Monsanto Company 11-SY-217U 1 of 467 

### Petition for the Determination of No nregulated 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 Pr incipal 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. Ge ng, 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. Ta uchman, 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 adva nce 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 a ny information based on appropriate legal grounds, e.g ., responsiveness, confiden tiality, and/or competitiv e 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 (incl uding website posting) without Monsanto's prior notice and consent.

©2011 Monsanto Company. All Rights Reserved.

This document is protected under copyright la w. 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 Co mpany. 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 propert y 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 resp onsibility 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 pest s in the United States (U.S). APHIS regulation 7 CFR § 340.6 provides that an ap plicant 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 requ est to APHIS for a determination of nonregulated status for the new biotec hnology-derived soybean product, MON 87712, any progeny derived from crosses betw een MON 87712 and conventional soybean, and any progeny derived from crosses of M ON 87712 with biotechnology-derived soybean that have previously been granted no nregulated 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 incr ease and as a major global supplier of soybean, th e U.S. must increase soybean production to help meet growing demand. Increased s oybean productivity in the U.S. has been accomplished by both increasing the area under cultivation a nd through yield increases per unit area.

Based on recent trends in farm production and land area, mo st OECD (Organization for Economic Cooperation and Development) count ries, including the U. S. and Canada, are predicted to face the challenge of expandi ng 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. Breede rs 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 targetin g some of these same major characteristics

as traditiona l breeding.

Crop yield results from a sequ ential growth and developmen t process – first the plant grows vegetatively and produces photosynthetic tissue, followed by flowering and the production of seeds, and finally seed filling a nd maturation. Actual yield performance is a complex parameter that is dependent on a num ber of genetic and e nvironmental factors, that influence a crop’s opportuni ty to realize its full yield po tential. Improvements in crop yield have been a primary focus of conve ntional breeding. The genetic changes that resulted in crop domestication and yield improve ment in conventional varieties have beenMonsanto Company 11-SY-217U 5 of 467 

### shown by modern molecular biology analysis to have been typical ly achieved through the selection and safe use of plant genes en coding transcriptional regulator proteins. Agricultural biotechnology provi des the opportunity to fu rther enhance crop yields through the introduction of new ge netic elements that use or modify existing pathways in the plant.

Monsanto Company has developed the biot echnology-derived soybean line MON 87712, which will be used in traditional breeding programs to produce commercial soybean varieties with increased yi eld opportunity. The yield incr ease 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 transc ription factors to regulate the plant’s day/night processes and results in increased availability of assimilates 1 

### 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 th is petition demonstrate that MON 87712 is unlikely to pose an increase in plant pest potential, including w eediness 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 prot ein in MON 87712 is unlikely to be a toxin or allergen based on ex tensive information collected.

4. A compositional assessment of harveste d seed and forage confirmed that MON 87712 is compositionally equiva lent 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 imp act compared to conventional soybean.

1 An Assimilate is a product of plant metabo lism (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 cu ltivation 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 Cr op 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 earl y as 1000 B.C. and soyb ean 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 repr esented 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 co mmercial soybean species in the U.S. ( Glycine

max L. Merr.) does not exhibit weedy char acteristics, 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, su ch as the ability to disperse, invade, or become a dominant species in new or divers e 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. Ho wever, 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 othe r 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 outcr oss 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, t hus, the effect of the BBX32 expression cassette and the expressed BBX32 protein can be assessed in an unbiased manner using a comparative safety assessment.Monsanto Company 11-SY-217U 7 of 467 

### Molecular Characterization Verified the In tegrity and Stability of the Inserted DNA

in MON 87712

MON 87712 was developed through Agrobacterium -mediated transformation of conventional soybean A3525 meristem tissu e with plasmid vector PV-GMAP5779. PV-GMAP5779 contains two T-DNAs, each delin eated 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-1 α promoter, EF-1 α leader, EF-1 α 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 transfor med 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 pr oduction 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 sequenc e 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 j unctions. Additionally, Southe rn 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 th at 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. BBX 32 is a protein that ha s 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 characterizati on and assessment confirmed that the BBX32 protein in MON 87712 is safe for human and animal consumption. The assessment involved: 1) characterization of the physicoc hemical and functional properties of BBX32; 2) quantification of BBX32 leve ls in plant tissues; 3) comparison of the amino acid sequence of BBX32 in MON 87712 to known alle rgens, gliadins, glutenins, toxins, and other biologically-active pr oteins known to have advers e effects on mammals; 4) evaluation of the digestibility of BBX32 protei n in simulated gastric and intestinal fluids; 5) documentation of the presen ce of related proteins in se veral plant species currentlyMonsanto Company 11-SY-217U 8 of 467 

