U.S. organic soybean production peaked in 2001, then declined as a result of 

competition from overseas. A 2006 study found that the price premiums organic farmers 

receive make up for higher production costs and lower yields compared to nonorganic 

soybeans. 

Agricultural Practices: 

Variety selection is critical to optimize yields and growers select varieties of the correct 

maturity group that yield well and are adapted to the length of the growing season in their 

geographical location. Tillage and no-till or con-till methods may be used for production 

of soybean. Soybean requires 16 essential elements for growth and development. The 

primary or major essential elements are nitrogen, potassium and phosphorous. Nitrogen 

is seldom applied to soybean fields because soybean is a member of the legume family 

and fixes its own nitrogen. Potassium and phosphorous are also seldom applied because 

sufficient amounts of these two elements remain in the soil from the previous season’s 

application to well fertilized crops like corn and wheat which are typically rotated with 

soybean. 

Within the U.S., 95% of the soybean-planted acreage has been in some form of a crop 

rotation system since 1991, with the majority being rotated to corn. Rotation has a 
Monsanto Company 11-SY-217U 413 of 467 

number of benefits, including increased yields, decreased fertilizer needs, and reductions 

in disease and weed losses. 

Insect damage in soybeans usually does not reach levels that cause economic loss in the 

U.S.. Major diseases resulting in yield loss include, in order of economic importance, 

soybean cyst nematode (Heterodera gylcines), Phytophthora root and stem rot 

(Phytophthora sojae), seedling diseases, charcoal rot (Macrophomina phaseolina) sudden 

death syndrome (Fusarium virguliforme), and sclerotinia stem rot (Sclerotinia 

sclerotiorum). Soybean cyst nematode causes by far the greatest soybean yield losses. 

Selecting resistant varieties and crop rotation are the primary tools growers have for 

disease control. 

When weeds are left to compete with soybean for the entire growing season, yield losses 

can exceed 75%. Cultural (e.g. crop rotation, narrow row spacing and planting date) and 

mechanical weed control practices (tillage) can be important components of an effective 

weed management program. Nearly all soybean fields receive some type of herbicide 

treatment and approximately 90% of soybeans planted are genetically modified for 

herbicide tolerance. 

Human and Animal Health: 

Both humans and animals have consumed soybean in various forms for thousands of 

years. Soybean improved with new traits produced by biotechnology pose no unique 

risks relative to other soybean and have been extensively evaluated for their safety prior 

to introduction. 

Animal and Plants: 

The affected environment for growing soybean plants can generally be considered the 

agroecosystem (managed agricultural fields) plus some area extending beyond the 

intended plantings that might be affected by agricultural operations. Mammals and birds 

may seasonally consume seed, and invertebrates can feed on the plant during the entire 

growing season. Plants growing in this adjacent area can be affected by fertilizer runoff, 

water runoff and/or herbicide drift. 

Physical Environment: 

Surface water may be impacted from soybean production, primarily by runoff of soil 

particles from soybean fields, but also by herbicides or other pesticides that may make it 

to streams, rivers, lakes, wetlands and other water bodies either through runoff or drift. 

Groundwater impacts may be of potential concern in some areas where nitrogen levels 

are either approaching or have exceeded the maximum contaminant level; however, 

soybean generally does not require addition of nitrogen fertilizer. 

Adjacent Agricultural Crops and Non-Agricultural Plants: 

Soybean is primarily grown adjacent to other large acre crops such as corn, wheat, and 

alfalfa. Soybean is highly self-pollinating and exhibits a very low level of outcrossing. 
Monsanto Company 11-SY-217U 414 of 467 

Soybean does not have any related wild relatives in the U.S. with which it can hybridize. 

Soybean is not found outside of cultivation and is not invasive or weedy. 

Environmental consequences 

Potential environmental impacts of the no action and deregulation in whole alternatives 

are summarized below: 

Attribute/Measure No Action Deregulation in Whole

Commercial 

Production and Use 

No change to the affected 

environment. MON 87712 not 

available for wide scale 

production, other 

biotechnology-derived soybean 

would be available. 

No change except for the potential 

for higher yields. Composition 

comparable to conventional control; 

widespread use of biotechnologyderived soybean. 

Economic No change to the affected 

environment. Higher yielding 

soybean varieties continue to be 

introduced, providing economic 

benefit. 

Higher yielding soybean varieties 

more readily available than if 

developed using conventional 

breeding programs. 

Land Use No change to the affected 

environment. Soybean will 

continue to be grown on land 

devoted to crop production – 

acreage driven by soybean 

demand. 

Not different from no action 

alternative. Soybean acres could 

potentially decrease due to higher 

soybean yields - acreage driven by 

soybean demand. 

Seed Production No change to the affected 

environment. Current 

production practices in place to 

handle specialty, organic and 

seed production for soybean 

would continue. 

Not different from no action 

alternative – similar production 

practices would be used to produce 

MON 87712 seed. 

Organic Soybean 

Production 

No change to the affected 

environment. Biotechnologyderived soybean has been on 

the market for over 14 years 

and co-existence practices are 

established – these practices 

will continue. 

Not different from no action 

alternative – MON 87712 will 

utilize similar production practices 

already established for commodity 

biotechnology-derived soybean. 

Cropping Practices 

(tillage, irrigation) 

No change to the affected 

environment. Growers will 

continue to use cropping 

Not different from no action 

alternative - No change in cropping 

practices expected based on 
Monsanto Company 11-SY-217U 415 of 467 

Attribute/Measure No Action Deregulation in Whole

practices and variety selection 

to maximize yield and return. 

observations of MON 87712 during 

confined release field trials. 

Insect & Disease 

Management 

No change to the affected 

environment. Insect & disease 

management practices will 

continue to be driven by pest 

and disease pressure. 

Not different from no action 

alternative - MON 87712 shows no 

change in insect & disease 

susceptibility. 

Variety 

Development 

No change to the affected 

environment. Current breeding 

practices and variety selection 

continue. 

Not different from the no action 

alternative. Slight delay in 

physiological maturity will be 

compensated for during new variety 

development and selection process. 

Weed Management No change to the affected 

environment. Current weed 

management practices expected 

to continue. 

Not different from no action 

alternative – MON 87712 will 

likely be bred with herbicide 

tolerant soybean and weed control 

practices used with herbicide 

tolerant soybean will be used. 

Human Health and 

Worker Safety 

No change to the affected 

environment. MON 87712 not 

available for wide scale 

production and soybean 

currently on the market are 

consumed. 

Not different from no action 

alternative. Soybean produced by 

MON 87712 are compositionally 

comparable to conventional 

soybean and the BBX32 protein is 

safe for consumption, therefore, 

MON 87712 will not impact human 

health and worker safety. 

Plant & Animal 

Communities; TES 

No change to the affected 

environment. MON 87712 

would be grown in isolation on 

small acreages. Higher 

yielding soybean will continue 

to be introduced through 

conventional breeding.. 

Not different from no action 

alternative. MON 87712 would 

potentially increase yield in 

soybean varieties. The increase in 

yield associated with MON 87712 

is unlikely to impact plants, animals 

or TES. 

Soil Microbes No change to the affected 

environment. Higher yielding 

soybean will continue to be 

introduced. 

Not different from no action 

alternative. Exposure to BBX32 

protein not expected to change soil 

microbial populations. 
Monsanto Company 11-SY-217U 416 of 467 

Attribute/Measure No Action Deregulation in Whole

Non-crop & NonAgricultural Areas No change to the affected 

environment. High yielding 

soybean would continue to be 

introduced 

Not different from no action 

alternative - MON 87712 has no 

change in invasive characteristics 

from conventional soybean. 

Cumulative Impact 

Due to Breeding 

with Previously 

Deregulated Events 

Stacking 

No change to the affected 

environment. MON 87712 not 

available for stacking with 

previously deregulated events 

under this alternative. 

MON 87712 available for breeding 

with previously deregulated events 

and potentially events under review. 

The stability of MON 87712 over 

multiple generations has been 

demonstrated. No cumulative 

impacts are expected. 
Monsanto Company 11-SY-217U 417 of 467 

M.1. Introduction 

This environmental report (ER) has been prepared for APHIS to facilitate the agency’s 

compliance with the National Environmental Policy Act (NEPA), including compliance 

with the Council on Environmental Quality’s (CEQ) regulations that implement NEPA.8

M.1.1. Background and Rationale 

Monsanto Company has developed the biotechnology-derived soybean line MON 87712, 

which will be used in traditional breeding programs to produce commercial varieties with 

increased yield opportunity. The yield increase in MON 87712 is achieved using the 

BBX32 gene from the plant Arabidopsis thaliana that produces a protein that interacts 

with one or more endogenous transcription factors to regulate the plant’s day/night 

processes and results in increased availability of assimilates (products of plant 

metabolism) in MON 87712 compared to an appropriate comparator without this gene. 

Increased plant nutrient availability in MON 87712 is supported by the measurement of 

factors indicative of an extended period of photosynthetic activity in MON 87712 and 

evidence of changes in diurnal metabolism during the reproductive phase of the soybean 

plant, as well as by the significantly higher yield of MON 87712 when compared to 

control, as observed in multisite field studies in the U.S. Higher yielding soybeans offer 

the opportunity for benefits to growers and the soybean food and feed chain, and help 

meet global demand for soybean. 

M.1.2. Purpose and Need for Action 

M.1.2.1 Regulatory Authority 

"Protecting American agriculture" is the basic charge of the U.S. Department of 

Agriculture's (USDA) Animal and Plant Health Inspection Service (APHIS). APHIS 

provides leadership in ensuring the health and care of plants and animals. In 1986, the 

Federal Government’s Office of Science and Technology Policy (OSTP) published a 

policy document known as the Coordinated Framework for the Regulation of 

Biotechnology. This document specifies three Federal agencies that are responsible for 

regulating agricultural biotechnology in the U.S.: USDA APHIS, the U.S. Department of 

Health and Human Services’ Food and Drug Administration (FDA), and the 

Environmental Protection Agency (EPA). APHIS regulates biotechnology-derived 

organisms under the Plant Protection Act of 2000. FDA regulates food and feed derived 

from biotechnology-derived organisms under the authority of the Federal Food, Drug, 

and Cosmetic Act. The FDA policy statement concerning regulation of products derived 

from new plant varieties, including those biotechnology-derived, was published in the 

Federal Register on May 29, 1992.9

 Under this policy, FDA uses a consultation process 

to ensure that human food and animal feed safety issues or other regulatory issues (e.g., 

labeling) are resolved prior to commercial distribution of food. 

 

8

 Title 40 of the Code of Federal Regulations (40 CFR) Parts 159901508. 

9

 57 FR 22984- 23005 
Monsanto Company 11-SY-217U 418 of 467 

M.1.2.2. USDA Regulation of Biotechnology-derived Organisms 

The APHIS Biotechnology Regulatory Service’s (BRS) mission is to protect America’s 

agriculture and environment using a dynamic and science-based regulatory framework 

that allows for the safe development and use of biotechnology-derived organisms. APHIS 

regulations,10 which were promulgated pursuant to authority granted by the Plant 

Protection Act, as amended,11 regulate the introduction (importation, interstate 

movement, or release into the environment) of certain biotechnology-derived organisms 

and products. A biotechnology-derived organism is considered a regulated article if the 

donor organism, recipient organism, vector, or vector agent used in engineering the 

organism belongs to one of the taxa listed in the regulation (7 CFR 340.2) and is also 

considered a plant pest. A biotechnology-derived organism may also be regulated under 

Part 340 when APHIS has reason to believe that the biotechnology-derived organism 

may be a plant pest or APHIS does not have sufficient information to determine if the 

biotechnology-derived organism is unlikely to pose a plant pest risk. 