### consumed; and 6) investigation of the pot ential mammalian toxic ity 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 detec tion in harvested seed and forage. Bioinformatics analysis determined that BBX32 does not share amino acid sequence similarities with known allergen s, gliadins, glutenins, or pr otein toxins. In addition, BBX32 was rapidly digested in in vitro assays using simulated gastric and intestinal fluids and did not show any adverse effects wh en 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 a ssessment 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 c onducted in accordance with soybean-specific OECD guidelines to assess le vels of key nutrients a nd anti-nutrients in MON 87712 compared to levels present in the parental conventional soybean cont rol A3525 as well as

conventional commercial reference varietie s. These compositional comparisons were made by analyzing the harvested seed and fora ge 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 esta blish a range of natural variab ility for the key nutrients and

anti-nutrients in conventional commercial soybean varieties have a history of safe consumption. Nutrients assessed in this st udy included proximates, fiber, amino acids, fatty acids, and vitamin E in harvested see d, and proximates and fiber in forage. The

anti-nutrients assessed in harvested seed in cluded raffinose, stachyos e, lectin, phytic acid, trypsin inhibitors, and isoflavones.

The combined-site analysis was conducted to id entify statistically si gnificant differences ( α =0.05) between MON 87712 and the conventiona l control A3525. The results from the combined-site data were reviewed using consid erations relevant to food and feed safety and nutritional quality. These considerations included assessm ents of: 1) the relative magnitude of the differences in the mean va lues of nutrient and an ti-nutrient components

of MON 87712 and the conventional contro l, 2) whether the MON 87712 component mean value was within the range of natural va riability of that component as represented by the 99% tolerance interval of the conven tional commercial reference varieties grown concurrently in the same field trial, 3) th e reproducibility of the statistically significant combined-site component differences at individu al sites, and 4) the differences within the

context of natural variabil ity of commercial soybean composition published in the

scientific literature and in the Internati onal Life Sciences In stitute (ILSI) Crop Composition Database.

Assessment of the analytical results conclu ded that the differences observed in the combined-site analysis were not meaningful to food and f eed 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 theMonsanto Company 11-SY-217U 9 of 467 

### range of variability of the conventional comm ercial 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 imp act on the composition and therefore on the food and feed safety, nutritional quality, a nd familiarity (as discussed below) of MON 87712 compared to conventional soybean.

MON 87712 Does Not Change Soybean Plan t 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 unde rlying 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. Familiar ity considers the biology of the plant, knowledge of the introduced tr ait, the receiving environmen t, and the interactions among these factors. This provides a basis fo r comparative risk assessment between a biotechnology-derived plant and the convent ional control. Thus, the phenotypic, agronomic, and environmental interactio n assessment of MON 87712 included the parental conventional control A3525 as a compar ator. This evaluation used a weight of evidence approach and considered statis tical differences between MON 87712 and the conventional control A3525 with respect to re producibility, magnitude, and directionality of any detected differences. Conventional co mmercial reference varieties were used to establish a range representative of natural variability in soybean for each characteristic assessed, and provided a context from whic h to further evaluate any statistical

differences. Characteristics assessed incl uded: seed dormancy and germination, pollen morphology, and symbiont interactions in th e greenhouse, plant phenotypic observations, environmental interaction evaluations, and vo lunteer potential and persistence outside of

cultivation evaluations conducted in th e field. The phenotypic, agronomic, and

environmental interaction assessment demons trated that MON 87712 does not differ from the conventional control A3525 in any biol ogically 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 s eed germination, supports a conclusion of no increased weediness of MON 87712 when comp ared 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 shoo t 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, agr onomic, 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 in cluded 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 statisti cally significant differe nces were detected

between MON 87712 and the conventional cont rol A3525 for seedling vigor, days to 50% flowering, days to 50% end of floweri ng, plant height, lodging, pod shattering, grain moisture, or 100 seed weight. Statistically si gnificant differences were observed between

MON 87712 and the conventional control A352 5 for early stand c ount, days to 50% senescence, days to physiol ogical maturity, final stand count, and yield. Although significantly different from the conventi onal 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 charac teristic and thus woul d not be adverse in terms of pest potential. Differences in days to 50% senescence, days to physiological maturity, and yield were consistent with th e mode of action of the introduced trait in MON 87712. The increase in yield is agronomi cally desirable and would not contribute to increased weediness potential of MON 87712 without changes in a combination of

other characteristics associ ated with weediness.