Under 7 CFR 340.6 entitled “Petition for Determination of Nonregulated Status”, A 

person may petition the agency to evaluate submitted data and determine that a particular 

regulated article is unlikely to pose a plant pest risk, and, therefore, should no longer be 

regulated. The petitioner is required to provide information under § 340.6(c)(4) related to 

plant pest risk that the agency may use to determine whether the regulated article is 

unlikely to present a greater plant pest risk than the unmodified organism. A 

biotechnology-derived organism is no longer subject to the regulatory requirements of 7 

CFR part 340 when APHIS determines that it is unlikely to pose a plant pest risk. 

M.1.2.3. Petition for Determination of Nonregulated Status: MON 87712 

Monsanto has submitted a petition to APHIS seeking a determination of non-regulated 

status for MON 87712 (petition; #11-SY-271U). As detailed in the petition, MON 87712 

contains a gene, BBX32, encoding for the production of a transcriptional accessory 

protein that modulates existing diurnally regulated processes resulting in increased 

incorporation of nutrients into the plant. Carbon and nitrogen metabolism is a diurnally 

regulated process; altering a plant’s response to the day/night cycle can thus affect carbon 

and nitrogen metabolism. Alteration of a plant’s reproductive responses to day/night 

length is a known mechanism for increasing yield components (Kantolic and Slafer, 

2005). The BBX32 activity shifts the diurnally regulated gene transcription, thereby 

influencing the plant’s day/night cycle. This modulation of the diurnally regulated 

carbon and nitrogen metabolism leads to increased availability of assimilates and hence 

increased yield. Interstate movements and field trials of MON 87712 have been 

conducted under permits issued or notifications acknowledged by APHIS since 2006. 

The petition includes information that has been collected from field trials, laboratory and 

greenhouse studies, and published literature to assess whether the increase in yield 

through production of the BBX32 protein and/or the gene insertion process has altered 

 

10 7 CFR § 340 11 Title IV Pub. L. 106-224, 114 Stat. 438, Title 7 of the U.S. Code (7 USC) § 7701-7772[font] 
Monsanto Company 11-SY-217U 419 of 467 

MON 87712 in any way that would make these plants more of a plant pest compared to 

conventional soybeans, or cause significant environmental impacts, including cumulative 

impacts. 

M.1.2.4. APHIS Action 

Under the authority of 7 CFR part 340, APHIS has the responsibility for the safe 

development and use of biotechnology-derived organisms under the provisions of the 

Plant Protection Act. APHIS must respond to petitioners that request a determination of 

the nonregulated status of biotechnology-derived organisms, including biotechnologyderived crop plants such as MON 87712. If a petition for nonregulated status is 

submitted, APHIS must make a determination if the biotechnology-derived organism is 

not likely to pose a plant pest risk. 

MON 87712 has been field tested in the U.S. since 2006 in APHIS authorized trials. 

Associated notifications acknowledged by APHIS are listed in the petition in Appendix 

A. Field tests conducted under APHIS oversight allow for evaluation in agricultural 

settings under confinement measures designed to minimize the likelihood of persistence 

in the environment after completion of the field trial. Under confined field trial 

conditions, data are gathered on multiple parameters and used by applicants to evaluate 

agronomic characteristics and product performance. These data are also valuable to 

APHIS for assessing the potential for a biotechnology-derived plant to pose a plant pest 

risk. 

As a Federal agency subject to compliance with the National Environmental Policy Act 

(NEPA),12 APHIS must consider the potential environmental effects of its 

actions/decisions and reasonable alternatives to those actions, consistent with NEPA 

regulations13 and the USDA and APHIS NEPA implementing regulations and 

procedures.14 This environmental report (ER) evaluates the potential impacts that may 

result from deregulation of MON 87712 and has been prepared to support APHIS’ 

compliance with NEPA. 

M.1.2.5. Submissions to Other Regulatory Agencies 

In accordance with FDA’s consultation policy (discussed in Appendix M.1.2.1), 

Monsanto will submit a food and feed safety assessment and nutritional assessment 

summary for MON 87712. The EPA regulates plant-incorporated protectants (PIPs) 

under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). MON 87712 is 

not a PIP and does not have pesticidal activity and therefore is not within the scope of 

EPA regulations for pesticides. In addition, the MON 87712 trait does not provide 

herbicide tolerance or otherwise impact herbicide use. No submissions to EPA are 

required. 

 

12 42 USC §4321 et seq. 13 40 CFR Parts 1500-1508 14 7 CFR 1b and 7 CFR Part 372[font] 
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To support commercial introduction of MON 87712 in the U.S., regulatory submissions 

will be made to countries that will eventually commercialize or import significant 

quantities of soybean and/or soybean products from the U.S. These will include 

submissions to a number of foreign government regulatory authorities, including: 

Ministry of Agriculture, People’s Republic of China; Japan’s Ministry of Agriculture, 

Forestry, and Fisheries, Ministry of Environment, and the Ministry of Health, Labor, and 

Welfare; the Canadian Food Inspection Agency and Health Canada; the Intersectoral 

Commission for Biosafety of Genetically Modified Organisms, Mexico; the European 

Food Safety Authority, as well as to 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. 

M.2. Affected Environment 

This section describes the environment in which soybeans are currently grown and 

utilized in the U.S. 

M.2.1. Commercial Soybean Production and Use 

Commercial soybean production and uses are discussed in Section VIII.B of the petition 

and summarized here; refer to the petition for more detail. 

Soybean was a relatively minor crop used mostly for forage in the U.S. from the late 

1700s to the 1920s and 1930s. When breeders developed cultivars with shatter-resistant 

seeds that allowed for use of mechanical harvesting equipment, acreage expanded 

rapidly, major investments were made in breeding in the 1930s, and today soybean is the 

world’s most important oilseed crop. 

Soybean is grown as a commercial crop in over 35 countries and is one of the most 

valued agricultural commodities because of its high protein and oil content. In 2009, 

soybean represented 59% of world oilseed production, and approximately 35% of those 

soybean were produced in the U.S. (ASA, 2008). In 2009, the U.S. exported 40.9 million 

metric tons (1.50 billion bushels) of soybean, which accounted for 44% of the world's 

export of whole soybean seeds (USDA FAS, 2011). Total U.S. soybean exports 

(including soybean meal and oil) were valued at $22.1 billion in fiscal year 2010 (USDA, 

2011). USDA world projections through 2020 show world soybean production and 

exports increasing greatly, but with the U.S. share slightly declining due to greatly 

increased production in Brazil (USDA, 2011). 

Currently, approximately 85% of the world’s harvested soybean supply is crushed to 

produce soybean meal and oil (Soyatech, 2010), 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. 

The U.S. soybean acreage in the past 10 years has varied from approximately 64.7 to 77.5 

million acres. Average soybean yields have varied from 33.9 to 44.0 bushels per acre 

over this same time period. According to data from USDA NASS (2011), soybean was 
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planted on approximately 77.5 million acres in the U.S. in 2009, producing 3.36 billion 

bushels of soybean with a value of $32.1 billion (USDA NASS, 2011). 

In the U.S., soybean production occurs throughout much of the eastern half of the U.S. 

and is concentrated in the Midwest (Table M-1). Table M-1 shows average state 

production by region in 2010. 

Table M-1. 2010 Soybean Productivity by Region 

Region 

2010 U.S. 

Soybean 

Acreage1

2010 Average 

Yield 

(bushels per acre)

Range of Average State 

Yields (bushels per acre) 

Midwest/Great 

Plains 

64.2 43.9 32.5 – 52.5 

Southeast 10.3 31.0 23.0 – 41.0 

Eastern Coastal 2.1 34.0 26.0 – 48.0 

1

U.S. soybean acreage – million acres 

Source USDA NASS (2011) 

M.2.1.1. Soybean Growth Stages 

Soybean growth stages are important to growers because different growth stages are 

critical for certain agronomic practices (e.g., weed control, application of 

fungicide/insecticide), and certain other stages are critical for yield determination (Koger 

2011, Pederson 2004, McWilliams et al., 2004). Growth stages are designated by two 

characters, beginning with either “V” for vegetative or “R” for reproductive. Some of the 

vegetative stage designations are VE for emergence, and V1 through Vn for the 

appearance of the sets of three-part (trifoliate) leaves. There are eight designated 

reproduction stages, for example, R1 for the appearance of the first flower, R3 for 

beginning of pod development, R5 for beginning of seed development, and R8 for full 

maturity (Pederson, 2004; Heatherly and Elmore, 2004). 

The time of onset and the duration of the various growth stages in soybean are highly 

dependent on photoperiod (hours of daylight and darkness) and temperature (Major et al., 

1975), and therefore, for the same soybean plant grown at different latitudes, the onset 

and duration of the growth stages and the total time from planting to maturity would be 

different. Also, in contrast to most other temperate-season crops, soybean is a “shortday” plant, meaning that maturity is delayed by longer day length (Major et al., 1975). In 

soybeans, flowering is initiated only after the night is longer (and days grow shorter) than 

a critical length (Holshouser, 2010). Once flowering begins, temperature controls the 

duration of flowering time (Heatherly and Elmore, 2004). 

 
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M.2.1.2. Soybean Variety Development and Yield 

Maturity Groups. Because soybean growth is so dependent on day/night length, different 

varieties are developed for different latitudes. In the U.S. ten geographically-designated 

“maturity groups” originally defined by Scott and Aldrich (1970) are widely used (Zhang 

et al., 2007). These maturity groups are mapped as bands extending from north to south, 

beginning with Group 00 at the far north and ending with Group VIII in the far south.15 

Groups II, III and IV, which extend from approximately the northern border of Iowa to 

the southern tip of Illinois, account for approximately 76% (24%, 36%, and 16%, 

respectively) of the soybean planted in the U.S. (T. Schlueter, personal communication, 

August 2008). Because day length delays maturity, a soybean cultivar suited to a 

southern maturity group would mature too late if planted too far north. Conversely, a 

northern cultivar would mature too early if planted in the south (Heatherly and Elmore 

2004). 

Maturity groups are often designated by Arabic rather than Roman numerals, so the 

sequence is 00 to 8, and there are subdivisions within the major maturity groups. These 

are designated by a decimal value. For example, a variety with maturity group 

designation 2.9 would be at the southern end of Group II. 

Variety Development. Crop domestication and improvement through breeding has been 

largely achieved through selection of genes that regulate the expression of desirable 

traits, such as those associated with higher yields or disease resistance. Once plants with 

the desired traits have been selected, a population of those plants with similar 

characteristics are classified as varieties. Historically breeders have developed desirable 

varieties by retaining for further breeding those plants that possess the desirable traits, as 

determined by visual inspection or by testing. In recent years breeders have used the 

more direct methods of molecular breeding techniques, such as marker assisted breeding, 

to accelerate the process of identifying breeding lines containing a desired set of positive 

traits. These techniques rely on inventories of genomic regions or genetic markers that 

have been positively associated with the desirable traits. Once the genetic markers 

associated with the desired traits have been identified, molecular breeders can quickly 

select the offspring inheriting the genes for further development and testing in the field 

(Voosen, 2009). 

Hundreds of soybean varieties are tested each year in performance trials (variety trials) 

conducted by universities and private companies in all the major soybean growing states. 

The following information can typically be obtained from the results of variety trials: 

maturity group, disease resistance, yield, maturity date, percent lodging (plants fallen 

over on the ground), height, and herbicide resistance (Tylka et al., 2010). In different 

parts of the country and/or in other trials, additional characteristics may be identified, 

such as iron deficiency tolerance or protein or oil content (Pederson, 2008a). 

 

15 They were originally designated north to south as Groups I through VIII. Groups 0 and 00 were later 

added to the north. 
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M.2.1.3. Yield Increases in U.S. Soybean Production 

As a result of new varieties developed through selective breeding, improved fertilizer and 

pesticide applications, and improved management practices, soybean yields have steadily 

increased over the past century (Ash et al., 2006). Figure M-1 shows the average annual 

soybean yield for the U.S. from the mid-1920s through 2010. As shown in the figure, 

from the mid-1920s to 2010, the average annual yield in the U.S. increased from 

approximately 11 to 44 bushels per acre (bu/acre), or a rate of 0.35 bu/acre, equivalent to 

a yield increase of approximately 400%. 

There have been many factors that have led to this astounding increase in soybean 

productivity. Specific major management decisions that positively impact yield for 

growers in recent years include variety selection, management of soybean cyst nematode, 

early planting, narrow rows, elimination of weed competition, keeping insect pressure 

below economic thresholds, and crop rotation (Pederson, 2010). Some university 

agronomists believe there is still substantial yield potential that can be realized through 

implementation of good management decisions (Pederson, 2011; Pederson, 2010), 

although others believe a yield plateau has been reached (Moore, 2009). Record 

maximum yields for soybean are over 160 bu/acre for irrigated soybean and nearly 100 

bu/acre for non-irrigated soybean (Alsager 2010). USDA projections through 2020 show 

an average annual rate of increased yields of 0.33 bu/acre for the period 2010 to 2020, 

which results in an average U.S. yield of 47.6 bu/acre in 2020/21 (USDA, 2011). While 

USDA projects increasing yields, the projected rate of increase is lower than the past rate. 

Current and future factors that negatively affect yield increases are the expansion of 

soybean production into northern and western parts of the country, where yields are 

typically lower than in the core Midwestern production acre, and a shift in some areas 

away from narrow rows to improve air circulation, which helps combat disease (USDA, 

2010). 

 
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Figure M-1. Annual U.S. soybean yield 1924 – 2010. 

Soybean yield rose at an annual average rate of 0.35 bu/A between 1924 – 2010. Linear 

regression analysis was conducted on data from the USDA National Agricultural 

Statistics Service (USDA-NASS, 2011, http://www.nass.usda.gov). 

M.2.1.4. Regional and Temporal Yield Variations 

As shown in Figure M-1, soybean yields can be highly variable from year to year. For 

example, between 1987 and 1988 (a drought year in the Midwest), average U.S. yields 

dropped over 20%, from 33.9 to 27.0 bu/acre; between 1993 and 1994, average yields 

increased 27%, from 32.6 to 41.4 bu/acre. Yields also vary geographically. For example, 

in 2010, the average yield in Nebraska (55 bu/acre) was 139% higher than the average 

yield in South Carolina (23 bu/acre). Within a state there is considerable local variability, 

as, for example, in eastern Nebraska, where the 2010 yields for Lancaster and Hamilton 

counties were 43.6 bu/acre and 64.2 bu/acre, respectively (USDA NASS, 2011). For 

another example, in two adjacent counties in Iowa, Hardin and Wright, in 2010 the 

average yield in Hardin County (55.3 bu/ac) was 33% greater than the 2010 yield in 

Wright County (41.7 bu/acre). In 2009, on the other hand, the average yield in Wright 

County (52 bu/acre) was 13% greater than the average yield in Hardin County (46 

bu/acre). Within each county between 2009 and 2010, the average yield in Hardin 

increased by 20% and the average yield in Wright decreased by 20% (USDA NASS, 

2011). Since these values represent averages over counties, variation from farm to farm 

and within a farm from year to year would be expected to be even greater. The current 

world record for yield is 160.6 bushels per acre in 2010, on a farm in Missouri, where the 

statewide average yield in 2010 was 42 bushels per acre (Alsager 2010, 2010). Thus, 
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even with the positive management decisions that were previously discussed, observed 

yield is highly variable, is dependent on many factors and likely rarely reaches the 

theoretical maximum yield potential. 

Growers are accustomed to this regional and temporal variability in yield, influenced by 

variable environmental conditions and the genetics of the varieties they select for 

planting. However, growers are also accustomed to the steady improvements of average 

soybean yield observed over past decades and generally continue to pursue varieties that 

further increase productivity and profitability on their farm. Within this context, U.S. 

farmers will understand the value of the trait MON 87712 that offers the opportunity to 

increase the yield of their soybean production. 

M.2.1.5. Crop Storage and Transportation 

Once soybean are harvested from a field, they may be stored directly on farm, stored off 

the farm at another location, delivered to a grain elevator or directly to a soybean crusher. 

Grain elevators play an important role 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. 

A larger than normal crop can stress the storage and transportation system for the crop. 

Because of the very high variability in crop production, storage facilities are not always 

adequate. Soybeans and other grain must sometimes be stored in temporary structures or 

in other existing buildings if storage facilities are overloaded, and this may result in 

additional costs for constructing or renting temporary facilities and/or potential losses 

from exposure (Hellevang, 1998; Dorn, 2011). The same conditions can result when 

prices are low and growers want to hold on to their crops in the hopes of selling at higher 

prices (Maier and Wilcke, 1998). Soybeans can also compete with corn for available 

storage space; and while corn can be stored on the ground, soybeans rarely are 

(Hurburgh, 2005). University extension services provide practical guidelines for 

temporary storage of soybeans and other crops (Harner et al., 1998; Hellevang, 1998; 

Dorn, 2011; Maier and Wilke, 1998; Hurburgh, 2005). 

Growers usually deliver their soybeans to the sale point using their own trucks. From the 

elevator or processing facility, the soybeans or oil and meal are shipped by rail, barge or 

truck. Approximately 24% of soybeans are transported by rail, although higher 

percentages of meal and soybean oil are transported by rail (STC, 2010). More than half 

of U.S. soybean exports are first shipped by barge on the Mississippi River (Ash et al., 

2006). USDA reported in 2006 that recent record-large soybean harvests have tested the 

capacity of the U.S. bulk transportation system; however, no specifics were provided 

(Ash et al., 2006). Large crops can result in greater shipping competition and higher 

shipping costs, which translates to lower prices offered to growers (Ash et al., 2006). 

 
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M.2.1.6. Economics 

In the short term, individual growers’ decisions about whether to plant and which crop to 

plant are typically based on the relationship between operating costs and expected prices; 

i.e., on expected crop profitability (Ash et al., 2006). Managing input costs and 

managing the crop for yield are major components to the economics of producing a 

soybean crop. Growers’ costs include both overhead costs and operating costs. 

Overhead costs are those that are not associated with a particular crop and/or that are 

present whether or not a crop is grown, such as the cost of land and the depreciation of 

equipment. Operating costs are those associated with growing a particular crop in a 

given year, such as seed and fertilizer. A producer’s cost of growing a particular crop 

includes a proportional part of the overhead of his or her entire farming operation, plus all 

the operating costs associated with that crop. 

Figure M-2 shows the average per acre net value of soybean production in the U.S. from 

1975 to 2010, based on data compiled by the USDA Economic Research Service (ERS). 

The net value is the value of the soybeans produced less all costs of production, both the 

allocated overhead and the operating costs. For comparison, corn and wheat are also 

shown. USDA’s data does not include crop subsidies. Overhead costs represent well 

over half the total costs (up to 69%), with the “opportunity cost of land (rental rate)” and 

the capital recovery cost of machinery and equipment representing the bulk of the 

overhead costs. The largest single operating cost is seed (USDA ERS, 2011). As the 

data show, farming is often not profitable when all costs, including land value costs, are 

included. For example, USDA reports that in 2004, 70 percent of soybean-producing 

farm operations were considered profitable, not considering government payments. The 

percent profitable rises to 76 when government payments are included (Ash et al, 2006). 

Figure M-2. U.S. Net Value of Soybean Production, Dollars Per Acre 

Inflation Adjusted (to 2011) Values from 1975-2009, and does not include Government Subsidies 

Sources: USDA ERS 2011, U.S. Bureau of Labor Statistics 2011 

‐$200

‐$150

‐$100

‐$50

$0

$50

$100

$150

$200

1975 1980 1985 1990 1995 2000 2005

Net Value (dollar per acre)

Year

Soybean

Corn

Wheat
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While an individual grower typically makes planting decisions based on the relationship 

between operating costs and expected prices (Ash et al., 2006), many factors influence 

both operating costs and expected prices. Government price supports can have a large 

effect on costs, and supply and demand governs prices. These are discussed in more 

detail in the next section. 

M.2.1.7. Land Use 

Land Used for Growing Soybeans. U.S. soybean planted acreage has increased since the 

1930s, with the 10 million acre mark exceeded in 1940, 20 million acres exceeded in 

1956, 30 million in 1964, 40 million in 1967, and 50 million in 1973. Planted soybean 

acreage has not dropped below 60 million since 1993 (USDA NASS, 2011). Soybean 

acreage rose rapidly from the end of World War II to the late 1970s, based on increased 

demand for vegetable oil and higher meat consumption (Ash et al., 2006). U.S. soybeans 

acres stagnated in the 1980s largely due to farm programs for other corps (Ash et al., 

2006). In the 1990s, changes in farm programs, overseas demand, lower production costs 

associated with herbicide-tolerant crops, increased yields and increased rotations with 

corn resulted in increased acreage planted to soybeans (Ash et al., 2006). 

USDA projections to 2020 show a 2.7% increase in acres planted to soybeans from 

2009/10 (77.5 million acres) to 2020/21 (78.5 million acres), with a corresponding 

increase in production of approximately 11%, based on expected yield increases (USDA, 

2011). The expected increase in production is substantially less than occurred in the 

previous 11 years (1999 to 2000), when U.S. soybean production increased by 25% 

(USDA NASS, 2011). Major factors affecting future U.S. soybean production include: 

competition with other crops, especially corn (reduces demand/production); consumer 

preference for other oils (reduces demand/production); soybean use for biodiesel 

(increases demand/production), government farm programs, both for soybeans and for 

other competitor crops (may result in increases or reductions); increases in overseas 

demand, especially in China (increases production); and competition from major overseas 

growers, especially in Brazil and Argentina (decreases U.S. demand/production) (USDA 

ERS, 2010). 

During the same time period that soybean acreage has increased, wheat, barley and 

sorghum acreages have all greatly decreased. USDA attributes a shift to soybeans from 

wheat, the largest of these three crops, to stagnant yields in spring wheat and the 

development of better-yielding short season soybeans adapted to more northerly climates 

(USDA ERS, 2010). As shown in Figure M-2, wheat, the largest of these three crops, has 

been consistently less profitable than soybeans for many years. 

Total U.S. Cropland. While the specific crops vary, and total acreage changes from year 

to year, total harvested cropland in the U.S. in recent times is about the same as it was 

100 years ago (USDA ERS, 2007). In 2006, the most recent year for which data are 

available, the total agricultural land used for crop in the U.S. was 330 million acres - the 

same as it was the first year USDA began tracking, in 1910. Peaks occurred in the early 

1930s, in 1949, and in the early 1980s, when the area of cropland used for crops reached 

over 380 million acres (USDA ERS, 2007). 
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The combined acreage planted to the two largest U.S. crops, corn and soybeans, was at an 

all-time high of 168.8 million acres in 2011 (USDA NASS, 2011). This was achieved 

through maximization of existing cropland, reduction of acreage sown to other grain 

crops and hay, and an increase in the rate of double-cropping (raising two crops in one 

year on the same land) (Ash, 2011). 

 M.2.2. Speciality Soybean Production 

M.2.1. Certified Seed Production 

Certified seed production is discussed in Section VIII.B.2 of the petition and summarized 

here. Standardized seed production practices are responsible for maintaining high-quality 

seed stocks, an essential basis for U.S. agriculture. The value of seed quality (including 

genetic purity, vigor, and minimizing presence of weed seed, seed-borne diseases, and 

inert materials, such as dirt) has been identified as a major factor in determining crop 

yields (Oplinger 1986, Pederson 2008a, USDA, 2000). 