Evaluations of volunteer potential and persistenc e outside of cultivation from field-grown plants provide information useful in assessi ng potential weedine ss characteristics of MON 87712 compared to the conventional control A3525. Evaluation of volunteer potential demonstrated no st atistically significant diffe rences between MON 87712 and

the conventional control A3525. Evaluation of persistence outside of cultivation demonstrated a few statistically signif icant differences between MON 87712 and the conventional control A3525, however these differe nces were not seen across all sites and were small in magnitude. Taken together, th ese comparative assessments indicate that MON 87712 is not likely to have increased weediness or pl ant pest potential compared to conventional soybean.

In summary, the phenotypic, agronomic, and environmental interaction data were generated and evaluated to characte rize 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 direc tion of detected differences between MON 87712 and the conventional control, and comparison to the range of the

commercial reference varieties. The resu lts indicated that MON 87712 does not possess weediness characteristics, increased susceptibi lity or tolerance to specific abiotic stress, diseases, or arthropods, or characteristics th at would confer a plant pest risk or a significant adverse environmental impact compared to conventional soybean.Monsanto Company 11-SY-217U 11 of 467 

### 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 intera ctions, including beneficial arthropods, or disease susceptibility compared to the conventional control A3525.

The biochemical information and experime ntal data for eval uation of MON 87712 included molecular characterization, BBX32 pr otein safety assessments, information

from the environmental interaction asse ssment, 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 conclusi on that MON 87712 has no reasonable mechanism to harm NTOs, nor does it pose an additional risk to threatened and endangered species compared to th e cultivation of c onventional 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. Fu rthermore, should cross-pollination occur, MON 87712 and its progeny are not expected to exhibit a signif icant environmental impact because, as described above, evalua tions have shown that the presence of BBX32 is not likely to enhance weediness or plant-pe st potential. Therefor e, the likelihood and environmental consequence of pollen transfer from MON 87712 to other Glycine species is considered negligible.

Deregulation of MON 87712 Will Not Sign ificantly Impact Soybean Agronomic

Practices or Land Use

Introduction of MON 87712 is expected to ha ve minimal impact on current cultivation and management practices for soybean. MO N 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 su sceptibility to insects and diseases as commercial soybean. MON 87712 did not requi re any additional inputs to produce a crop. The intended increase in yield is not ex pected to significan tly impact any of the agricultural practices farmers use to produ ce 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 accustomedto experiencing field-to-field and yearto-year yield variation based on environmental conditions and the genetics of the varieties they select for planting. Therefore, grow ers 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 Determ ination 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 Fore ign 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 Phyl ogenetics 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 TH E 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 .....................46Monsanto 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 De termine 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 De termine 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 Ch aracterization 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 Equivale nce of BBX32 Protein from MON 87712 ...........76

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

V.D. Assessment of Potential A llergenicity of the BBX32 Protein .............................81

V.E. Safety Assessment Summar y 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 M ON 87712 is Not Acutely Toxic ..............................82

V.F. BBX32 Protein Characteri zation and Safety Conclusion ....................................83Monsanto 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, AGRONO MIC, 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 Po tential 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 ..............................................................162Monsanto Company 11-SY-217U 16 of 467 

### 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. ENVIRONMENTA L CONSEQUENCES ...........................................................167

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

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

IX.B.1. Characteristics of the Gene tic 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 W ild Perennial Species of Subgenus Glycine .................................................................................................................175

IX.D.4. Transfer of Genetic Informa tion 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 CONSEQUENC ES 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, a nd Individual-Site Results for Seed Dormancy and Germination Assessment of MON 87712 ..........................................339Monsanto Company 11-SY-217U 17 of 467 