Soybean seed has four classes: 1) breeder, 2) foundation, 3) registered, and 4) certified 

(Association of Official Seed Certifying Agencies [AOSCA], 2009). Breeder seed is 

seed directly controlled by the originating or sponsoring plant breeding organization or 

firm. Foundation seed is first-generation seed increased from breeder seed and is handled 

to maintain purity and identity of a specific variety. 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 typically 

two generations from foundation seed. Not all soybean seed sold is officially certified; 

however, commercial soybean seed sold and planted for commodity soybean production 

typically meets or exceeds certified seed standards. 

Seed certification programs were initiated in the early 1900s in the U.S. to preserve the 

genetic identity and variety purity of seed. The federal government passed the U.S. 

Federal Seed Act of 1939 to recognize seed certification and the establishment of official 

certifying agencies. Regulations first adopted in 1969 under the Federal Seed Act 

recognize land history, field isolation, and varietal purity standards for foundation, 

registered, and certified seed. Seed certification services are available through various 

state agencies affiliated with the AOSCA. 

Soybean seed is produced throughout most of the U.S. soybean-growing regions by 

companies that produce and sell seed, and by toll seed producers, or tollers, which are 

companies that produce but do not sell certified seed. Seed companies and tollers in turn 

contract acreage with growers to produce the desired amount of soybean seed for 

particular varieties. Production or processing plants at these seed companies clean, 

condition, and bag the harvested soybean seed; verify the seeds meet all state and federal 

seed standards and labelling requirements; as well as monitor and inspect all the 

processes at the plant. Verification that standards are met includes carrying out certain 

tests such as germination tests. 
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The entire seed production process at the majority of the seed companies and tollers 

operates under standards established by the International Organization for 

Standardization (ISO), and includes internal and external audits (ISO, 2009). Field 

inspections are conducted on seed production fields throughout the soybean growing 

season to evaluate variety purity and ensure soybean plants are developing properly. 

Management practices in the field are designed with the intent of keeping the fields free 

of weeds, insects, and diseases. The seed production fields are also mapped to ensure the 

seed field has the minimum isolation requirement to prevent mechanical mixing of other 

soybean varieties (AOSCA, 2009). The American Seed Trade Association (ASTA), to 

which most major seed producers belong, also provides best management practices for 

seed production (ASTA, 2010). 

The field operations and management practices for producing soybean seed are similar to 

commodity soybean production. However, special attention is needed in certain 

production practices to produce seed with low levels of weed seed and other foreign 

material and with high germination rates, and high genetic purity (Helsel and Minor, 

1993). 

M.2.2.2. Organic Soybean Production 

U.S. Organic Soybean Production. Organic soybean acreage by state is reported by 

USDA AMS from 1997 through 2008 (USDA ERS, 2010). Acreage ranged from 82,143 

in 1997 to a high of 174,467 in 2001. In the most recent year available, 2008, USDA 

ERS reported 125,621 acres of organic soybeans (USDA ERS, 2010); however, the 

Census of Agriculture reported 98,199 harvested acres of organic soybeans in 2008, 

yielding 2.58 million bushels, for an average yield of 26 bushels per acre and an average 

value of $19.45 per bushel (USDA NASS, 2010). 

The decline in U.S. organic soybean production from the high in 2001 is likely a result of 

competition from lower-cost organic soybeans produced primarily in China. The 

Japanese market for U.S. organic soybeans was largely lost to China; and Chinese 

imports became more readily available in the U.S. after USDA streamlined the process 

for other countries to export certified organic products to the U.S. (Organic and NonGMO Report, 2007). 

In 2008, organic soybeans were produced in 28 states, with the largest acreage in 

Minnesota and Iowa. In 2010, Minnesota and Iowa were the first and fourth largest 

producers of soybean. The majority of organic soybeans are used for tofu and other soy 

products in the U.S. Some organic soybean are still exported to Japan and other 

countries, while other markets for organic soybeans include soybean oil and meal for 

livestock (ISU, 2003). 

In a comparison of organic and non-organic soybean production in 2006, McBride and 

Greene found that the total economic costs of producing organic soybeans were $6.20 per 

bushel higher than non-organic, that organic yields were much lower than non-organic, 

and that the average price premium received for organic soybeans was $9.16 per bushel, 

which more than offset the added production costs (McBride and Greene, 2009). Other 
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researchers have found that organic soybean price premiums in 2009 ranged from 65 to 

139% and averaged 107% (Clark and Alexander, 2010). 

Organic producers must use organic seed if it is available. However, there are exceptions 

to this rule when comparable organic seed is not available and that exception can apply to 

a whole range of characteristics including disease resistance profile, quality aspects and 

fit to a local growing region (USDA AMS, 2011). Several companies produce 

organically certified soybean seed as well as conventional non-treated soybean seed that 

can be used by organic growers (Coulter et al., 2010). Many crop management practices 

are similar to those used by non-organic soybean growers, except that organic growers 

may not use synthetic fertilizers or pesticides. Because synthetic herbicides are not 

allowed, organic growers generally need to use more tillage to control weeds (Coulter et 

al., 2010; Kuepper, 2003). 

The National Organic Program. Organic farming operations as described by the 

National Organic Program (NOP), which is administered by USDA’s Agricultural 

Marketing Service (AMS), requires organic production operations to have distinct, 

defined boundaries and buffer zones to prevent unintended contact with prohibited 

substances (such as pesticides and synthetic fertilizers) or products of excluded methods 

from adjoining land that is not under an organic production management plan.16 

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 87712 is an excluded method under the National Organic Program.17 

On April 15, 2011, USDA AMS released a Policy Memorandum regarding “Clarification 

of Existing Regulations Regarding the Use of Genetically Modified Organisms in 

Organic Production and Handling” (USDA AMS, 2011). This memo was intended to 

answer questions that have been raised concerning biotechnology-derived crops and 

organic production and handling. The memorandum reiterates that organic operations 

must follow a set of production standards and practices which meet the requirements of 

the Organic Foods Production Act of 1990 and National Organic Program (NOP) 

regulations. NOP regulations prohibit the use of excluded methods (i.e., “GMOs”) in 

organic operations. Therefore, the use of biotechnology-derived crops remains an 

excluded method but not a prohibited substance. 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 avoidance of prohibited 

substances and the use of excluded methods.18 

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 

 

16 7 CFR 205.202(c) 

17 7 CFR § 205.2. 

18 7 CFR Part 205. 
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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 (USDA AMS, 2011). 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 certification indicates that organic production and handling 

processes have been followed, not that the product itself is “free” from any particular 

substance. As USDA AMS has recently re-iterated in a policy memorandum, organic 

certification is processed based. The NOP regulations do not allow the use of excluded 

methods such as biotechnology; however, the inadvertent presence of products of 

biotechnology “does not constitute a use because there was no intent on the part of the 

certified operator to use excluded methods” (USDA AMS, 2011). 

Organic soybean producers use production practices designed to prevent commingling of 

their crop with neighboring crops treated with herbicides and other pesticides (spray 

drift), or that may be using plant varieties produced using excluded methods (pollen 

movement from biotechnology-derived crops). These well established practices include 

isolation zones, use of buffer rows surrounding the organic crop, adjusted planting dates, 

and varietal selection (Kuepper, 2006). 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. Soybean is a 

highly self-pollinated species and exhibits a very low level of outcrossing (see Section 

IX.D). Outcrossing is the genetic transmission of a defined heritable characteristic from 

one group of individuals (population, crop variety) to another. Outcrossing most 

commonly results from cross-pollination. Since soybean is highly self-pollinating, 

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 reasonably avoid 

biotechnology-derived soybean and maintain organic or conventional production status. 

 M.2.3. Agronomic Practices for Soybeans 

M.2.3.1. Production Management Considerations 

Production management considerations are described in detail in Section VIII.D of the 

petition and are summarized here. Refer to Section VIII.D for more detail. 

Pre-Season. Well in advance of planting a soybean crop, decisions are made regarding 

the planned crop rotation, the tillage system and row spacing that will be implemented, 

the planting equipment that will be used, the seed or variety that will be planted, and soil 

fertility management requirements. Soybean rotation practices are summarized below. 

Variety selection has been discussed previously. Soybean requires 16 essential elements 

for growth and development. Deficiencies in any of these elements can reduce yields 
Monsanto Company 11-SY-217U 432 of 467 

(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. Nitrogen fertilizer applications at planting generally 

do not improve yield and decrease nodulation while increasing the plant’s dependency on 

the soil for nitrogen (Pedersen, 2008d). Therefore, nitrogen fertilizer is seldom applied 

prior to planting a soybean crop. Soil tests are used to determine the pH, phosphorus, and 

potassium levels in the soil and assess whether any adjustments need to be made. 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. 

Planting and Early Season. An understanding of the growth stages of soybean is 

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, 2004). 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. 

The time of onset and the duration of the various growth stages in soybean are highly 

dependent on photoperiod (hours of daylight and darkness) and temperature (Major et al., 

1975), and therefore, for the same soybean plant grown at different latitudes, the onset 

and duration of the growth stages and the total time from planting to maturity would be 

different. 

Good seedbed preparation, soil temperature, soil moisture and planting date impact yield. 

With planting date having the greatest impact. Highest yields are generally achieved 

when planting in early to mid-May. Row spacing influences yield because it impacts 

canopy development and research conducted in the Midwest shows that row spacing of 

less than 20 inches is optimum. 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). 

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. 

Mid to Late Season. Weather, day length and management of diseases and insects are the 

greatest factors impacting yield during the mid to late season stages. Ideal daytime 

temperatures for soybean growth are between 75oF and 85oF (Hoeft et al., 2000). 

Soybean is photoperiod sensitive, which means that it transitions from vegetative to 
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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. 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 requirement during the seedfilling stages is a high rate of photosynthesis to maximize yield. 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 soybean 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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M.2.3.2. Crop Rotation 

The use of crop rotation in soybean production is discussed in Section VIII.G of the 

petition. The well-established farming practice of crop rotation is a key management tool 

for soybean growers. The purposes of growing soybean in rotation include, from 

Sandretto and Payne (2006) unless otherwise noted: 

 improving yield and profitability of one or both crops over time; 

 decreasing the need for nitrogen fertilizer on the crop following soybean; 

 mitigating or breaking disease, insect, and weed cycles; 

 improving soil tilth and soil physical properties; 

 increasing residue cover; 

 reducing soil erosion; 

 increasing soil organic matter; and 

 reducing runoff of nutrients, herbicides, and insecticides (Heatherly and Elmore, 

2004; Al Kaisi et al., 2003). 

According to the USDA Economic Research Service, 95% of the soybean-planted 

acreage has been in some form of a crop rotation system since 1991, and 5% of soybeanplanted acreage is grown in continuous soybean (USDA ERS, 2005). Corn- and wheatplanted 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 of various 

cropping systems. Crop rotations also afford growers the opportunity to diversify farm 

production in order to minimize market risks. 

Agronomic practices such as rotation patterns for soybean vary from state to state. 

However, there are similarities among states within certain growing regions. The 

majority of the U.S. soybean acreage (68.6%) is rotated to corn with approximately 

14.5% of the subsequent corn acreage rotated back to soybean in the third year of the 

rotation (soybean-corn-soybean). Wheat follows soybean on approximately 11.2% of the 

U.S. soybean acreage. 

Continuous soybean production is uncommon in the Midwest. Soybean extension 

specialists encourage growers to avoid the practice as a way to reduce the risk of damage 

from 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. 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). Long-term studies in the Midwest indicate 
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that the corn-soybean rotation improves yield potential of no-till systems compared to 

continuous corn production (Al-Kaisi, 2001). 

 M.2.3.3. Irrigation 

The use of irrigation in soybean production is discussed in Section VIII.B of the petition. 

The productivity 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 Midwestern, Eastern and portions of the Great Plains regions 

of the U.S. provide sufficient water 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 

deeper in medium-textured soils than in clay soils. Irrigation is used on approximately 

9% of the soybean acreage in the U.S. to supplement the water supply during dry periods 

in the Western and Southern soybean growing regions (USDA ERS, 2008). 