### 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 .....................................................411Monsanto 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 Geneti c Elements in the PV-GMAP5779 ...........................42

Table IV-1. Summary Chart of th e Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used in MON 87712 Analysis ...........50

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

Table IV-3. Segregation of the Expre ssion 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 Difference s (p<0.05) for the Comparison of Soybean Component Levels for MO N 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-sit e 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. Fi eld Trials, Laboratory or Greenhouse

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

Table VII-2. Germination Char acteristics of MON 87712 and the

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

Table VII-3. Field Phenotypic E valuation 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 Characteristic s of MON 87712 Compared to the

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

Table VII-6. Symbiont Interact ion Assessment of MON 87712 and

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

Table VIII-1. Soybean Production in the U.S, 2001 – 2010 1 

### .....................................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 2010 1 

### .......................149Monsanto Company 11-SY-217U 19 of 467 

### Table VIII-4. Ratings for Postemergence Control of Volunteer Soybean in Labeled Rotational Crops 1 

### ............................................................................................164

Table IX-1. Summary of Published Lit erature 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 significan t differences between MON 87712 soybean and control (MON 87712(-) and A3525) le af tissue at the R1 developmental
stage ......................................................................................................................... ........220

Table B-5: Summary of significan t 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 MO N 87712-Produced BBX32 Protein .............................242

Table F-1. Commercial Ref erence 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 fr om 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 .........................................................................275Monsanto Company 11-SY-217U 20 of 467 

### 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 Us ed in Dormancy Assessment .............................342Monsanto Company 11-SY-217U 21 of 467 

### Table G-2. Dormancy and Germinatio n Characteristics of MON 87712 and

the Conventional Control A3525 Seed Pr oduced at each of the Three Field

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

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

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

Table H-2. Field and Pl anting Information ................................................................349

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

Table H-4. Individual-Site Phenot ypic 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 Respon se Evaluations of MON 87712 and Conventional Control A3525 Using an Observational Severity Scale ......................374

Table H-7. Disease Damage Evalua tions of MON 87712 and Conventional Control A3525 Using an Observational Severity Scale ..............................................375

Table H-8. Arthropod-Related Da mage Evaluations of MON 87712 and Conventional Control A3525 Using an Observational Severity Scale ......................376

Table H-9. Abundance of Pest Arthro pods in Samples Collected from MON 87712, Conventional Control A3525, and the Commercial Reference

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

Table H-10. Abundance of Beneficial Ar thropods in Samples Collected from MON 87712, Conventional Control A3525, and the Commercial Reference

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

Table J-1. Starting Seed of MO N 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 Persiste nce Outside of Cultivation Assessment .........399

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

Information for Persistence Outsid e 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 Eval uated 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 .......................................................407Monsanto Company 11-SY-217U 22 of 467 

### Table L-7. Plant Vigor Monitoring of MON 87712, the Conventional Control A3525, and the Commercial Ref erence Varieties in 2009 ..........................................408

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

Each Site in 2009 ............................................................................................................4 09

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

Table M-2. Deregulated or Submit ted Biotechnology-derived Soybean

Products ...................................................................................................................... ....458Monsanto Company 11-SY-217U 23 of 467 

### 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 Sequen ces 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 Sequen ces in MON 87712: Probe 8 .............................64

Figure IV-9. Overlapping PCR Analys is 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 Ge nerating 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 yiel d in conventional soybean. ....................................204Monsanto Company 11-SY-217U 24 of 467 

### Figure B-3. Diurnal cycling of proce sses such as nitrogen and carbon metabolism in plants is achieved th rough 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 Pattern s 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 ..............................................................................................................24 1

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 Soyb ean 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 histor ical U.S. fertilizer application to corn crop and corn and soybean yields over time. ...............................................................................455Monsanto Company 11-SY-217U 25 of 467 

### ABBREVIATION AND DEFINITIONS 2



### ~ 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, μ g, 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) benzenesulf onyl 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-phospha te synthase protein from Agrobacterium sp. strain CP4 Continuing Survey of F ood 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 desorpti on/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-polyacr ylamide gel electrophoresis Simulated gastric fluid

Simulated intestinal fluid Standard operating proceedure Sucrose Phosphate Synthase Tris-buffered saline containing Tween-20 Tris(2-carboxyet hyl)phosphine

test/control/reference Trypsin Inhibitor Unit melting temperature tissue-specific site pool