M.2.3.4. Management of Insects 

The management of insects in soybean production is discussed in detail in Section VIII.D 

and summarized here. 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). Insect 

injury can impact yield, plant maturity, or seed quality. Insect injury in soybean seldom 

reaches levels to cause an economic loss in the primary soybean production areas, 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 pests 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 leaf-feeding insects comprise the biggest group of soybean insect pests, but 

not necessarily the most economically damaging insects. Recent research on defoliation 

has determined that a major effect of leaf 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. Insect damage occurring in the full 

pod stage (R4) or seed-filling stage (R5) would have the most impact on yield 

(McWilliams et al 2004). 
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 M.2.3.5. Management of Diseases and Other Pests 

The management of diseases and other pests in soybean production is discussed in detail 

in Section VIII.E. More than 100 pathogens are known to affect soybean, of which 35 

are considered to be of economic importance (Heatherly and Hodges, 1999). 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 severity of the disease on individual plants, and the number of 

plants affected (Heatherly and Hodges, 1999). 

According to field surveys conducted in soybean-producing states during 1996 to 2009, 

soybean cyst nematode (Heterodera gylcines) caused by far the greatest soybean yield 

losses (Figure M-3) (Wrather et al., 2000). Phytophthora root and stem rot 

(Phytophthora sojae), seedling diseases, charcoal rot (Macrophomina phaseolina), 

sudden death syndrome (Fusarium virgulifome), and sclerotinia stem rot (Sclerotinia 

sclerotioru) followed in economical importance. Brown stem rot (Phialophora gregata), 

viruses, anthracnose (Colletotrichum truncatum), and pod and stem blight (Diaporthe 

phaseolorum) caused the remainder of the top ten soybean yield losses during 1996 to 

2009. As expected, yield losses due to diseases and nematodes varied by region. 

 
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Figure M-3. Soybean Yield Losses in the U.S. Averaged Over the Period 1996 to 

2009 Attributable to Pests and Diseases 

Source: Wrather and Koenning, 2010 

Selection of varieties that are resistant to disease is the primary tool growers have for 

disease control in soybean (Heatherly and Hodges, 1999). Cultural practices can also 

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 fungalresting structures. Crop rotation is often recommended as a disease-management 

strategy. Rotating crops interrupts the disease cycle and allows time for the 

decomposition of inoculums. One exception is Rhizoctonia, a soil-inhabitant pathogen 

that grows on a wide variety of crops and can survive sufficiently in the soil to make crop 

rotation an impractical means of controlling this pest. Row spacing, plant population, 

and planting date can also be changed to manage soybean diseases. 

Soybean cyst nematode (SCN), Heterodera glycines, is one of the most damaging 

pathogens of soybean throughout the soybean growing regions of the U.S. with losses 

estimated to be about $1.5 billion (Pedersen, 2008b). 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. However, planting resistant varieties is regarded as the 

best and most effective management practice to prevent losses from this pest. 

100000

600000

1100000

1600000

2100000

Soybean Yield Loss (Thousand Bushel)
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High-quality seed is essential for controlling seedling diseases. The most important 

seedling diseases in soybean are Phytophthora, Pythium, Rhizoctonia, and Fusarium

(Pedersen, 2008c). Many soybean varieties demonstrate resistance to specific taxonomic 

races of Phytophthora. Treating soybean seed with a fungicide (e.g., metalaxyl or 

mefenoxam) is effective against damping-off disease (seedling blight) caused 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 (LSU, 2010). At this time, foliar application of fungicides is the 

standard disease-management practice to limit yield losses due to soybean rust. 

M.2.3.6. Weed Management 

The management of weeds in soybean production is discussed in Section VIII.F. Weed 

control in soybean is essential to optimizing yields because weeds compete with soybean 

for light, nutrients, and soil moisture. Weeds can also harbor insects and diseases, and 

also can interfere with harvest, causing extra wear on harvest equipment (Pedersen, 

2008). Approximately 98% of the soybean acreage received an herbicide application in 

2006, indicating the importance of excellent weed control in maximizing soybean yield 

(USDA NASS, 2007). 

Herbicide-tolerant soybean was introduced to provide growers with additional options to 

improve crop safety and/or improve weed control. The Roundup Ready soybean system 

(planting Roundup Ready soybean and applying glyphosate in crop to provide primary 

weed control) was introduced in 1996 and has become the standard weed control program 

in U.S. soybean production and was utilized on 91% of U.S. soybean acreage in 2008 

(USDA-NASS, 2009). 

The primary factors that affect a potential yield loss in soybean 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). 

Cultural and mechanical weed control practices can be important components of an 

effective weed management program (Loux et al., 2010). Cultural practices such as 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. 

Mechanical methods of weed control including tillage have been used for centuries to 

control weeds in crop production. Spring or fall preplant tillage and in-crop shallow 

cultivation can effectively reduce the competitive ability of weeds by burying the plants, 

disturbing or weakening their root systems, or causing sufficient physical injury to kill 

the plants. A consequence of in-crop cultivation for weed control is that it can injure 

crop roots and cause moisture loss. 
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M.2.4. Human Health and Worker Safety 

Humans safely consume soybean and have done so for thousands of years. The current 

primary food use is soybean oil (OECD, 2001). Nutrients in soybeans and soy products 

include protein, fats, carbohydrates and fiber (OECD, 2001). The essential amino acids 

required by humans are contained in soybeans (OECD, 2001). Anti-nutrients include 

trypsin inhibitors (interfere with digestion), lectins (inhibit growth), isoflavones (also 

referred to as phytoestrogens that may affect reproduction), stachyose and raffinose 

(produce gas), and phytic acid (binds mineral nutrients, making them unavailable) 

(OECD, 2001). Soybeans also contain allergenic proteins that may cause reactions in 

hypersensitive individuals (OECD, 2001). Toasting or heating the soybeans during 

processing reduces the content of trypsin inhibitors and lectins (OECD, 2001). All 

biotechnology-derived soybean with new traits produced by biotechnology have been 

reviewed by the FDA through FDA’s consultation process. Biotechnology-derived 

soybean on the market pose no unique risks relative to other soybean developed using 

traditional breeding methods. Most soybeans currently grown are herbicide tolerant and 

have been shown to be as safe and nutritious as their conventional counterparts. 

Agriculture ranks among the most hazardous industries in the nation (USBLS, 2011) 

Fatal injuries constitute a significant burden on the agricultural sector, as indicated by the 

456 farmers and farm workers who died from a work-related injury for a fatality rate of 

25.1 deaths per 100,000 workers during that same year, compared to the 5,214 fatal 

injuries (for a fatality rate of 3.7 deaths per 100,000 workers) that happened across all 

industries in 2008 (USBLS, 2011 and USDOL-OSHA, 2011). No fatalities were reported 

in 2008 directly associated with soybean farming, although four fatalities were reported 

in 2007 associated with soybean farming (USBLS, 2011). According to OSHA in 1986, 

farm tractors (rollovers and run-overs) represented 51% of all farm related fatalities, 

followed by fatalities associated with buildings and structures (including grain 

suffocation and silo-gas), and farm trucks (non-highway accidents), with 11% and 6%, 

respectively. Other farm related hazards related to crop workers (including soybean) 

include injuries related to ergonomics, noise, respiratory (including dust hazards), and 

chemicals (including pesticides and fertilizers), These hazards can be minimized, among 

other things, through the use of hazard recognition, hazard control and the use of 

personnel protective equipment (USDOL-OSHA, 2011). Genetically engineered soybean 

with new traits produced by biotechnology pose no unique worker safety issues relative 

to other soybean developed using traditional breeding methods. 
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M.2.5. Animal Health 

Animals have consumed soybeans as forage and meal for many years. Soybean meal is 

currently the primary animal feed from soybeans and is fed to animals primarily as a 

protein source. Soybean meal contains relatively high levels of essential amino acids that 

are deficient in other common feed (OECD, 2001). Trypsin inhibitors, lectins and phytic 

acid are the primary anti-nutrients in soybeans that should be minimized in animal diets. 

Toasting or heating the soybeans during processing reduces the content of trypsin 

inhibitors and lectins (OECD, 2001). All biotechnology-derived soybean with new traits 

produced by biotechnology have been reviewed by the FDA through FDA’s consultation 

process. Biotechnology-derived soybean with new traits produced by biotechnology pose 

no unique risks relative to other soybean developed using traditional breeding methods. 

Soybeans currently grown are largely herbicide tolerant and have been shown to be as 

safe and nutritious as their conventional counterparts. 

M.2.6. Animal and Plant Communities 

M.2.6.1. Animal Communities 

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. The USDA reports that soybean has 

minor to moderate value as a food source for large animals; low/minor value as a food 

source for small mammals, water birds and terrestrial birds; and moderate value as cover 

for terrestrial birds (USDA NRCS, 2011). 

M.2.6.2. Plant Communities 

The affected environment for growing soybean plants can generally be considered the 

agroecosystem (managed agricultural fields) plus some area extending beyond the 

intended plantings that might be affected by agricultural operations. Plants, extraneous to 

the crop, which grow in planted fields can be considered weeds and are dealt with in a 

separate section in this document. Plants not growing in a field amongst the soybeans 

would be considered in this section. These plants could be in ditches, hedge rows, fence 

rows, wind breaks, yards, etc. These plants could be annuals, biennials or perennials. 

Regardless of the agricultural operation, these plants may be impacted, both positively 

and negatively, by agricultural operations. Fertilizers and/or water may run off into 

adjacent lands, resulting in increased plant growth outside the agroecosystem. Negative 

impacts on plants adjacent to production fields can occur from herbicide drift, however, 

measures can be implemented to minimize drift. 

 
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M.2.7. Physical Environment 

M.2.7.1. Surface Water and Groundwater Quality 

Surface water may be impacted from soybean production by runoff from soybean fields 

that carries soil particles and herbicides or other pesticides to streams, rivers, lakes, 

wetlands and other water bodies. As discussed below, based on existing data, the soil 

component of runoff is a much more important contributor to surface water impacts than 

is the pesticide component. 

Tillage causes widespread soil disturbance. Thus, erosion, topsoil loss and the resulting 

sedimentation and turbidity in streams are likely to increase with increased tillage. In 

2009, based on the states’ water quality reports, EPA identified sedimentation and 

turbidity as two of the top 10 causes of impairment to surface water in the U.S. in 

general; in 2007, EPA identified sedimentation/siltation as a leading cause of impairment 

to rivers and streams in particular (U.S. EPA, 2009; U.S. EPA, 2007). Although a 

comprehensive data set has not yet been developed to confirm its assertion. EPA has 

projected conservation tillage to be “the major soil protection method and candidate best 

management practice for improving surface water quality” (U.S. EPA, 2002). EPA 

identifies conservation tillage as the first of its CORE4 agricultural management practices 

for water quality protection (U.S. EPA, 2008a). 

Based on the states’ water quality reports to EPA, which EPA makes available through its 

National Assessment Database, pesticides in general and herbicides in particular are a 

relatively minor contributor to impairment of surface water in the U.S., compared to 

sedimentation/siltation and turbidity (U.S. EPA, 2008b). Pesticides accounted for less 

than one percent of reported causes of surface water impairment in all but four of the 17 

leading U.S. soybean-producing states. In those four states, pesticides accounted for two 

to eight percent of reported causes of impairment. Of the pesticides that were reported as 

contributing to impairment among the 17 leading soybean-producing states, almost all are 

previously used, highly persistent chemicals that are no longer registered for use in the 

U.S. (U.S. EPA, 2008b). 

Groundwater impacts may be of potential concern in some areas where nitrogen levels 

are either approaching or have exceeded the maximum contaminant level (10 

milligrams/liter) (Klocke et al., 1999). In areas, such as Nebraska, where soybean and 

corn are grown in rotation and where ground water is a principle source of water for 

human consumption, this can be an important issue. However, as a legume, soybean 

generally does not require addition of nitrogen fertilizer, and therefore does not 

contribute appreciably to nitrogen levels in groundwater. 

M.2.7.2. Air 

Many agricultural activities, including those associated with soybean production, affect 

air quality including exhaust emissions from tillage, planting, and harvesting equipment; 

and nitrous oxide emissions from the use of nitrogen fertilizer. Approximately 80% of 

the global CO2 emissions attributable to human activity are derived from the combustion 
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of fossil fuels and cement production, and ~20% are derived from land use change and 

deforestation related to agriculture (Burney et al., 2010; IPCC, 2007). 

M.2.7.3. Soil and Soil Microorganisms 

Microbial populations and associated biochemical processes are critical to maintaining 

soil health and quality. Soil microbial communities are highly complex and are often 

characterized by high microbial diversity (Tiedje et al., 1999). The occurrence and 

abundance of soil microorganisms are affected by 1) soil characteristics like tilth, organic 

matter, nutrient content, and moisture capacity, 2) typical physico-chemical factors such 

as temperature, pH, and redox potential, and 3) soil management practices. Agricultural 

practices such as fertilization and cultivation may also have profound effects on soil 

microbial populations, species composition, colonization, and associated biochemical 

processes (Buckley and Schmidt, 2001; Buckley and Schmidt, 2003). Consequently, 

significant variation in microbial populations is expected in agricultural fields. 

Maintaining soil pH in the range of 6.0 to 7.0 will enhance the availability of inherent and 

fertilizer nutrients; reduce the availability of toxic elements, particularly aluminium and 

manganese; and enhance microbial activity (Hoeft, 2000). The increased microbial 

activity that is associated with the optimum pH level results in oxidation of organic 

matter and increased release of nutrients from the organic matter. The increased 

microbial activity applies also to the rhizobia bacteria that are responsible for symbiotic 

nitrogen fixation in soybeans and other legumes (CAST, 2009). Properly nodulated 

soybeans grown at the proper pH will fix about two-thirds of the nitrogen contained in 

the harvested crop (Hoeft, 2002). Results of most studies show that application of 

nitrogen fertilizer does not improve yields in soybeans (CAST, 2009 p. 25). Soybean 

removes approximately 0.85 pounds of phosphate and 1.2 pounds of potash (Potassium) 

for each bushel of seeds harvested (CAST, 2009). Micronutrient deficiencies are 

uncommon and maintaining proper pH prevents most micronutrient problems (Heatherly 

and Elmore, 2004). 

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, which provide an environment in which differentiated bacteria called 

bacteroids are capable of reducing or “fixing” atmospheric nitrogen. The product of 

nitrogen fixation, ammonia, can then be utilized by the plant. As a result of this 

relationship, nitrogen inputs are typically not necessary for agricultural production of 

soybeans. 

 M.2.7.4. Adjacent Agricultural Crops and Non-Agricultural Plants 

Soybean is widely grown throughout the U.S. on land devoted to agricultural use. The 

biology of soybean is discussed in Section II. Soybean (Glycine max (L.) Merr.) 

originated in east Asia and is not native to the Americans (USDA ARS, National Genetic 

Resources Program [GRIN]). Glycine max is not listed as an invasive or noxious weed 

by USDA (USDA NRCS, 2011). Soybean does not have any related wild relatives in the 
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U.S. with which it can hybridize. Soybean is not found outside of cultivation and is not 

invasive or weedy (OECD, 2000). 

Soybean is primarily grown adjacent to other large acre crops such as corn, wheat, and 

alfalfa. It may also be grown near vegetables, orchards, pastures, and adjacent to nonagricultural lands, such as forests, grasslands, streams, lakes, rivers and occasionally near 

urban lands. 

Soybean is self-pollinating and shows low rates of outcrossing. The potential for crosspollination in soybean 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, 2006). Numerous studies on soybean cross-pollination have been 

conducted, and the published results, with and without supplemental pollinators, are 

summarized in Table IX-1, Section IX. Under natural conditions, cross-pollination 

among adjacent plants in a row or among plants in adjacent rows ranged from 0 to 6.3%. 

 
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M.3. Alternatives 

The decision-making process of deregulation is governed by 7 CFR § 340.6 (d)(3)(i) 

which states that APHIS may approve the petition in whole or in part, resulting in three 

possible outcomes in response to Monsanto’s petition, described below. 

M.3.1. Alternatives Studied in Detail 

M.3.1.1. Deregulation in Whole Alternative 

Under the “deregulation in whole” alternative, MON 87712 would no longer be a 

regulated article under 7 CFR Part 340 and would be widely available for planting, 

movement and importation without prior authorization in a permit or notification. 

MON 87712 is expected to be gradually adopted by many of those growers who are 

already growing biotechnology-derived soybeans. The rate of adoption will depend on 

the expectation of yield increase, the price of the seed, and other factors. The rate of 

adoption is expected to be gradual partly because of the difficulty to a grower of 

assessing a yield increase within the inherent temporal and spatial variability in yield due 

to many other factors, as discussed in Appendix M.2.1.2. Unlike some other traits (e.g., 

herbicide tolerance), where the trait and benefits are immediately obvious, the yield 

increase may not always be readily apparent and in some cases may be masked by other 

yield-inhibiting factors such as disease, drought stress or insect pressure. Maximum trait 

adoption is expected to be approximately 50 to 60%, based on Monsanto’s estimate. This 

estimate assumes that other seed companies will offer soybean varieties that do not 

contain MON 87712 and these varieties will compete for market share with varieties that 

contain MON 87712. In addition, it is assumed that there will be demand for 

conventional, organic soybean as well as other specialty soybean and these soybean 

varieties will also compete for acreage with MON 87712. 

No Action Alternative 

Under the “no action” alternative, MON 87712 would remain a regulated article under 7 

CFR Part 340. MON 87712 could be grown under USDA notification or permit under 

confined release conditions. However, currently deregulated biotechnology-derived 

soybean would continue to be available and would be expected to continue to be widely 

grown. It is expected that higher yielding soybean varieties developed through 

conventional breeding would continue to be introduced and that growers will continue to 

adopt these new varieties for use on their farm. 

M.3.2. Alternatives Considered and Dismissed 

M.3.2.1. Deregulation in Part 

Approval in Part Based on Plant Pest Risk 

The ‘approval in part” alternative would be dependent upon a finding of the potential for 

a plant pest risk for MON 87712 in certain geographies or under certain conditions. 

APHIS may impose conditions upon the cultivation or use of MON 87712 in specific 
Monsanto Company 11-SY-217U 445 of 467 

geographies or conditions to mitigate potential plant pest risk. For example, APHIS 

could impose conditions that require assurance of the integrity and purity of the material 

containing MON 87712, or conditions requiring the implementation of stewardship 

practices in the use of MON 87712 that formed the basis of an APHIS decision that 

MON 87712 does not pose a plant pest risk. MON 87712 has been thoroughly 

characterized and extensive information presented in Sections I through IX of 

demonstrates that MON 87712 does not present a plant pest risk in any of the 

geographies or under any conditions where MON 87712 may be grown. Therefore, from 

a plant pest risk perspective, there is no basis for imposing geographic or other 

restrictions on MON 87712. Monsanto has requested a determination of non-regulated 

status without conditions. 

M.4. Environmental Consequences 

This section describes the environmental consequences of the no action and the 

deregulation in whole alternatives. 

M.4.1.1. Commercial Soybean Production and Use 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. Also 

under the no action alternative, soybean acres planted in the U.S. are expected to 

experience modest increases, based on USDA projections (Appendix M.2.1). General 

locations of production and uses of soybean would be expected to continue as they are 

now, potentially with continued westward and northward expansion and slight decreases 

in production in the south and east. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. As summarized in the petition, MON 87712 soybean is agronomically, 

phenotypically, and compositionally comparable to conventional soybean with the 

exception of its yield trait. Introduction of MON 87712 is not expected to result in an 

increase in percent of soybeans planted that are biotechnology-derived, as over 90% of 

soybean currently grown are biotechnology-derived. Due to a lack of phenotypic 

characteristics that would make it suitable to be grown in regions outside of where 

soybeans are currently grown, the MON 87712 trait is not expected to have an impact on 

where and how soybean is grown in the U.S. 

M.4.1.2. Soybean Varieties and Maturity Groups 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. Also 
Monsanto Company 11-SY-217U 446 of 467 

under the no action alternative, soybean variety development, selection and evaluation 

will continue to be used, and may be adjusted based on new data and practices. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual commercial introduction 

of MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

increased yield resulting from use of commercial soybean varieties that contain 

MON 87712. Data presented in the petition showed that MON 87712 displayed a delay 

in days to 50% senescence and days to physiological maturity (Table VII-4). Farmers 

select soybean varieties for planting on their farm based upon the correct maturity group 

for their region. Leaf senescence and physiological maturity are two of several 

characteristics that breeders use to help assign a variety to a correct maturity group 

(Heatherly and Elmore, 2004). A delay in physiological maturity of a variety may 

impact when the soybeans can be harvested. Soybean yield and quality are affected if a 

season ending freeze occurs before a variety reaches its physiological maturity. 

It is not expected that the delay in physiological maturity or leaf senescence observed will 

impact the maturity group designation of soybean varieties that are commercialized using 

MON 87712 for the following reasons. The majority of soybean varieties on the market 

today are produced from a breeding process termed forward breeding. In this process 

new soybean varieties are created by crossing two existing varieties. The varieties 

crossed are not necessarily from the same maturity group. The progeny of the crosses are 

tested in field trials to evaluate a number of parameters including yield, maturity, 

flowering time, leaf senescence, overall plant health, and seed characteristics like protein, 

and oil levels (Heatherly and Elmore, 2004). Varieties that yield better than the checks of 

the same maturity group are selected for commercialization. The entire process from 

initial cross to introduction of a new variety usually takes several years during which 

each new variety is repeatedly evaluated against the best commercially available varieties 

of the same maturity group. When MON 87712 is used in soybean breeding programs, 

there may be an impact on leaf senescence and physiological maturity in new soybean 

varieties containing the trait. However, the system that breeders use to evaluate new 

varieties and assign a variety to a correct maturity group will be used to place the new 

variety into the correct maturity group. Breeders consider many characteristics of the 

new variety to determine a maturity group designation for a new soybean variety. As 

with any new soybean variety, whether it is biotechnology-derived or conventional, the 

interplay of numerous characteristics ultimately contributes to maturity group 

assignments and breeders will continue to rely on these characteristics during the variety 

development process.

M.4.1.3. Yield 

No Action Alternative Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Under the no action alternative, continued yield increases would be expected in soybean, 

at least for the foreseeable future, before a true yield plateau is reached (Appendix 
Monsanto Company 11-SY-217U 447 of 467 

M.2.1.3.). As discussed in Appendix M.2.1., USDA projects average annual yield 

increases from 2010 to 2021 of 0.33 bu/acre (USDA, 2011). These yield increases may 

be expected to result from development of higher-yielding biotechnology-derived and 

conventionally bred varieties, and from continued improvements in agronomic and 

cultural practices. 

Deregulation in Whole Alternative. The primary difference between the no action and 

the deregulation in whole alternative attributable to the introduction of MON 87712 into 

a broad base of soybean germplasm would be the opportunity for increased yield in 

commercial soybean varieties that contain MON 87712. The actual yield gain 

attributable to MON 88712 will continue to be evaluated as the event is utilized and 

introgressed into a broader range of elite germplasm and new varieties are developed and 

released. 

Yield is a complex parameter impacted by many variables and varies from year to year 

and by location. Because adoption of the technology is expected to occur over a period 

of several years and maximum market adoption is expected to reach 50 to 60% over time 

(Appendix M.3.1.1) the use of commercial soybean varieties containing MON 87712 

would likely result in a gradual increase in the slope of the average soybean yield curve 

shown in Figure M-1. The change would still be within the range of scatter shown in 

Figure M-1. As discussed in Appendix M.2.1.2, changes of 20% or more between one 

year and the next in U.S. average annual per-acre soybean yield are not uncommon, and 

large local temporal and geographic variation is common. As discussed in Appendix 

M.2.1.2, because of the high variability in crop production, producers, processors and 

transporters are accustomed to dealing with harvested crops in excess of system capacity. 

M.4.1.4. Economics 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Under the no action alternative, growers would be expected to continue to choose crops 

for planting based on expected profitability, and profitability would likely continue to 

vary from year to year and crop to crop (Appendix M.2.1.3). Seed prices are likely to 

continue to be a major operating cost in soybean production, with overhead cost the 

majority of the total costs. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The only difference 

between the no action and the deregulation in whole alternative attributable to the 

introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield from the use of commercial MON 87712 soybean 

varieties, in addition to the incremental yield opportunity resulting from the use of other 

technologies or conventional breeding. The economic benefit of higher yield is well 

understood and is compelling to growers as it helps them increase productivity and 

profitability on their farm. To the U.S. economy as a whole, increased productivity 

(increased value of production without increasing inputs) is of value as well as it helps 
Monsanto Company 11-SY-217U 448 of 467 

U.S. businesses to be more competitive in the global economy. How that value will be 

apportioned between consumers (in the form of reduced prices for soybeans or derived 

products) and growers (in the form of increased profits), will vary depending on the 

government policies and global market conditions, as described in Appendices M.2.1.3 

and M.2.1.4. 

M.4.1.5. Land Use 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. Also, 

under the no action alternative, based on USDA projections, acres planted in soybean in 

the U.S. are expected to experience a small increase over the next ten years. However, 

based on the historic stability of overall U.S. cropland acres, any increases in soybean 

acreage in the U.S. would likely occur with corresponding decreases in some other crops, 

and increases in the rate of double-cropping (Appendix M.2.1.4). Growers will produce 

the crops they anticipate to be most profitable. Any changes in soybean production 

would not be expected to expand outside the range of agricultural land used for crops in 

the U.S. for the past 100 years (Appendix M.2.1.4). As discussed in Appendix M.2.1.4, 

corn and soybean combined planted acreage is at an all-time high in 2011. This was 

achieved partly through maximization of existing cropland. Other factors included 

decreases in other crop acreage and double-cropping. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. The presence of the MON 87712 trait is not expected to affect the overall 

land used for crop production in the U.S., based on the historic stability of U.S. cropland 

acreage. While the yield increases expected with MON 87712 may encourage increased 

soybean planting to some extent, many factors other than yield affect planted acreage, 

and yield is not necessarily a good predictor of crop acreage. In the other major U.S. 

crops, corn, production has increased much more dramatically than soybeans in recent 

years, and has not affected the overall range of acreage of cropland; and corn acreage is 

well below historical corn acreage in spite of dramatically increased yields (USDA 

NASS, 2011). 

M.4.1.6. Seed Production 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Under the no action alternative, soybean seed production would be expected to continue 

as described in Appendix M.2.2.1. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 
Monsanto Company 11-SY-217U 449 of 467 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. MON 87712 will also be a trait in soybean grown for seed production. As 

summarized in Section VII, MON 87712 soybean is agronomically, phenotypically, and 

compositionally comparable to conventional soybean with the exception of its increased 

yield trait. It is expected that MON 87712 soybean grown for seed production purposes 

would yield more seed. Seed growers are accustomed to variations in seed yield due to 

the same factors that impact grain yield (e.g., climate, pest pressure, etc.). Therefore, 

seed production practices are not expected to change. 

M.4.1.7. Organic Soybean Production 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Under the no action alternative, organic soybean production would be expected to 

continue as described in Appendix M.2.2.2. and it is expected that biotechnology-derived 

soybean varieties will continue to be grown on over 90% of the soybean acres. As 

described in Appendix M. 2.2.2., organic soybean producers employ practices that allow 

them to reasonably avoid biotechnology-derived soybeans. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. As summarized in the petition, (Section VII), MON 87712 soybean is 

agronomically, phenotypically, and compositionally comparable to conventional soybean 

with the exception of its yield trait. Soybean is principally self pollinated; however, low 

levels of cross pollination occur. The pollen from MON 87705 has been characterized 

and compared to pollen produced by conventional soybean. No statistically significant 

differences were detected between MON 87712 and the conventional control A3525 for 

percent viable pollen or pollen grain diameter (Table VII-5). Furthermore, no visual 

differences in general pollen morphology were observed between MON 87712 and the 

conventional control A3525. Thus, the potential for cross pollination from MON 87712 is 

expected to be the same as conventional soybean and the practices that organic farmers 

are using to avoid biotechnology-derived traits are not expected to change with the 

introduction of MON 87712. MON 87712 is not expected to result in increases in 

biotechnology-derived soybean, as over 90% of U.S. soybean is currently biotechnologyderived. For these reasons, MON 87712 is not expected to have an impact on organic 

soybean production any different than the no action alternative. 

 
Monsanto Company 11-SY-217U 450 of 467 

M.4.1.8. Agronomic Practices 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Soybean breeders will continue to develop higher yielding soybean varieties and farmers 

will likely use these new varieties on their farm due to their economic returns. Under the 

no action alternative, agronomic practices are expected to continue as described in 

Section VIII. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. MON 87712 soybean is agronomically, phenotypically, and 

compositionally comparable to conventional soybean with the exception of its yield trait 

(See Section VII). Field assessments included observations for plant responses to abiotic 

stressors, disease, and plant-arthropod interactions. The observed phenotypic 

characteristics were similar between MON 87712 and the control (Section VII). 

Therefore, MON 87712 is not expected to result in changes in agronomic practices and 

no impacts on tillage, crop rotation, irrigation, management of insects, disease 

management, weed management or herbicide tolerant weeds are expected with the 

introduction of MON 87712. 

The expected yield increase associated with MON 87712 will predictably require more 

potassium and phosphorous from the soil. As mentioned previously, soybean fixes its 

own nitrogen and does not benefit from added nitrogen; therefore, no additional nitrogen 

inputs are expected for MON 87712. Potassium and phosphorous are rarely applied to 

soybean because soybean follows well fertilized crops like corn and wheat in a rotation 

and residual levels of potassium and phosphorous are sufficient to produce a soybean 

crop. The application of fertilizers to corn has not changed substantially from the late 

70’s even though higher yielding soybean varieties have continued to be adopted (See 

Figure M-2 and M-4). As noted in Section VII, detailed observations were taken of 

MON 87712 and the control over the growing season at multiple locations. No signs of 

nutrient deficiency were noted for MON 87712. Therefore, the use of MON 87712 in 

breeding programs and the opportunity to increase yield is not expected to change 

fertilization practices for soybean compared to the no action alternative. 

M.4.1.9. Human Health and Worker Safety 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Human health and worker safety issues are described in Appendix M.2.4. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 
Monsanto Company 11-SY-217U 451 of 467 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. As summarized in the petition, (Section VI) MON 87712 soybean is 

compositionally comparable to conventional soybean. Detailed compositional analyses 

were conducted in accordance with OECD guidelines to assess levels of key nutrients and 

anti-nutrients in MON 87712 compared to levels present in the parental conventional 

soybean control of a similar genetic background as well as conventional commercial 

reference varieties. These compositional comparisons were made by analyzing the seed 

and forage harvested from plants grown at each of eight field sites in the U.S. during the 

2009 field season. The conventional commercial reference varieties used to establish a 

range of natural variability for the key nutrients and anti-nutrients in conventional 

commercial soybean varieties have a history of safe consumption. Nutrients assessed in 

this study included proximates, fiber, amino acids, fatty acids, and vitamin E in seed, and 

proximates and fiber in forage. The anti-nutrients assessed in seed included raffinose, 

stachyose, lectin, phytic acid, trypsin inhibitors, and isoflavones.. 

Assessment of the analytical results confirmed that the differences observed in the 

combined-site analysis were not meaningful to food and feed safety or the nutritional 

quality of MON 87712 soybean. In addition, the levels of assessed components in 

MON 87712 were compositionally equivalent to the conventional control and within the 

range of variability of the conventional commercial reference varieties that were grown 

concurrently in the same field trial. Therefore, it is concluded that soybean seed and 

forage produced from MON 87712 are compositionally equivalent to that of the 

conventional soybean and that the high yield trait in MON 87712 does not have a 

meaningful impact on the composition and therefore on the food and feed safety or 

nutritional quality of MON 87712 compared to conventional soybean. 

The food, feed and environmental safety of the BBX32 protein has been addressed in the 

petition (Section V). The BBX32 protein is produced by a gene obtained from the 

Arabadopsis plant. The BBX32 protein is similar to other plant produced BBX proteins 

with know histories of food, feed and environmental safety. The B-box zinc finger 

family is found in many plant species including soybean, where the B-box family 

contains 61 genes. The protein represents an extremely minor fraction of the total protein 

produced by the plant and has no characteristics associated with or similar to toxic or 

allergenic proteins. 

Therefore, soybeans and the processed fraction produced from MON 87712 are expected 

to be as safe and nutritious as soybeans and processed fractions produced from existing 

commodity soybeans. Practices for use of herbicides and other pesticides are not 

expected to be affected. Therefore, MON 87712 is expected to have the same impact as 

commodity soybean to human health and worker safety. 

M.4.1.10. Animal Health 
Monsanto Company 11-SY-217U 452 of 467 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. As 

discussed in Appendix M.2.5., soybean meal is currently the primary animal feed from 

soybeans and is fed to animals as a protein source. Anti-nutrients of potential concern in 

soybean meal are trypsin inhibitors, lectins and phytic acid, which are generally 

controlled by heating during processing. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. As summarized in Section VII, MON 87712 soybean is agronomically, 

phenotypically, and compositionally comparable to conventional soybean with the 

exception of its high yield trait. As discussed above in Appendix M.4.1.6, MON 87712 

has been found to be compositionally equivalent to conventional soybeans in terms of 

both nutritional and anti-nutritional composition. Therefore, soybean as forage and 

soybean meal produced from MON 87712 are expected to be as safe and nutritious as 

soybean forage and meal produced from existing conventional and GE soybeans. As 

discussed above, the BBX32 protein is produced by a gene obtained from the 

Arabadopsis plant. The BBX32 protein is similar to other plant proteins with know 

histories of food, feed and environmental safety. The protein represents an extremely 

minor fraction of the total protein produced by the plant and has no characteristics 

associated with or similar to toxic or allergenic proteins. Therefore, MON 87712 is 

expected to have the same impact as commodity soybean on animal health. 

M.4.1.11. Animal and Plant Communities 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Animal and plant communities are discussed in M.2.6. There are no native species in the 

U.S. related to soybeans; therefore, outcrossing to native species is not a concern with 

soybean. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. As summarized in Section VII, MON 87712 soybean is agronomically, 

phenotypically, and compositionally comparable to conventional soybean with the 

exception of its increased yield trait. Agronomic practices that may affect animal and 

plant communities are not expected to change as a result of the introduction of 

MON 87712. Therefore, MON 87712 is expected to impact animal and plant 

communities the same as commodity soybean under the no action alternative. 
Monsanto Company 11-SY-217U 453 of 467 

M.4.1.12. Surface Water, Groundwater and Air Quality 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Surface water and groundwater are discussed in Appendix M.2.7.1. and air is discussed in 

Appendix M.2.7.2. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

increased yield in commercial soybean varieties that contain MON 87712. The presence 

of the MON 87712 trait is not expected to affect agronomic practices such as tillage or 

herbicide or pesticide application. As described in Appendix M.2.7.3., the increased 

yield that will occur with MON 87712, or increases in soybean yield from any other 

source, may result in the removal of more phosphate and potash from the soil, and 

therefore may require the replacement of these nutrients at a slightly higher rate than 

would be needed with lower-yielding soybeans. For the same volume of soybeans 

produced, however, the phosphate and potash needs would be the same. Because 

agronomic practices will be negligibly affected by the introduction of MON 87712, the 

introduction of MON 87712 is expected to have negligible impacts on surface water, 

groundwater or air quality, in terms of per acre impacts compared to existing soybean 

varieties. Because MON 87712 offers the potential to increase productivity of soybean, 

production a small positive impact to surface water and air quality may be expected in 

terms of impacts per bushel of soybean produced. That is, on average, the impacts as 

described in Appendices M.2.7.1. and M.2.7.2 will be essentially the same per acre while 

more soybeans may be produced on that acre. Therefore, the impact per bushel of 

soybean production will be slightly less than before the introduction of MON 87712. 

The introduction of MON 87712 is not expected to impact air quality differently from the 

no action alternative. In the future it is expected that more food will need to be produced 

to feed a growing and increasingly affluent global population. This can be accomplished 

by either increasing crop yield per acre, or expanding the amount of land used for 

agriculture. Under the no action alternative and deregulation in whole alternative, it is 

likely that crop yields will increase. The increased yield associated with MON 87712 is 

expected to allow for less land-use change compared to the no action alternative, and is 

not expected to have any direct impact on exhaust emissions or fertilizer applications. 

Therefore, MON 87712 is not expected to have a deleterious effect on air quality. Of the 

global anthropogenic CO2 emissions related to climate change, ~80% are derived from 

the combustion of fossil fuels and cement production, and ~20% are derived from land 

use change and deforestation related to agriculture (IPCC, 2007). Higher yielding crop 

varieties will likely help to meet the higher food demands in the 21st century without 

expanding the amount of land used in agriculture, therefore leading to less land-use 

change and potentially lower anthropogenic CO2 emissions (Burney et al., 2010). 

 
Monsanto Company 11-SY-217U 454 of 467 

M.4.1.13. Soil and Soil Microorganisms 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. Soil 

and soil microorganisms are described in Appendix M.2.7.3. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. One critical interaction between soybeans and microbes in the soil is in the 

development of a symbiotic relationship between soybean and rhizobia. As noted in 

Section VII.C, measurement of nodule number and mass along with plant growth and 

nitrogen status are commonly used to assess differences in the symbiotic relationship 

between a legume and its associated rhizobia. As described in Section VII.C, no 

statistically significant differences were detected (5% level of significance) between 

MON 87712 and the conventional control for each measured parameter related to 

assessing this relationship, including nodule number, shoot percent total nitrogen, shoot 

total nitrogen (g), and dry weight of nodules, shoot material, and root material. In 

addition, field assessments included observations for plant responses to abiotic stressors, 

disease, and the observed phenotypic characteristics were similar between MON 87712 

and the control. 

The introduction of MON 87712 is expected to have minmal impact on agronomic 

practices compared to the no action alternative. As described in Appendix M.2.7.3, the 

use of MON 87712 in soybean varieties offers the opportunity for increased yield. As 

with any higher yielding soybean variety, increases in soybean yield may result in the 

removal of more phosphate and potassium from the soil, and may require the replacement 

of these nutrients at a slightly higher rate than would be needed with lower-yielding 

soybeans. Soybean is typically rotated with corn in crop rotations and benefits from the 

fertilizers applied to corn requiring very little fertilizer inputs. According to the USDA 

NASS data base, fertilizer inputs on corn the primary rotational crop for soybean have 

leveled off or slightly decreased since the 1980’s (Figure M-4). In this same period, 

yields of soybean have continued to increase suggesting that higher yields in soybean are 

not related to increased fertilizer inputs. Similarly, the expected yield increase in 

MON 87712 soybean varieties is unlikely to change this trend. Rather, improved crop 

genetics and more precise farming methods are expected to provide increased yield on 

the same or reduced land base using the same or reduced inputs. 
Monsanto Company 11-SY-217U 455 of 467 

Figure M-4: Average historical U.S. fertilizer application to corn crop and corn and 

soybean yields over time. 

Source: USDA NASS 

 

0

20

40

60

80

100

120

140

160

1960 1970 1980 1990 2000 2010

Average Fertilizer Applied (lb/ac)

Year

Average U.S. Fertilizer Application on

corn from 1964‐2010

N P K

20

25

30

35

40

45

50

50

70

90

110

130

150

170

1960 1970 1980 1990 2000 2010

Average Soybean Yield (bu/ac)

Average Corn Yield (bu/ac)

Year

Average U.S. Corn and Soybean Yield

from 1964‐2010

Corn Soybean
Monsanto Company 11-SY-217U 456 of 467 

M.4.1.14. Adjacent Agricultural Crops and Non-Agricultural Plants 

No Action Alternative. Under the no action alternative, MON 87712 would continue to 

be regulated and would only be grown on limited acres under notification or permit. 

Adjacent agricultural crops and non-agricultural plants are discussed in Appendix M. 

2.7.4. 

Deregulation in Whole Alternative. The difference between the no action and the 

deregulation in whole alternative is expected to be the gradual introduction of 

MON 87712 into soybean varieties and use in soybean production. The primary 

difference between the no action and the deregulation in whole alternative attributable to 

the introduction of MON 87712 into a broad base of soybean germplasm would be the 

opportunity for increased yield in commercial soybean varieties that contain 

MON 87712. As summarized in Section VII, MON 87712 soybean is agronomically, 

phenotypically, and compositionally comparable to conventional soybean with the 

exception of its high yield trait. Because MON 87712 impacts the yield of soybean, it 

may be speculated that the trait could impact the invasiveness or persistence of soybean. 

The assessment of the volunteer potential of MON 87712 is presented in the petition 

(Section VII.C.5). Based on the assessed data, the results of the study support a 

conclusion that the introduction of the yield trait did not alter the volunteer potential of 

MON 87712 compared to conventional soybean. Furthermore, these results demonstrate 

that the increased yield trait in MON 87712 confers no biologically meaningful change 

to the invasiveness or potential for soybean to persist in the environment (petition p. 158). 

Therefore, MON 87712 is expected to have no impact on adjacent agricultural crops and 

non-agricultural plants. 

M.5. Cumulative Effects 

CEQ regulations define cumulative impacts as “the impact on the environment which 

results from the incremental impact of the action when added to other past, present, and 

reasonably foreseeable future actions regardless of what agency (federal or non-federal) 

or person undertakes such other action.” 19 Thus, cumulative impacts are assessed only 

for those resources that are impacted by the proposed action. 

As described in Appendix M.4.0, the primary impact of dereculation in whole of 

MON 87712 is increased yield in soybeans. Therefore, the only cumulative impacts 

assessed are those associated with increased yield. The no action alternative (Appendix 

M.4.1.3) already includes yield increases in soybean that will result from other past, 

present and reasonably foreseeable future actions , and based on the trend of incremental 

annual average soybean yield gains observed over several decades, these are expected to 

occur whether or not MON 87712 is introduced. Note that these projected yield increases 

in soybean are not based on specific events or activities by others, but are based on 

USDA ERS projections. The impacts as described in Appendix M.4.1.3 includes 

projected yield increases under the no action alternative plus those resulting from 

 

19 40 CFR 1508.7 
Monsanto Company 11-SY-217U 457 of 467 

deregulation of MON 87712; thus, there are no other foreseeable cumulative yield 

impacts to discuss. 
Monsanto Company 11-SY-217U 458 of 467 

Conventional Breeding with Other GE or Conventional Soybean 

As previously mentioned, several biotechnology-derived soybean products have been 

deregulated or are under consideration for deregulation, and a list of the events 

deregulated or under review by USDA is presented in Table M-2. 

Table M-2. Deregulated or Submitted Biotechnology-derived Soybean Products 

Phenotype ID Code(s) Institution Date Deregulated 

High Oleic Acid, Low 

Saturated Fat 

MON 87705 Monsanto Submitted 

Omega 3 Fatty Acid MON 87769 Monsanto Submitted 

Lepidopteran Resistant MON 87701 Monsanto Submitted 

Herbicide-tolerant 

(Glyphosate/Isoxaflutole)

FG72 Bayer 

Crop Sciences 

Submitted 

Herbicide-tolerant 

(Imidazolinone)

BPS-CV127-9 BASF 

Plant Science 

Submitted 

Dicamba-tolerant MON 87708-9 Monsanto Submitted 

High Oleic Acid DP-3Ø5423-1 Pioneer June, 2010 

Glyphosate- and ALStolerant DP-356Ø43-5 Pioneer July, 2008 

Glyphosate-tolerant MON 89788 Monsanto July, 2007 

Phosphinothricin-tolerant GU262 AgrEvo October, 1998 

Phosphinothricin-tolerant A5547-127 AgrEvo April, 1998 

Altered Oil Profile G94-1, G94-19, G168DuPont May, 1997 

Phosphinothricin-tolerant W62, W98, A2704-

12, A2704-21, 

A5547-35 

AgrEvo July, 1996 

Glyphosate-tolerant 40-3-2 Monsanto May, 1994 

Source is website: http://www.aphis.usda.gov/brs/not_reg.html. and 

http://www.aphis.usda.gov/brs/status/petday.html

 Once deregulated, MON 87712 may be bred with these deregulated biotechnologyderived soybean products as well as with conventional soybean, creating new improved 

varieties. APHIS has determined that none of the individual biotechnology-derived 

soybean products it has previously deregulated displays increased plant pest 

characteristics and that any progeny derived from crosses of these soybean 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 87712 is discussed in Section 

IV of and summarized here. Data have demonstrated that MON 87712 is stable in its 

progeny. Having established that the genetic material is stable and inherited in a 

Mendelian fashion, and based on data and observations taken on soybean varieties 
Monsanto Company 11-SY-217U 459 of 467 

containing MON 87712 in Monsanto’s plant breeding program, it is concluded that the 

phenotype of MON 87708 is likewise stable. Traditional soybean breeding has an 

established history of safe use, and use of MON 87712 in breeding programs is expected 

to behave in a manner similar to other conventional traits and biotechnology-derived 

traits. Given that there have been no plant pest characteristics associated with 

MON 87712, or with any of the previously deregulated events listed in Table 2, no 

significant impacts are expected to other soybean through the use of MON 87712 in 

breeding programs and in combination with any of the previously deregulated 

biotechnology-derived soybean 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 M-2). Since MON 87712 is expected to be utilized broadly in the future for 

Monsanto’s soybean products, it will likely be bred with other biotechnology-derived 

soybean products that Monsanto has petitioned APHIS for deregulated status (e.g., MON 

89788, MON 87701, MON 87705). 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 all completed a safety consultation with FDA and on 

the basis of knowledge of the type of modifications made to each of the deregulated 

events, and to the events under review, the biochemical pathways are not likely to 

unexpectedly interact or result in the production of novel constituents. 

The decision to deregulate MON 87712 would also allow for breeding of this product 

with conventional soybean of diverse genetic background. No impacts to public health 

(e.g., food or feed safety) or environmental safety are expected due to the breeding of 

MON 87712 with these other soybean 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 soybean to produce combined trait 

products is designed to identify and remove off-types (i.e., plants that lack the intended 

phenotype or that do not show expected agronomic or phenotypic characteristics) 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 trait products, in terms of phenotypes, agronomic characteristics, and the 

efficacy of the individual traits when present in combination. This screening process is a 

means to assure and confirm that the traits present in stacked products are performing as 

expected and thus do not display novel phenotypes. Given this evaluation it can be 

concluded that no significant impacts are expected through the use of MON 87712 in 

combination with previously deregulated biotechnology-derived soybean products. 
Monsanto Company 11-SY-217U 460 of 467 

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