United States United States Department of Agriculture Untranslated RegionMonsanto Company 11-SY-217U 27 of 467 

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

I.A. Basis for the Request for a Determ ination of Nonregulated Status under 7 CFR § 340.6

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (USDA) has res ponsibility, 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 pe sts 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 arti cle does not present a pl ant pest risk and no longer should be regulated. If APHIS determ ines that the regulat ed article does not present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction

of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status for the new biotec hnology-derived soybean product, MON 87712, any progeny derived from crosses betw een MON 87712 and conventional soybean, and any progeny derived from crosses of MON 87712 with any bi otechnology-derived soybean that have previously been gran ted nonregulated status under 7 CFR Part 340.

I.B. Rationale for the Development of MON 87712

Monsanto Company has developed the biot echnology-derived soybean line MON 87712, which will be used in traditional breeding pr ograms to produce commer cial varieties with increased yield opportunity. The yield incr ease 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 tr anscription factors to regu late the plant’s day/night processes and results in increased availability of assimilates (products of plant metabolism) in MON 87712 compared to an ap propriate 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 dur ing the reproductive phase of the soybean plant, as well as by the significantly hi gher yield of MON 87712 when compared to control, as observed in multisite field st udies in the U.S. Appendix B provides supplemental information on the function of the BBX32 protei n in MON 87712. The data demonstrate how the plant BBX32 protei n affects existing diurnal processes in soybean to increase yield in MON 87712. Highe r 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 av erage 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 suppl ier of soybean, theMonsanto 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 si nce 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 markthe third lowest level in 10 years (USDAERS, 2011). Such low inventories indicate that future soybean productivity must not only be large enough to refill pres ent stockpiles to levels that provide increased food and feed security, but must also be adequate e nough to accommodate end-user demand until the next crop is harvested the following year.

Increased soybean produc tivity in the U.S. has been acco mplished by both increasing the area under cultivation and through yield in creases 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 th e U.S. (USDA-NASS, 2011b). Annual improvement in U.S. soybean yield is attribut ed to rapid producer a doption of agricultural improvements such as genetic and agronomic innovations that provide producers with means for reducing “on-farm” yield constrai nts (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 producti on and land area, most OECD countries, including the U.S. and Canada, are predic ted to face the challenge of expanding agricultural output by raising pr oductivity on a stable or re duced land area (OECD-FAO, 2008). Therefore, much of the projected expa nsion in soybean producti on in the future is expected to come from in creased 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. Biotechnol ogical approaches complement traditional soybean breeding efforts by targeting some of the major characteristics as traditional breeding.

Crop yield results from a sequ ential growth and developmen t process – first the plant grows vegetatively and produces photosynthetic tissue, followed by flowering and the production of seeds, and finally seed filling an d maturation. Yield is a complex trait that is dependent on a number of genetic and envi ronmental factors, th at 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 c onventional varieties ha ve been shown by modern molecular biology analys is to have been typically ac hieved through the selection and safe use of plant genes encoding transcriptional regula tor proteins. Agricultural biotechnology provides the opportunity to fu rther 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 gr owers, 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 Regul ation of Biotechnology, the responsibility for regulatory oversight of biotechnology-derived crops fa lls 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). Deregulati on of MON 87712 by USDA constitutes only one component of the overall regulato ry 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 th e 1992 FDA policy statement concerning regulation of products derive d from new plant varieties, including those developed through biotechnology (U.S. FDA, 1992). In co mpliance with this policy, Monsanto will

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

assessment of MON 87712.

I.C.2. Submissions to Forei gn Government Agencies

To support commercial introduction of MON 87712 in the U.S., re gulatory 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, includi ng: the Ministry of Agriculture, People’s Republic of China; Japan’s Mini stry of Agriculture, Forestr y, and Fisheries, Ministry of Environment, and the Ministry of Healt h, Labor, and Welfare; the Canadian Food Inspection Agency and Health Canada; the Intersectoral Commissi on for Biosafety of Genetically Modified Organisms, Mexico; th e European Food Safety Authority, as well as to regulatory authorit ies in other soybean importi ng 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, biol ogy, 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 (OEC D, 2000), and 2) a summary prepared by USDA-APHIS (2006) and a biology documen t 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 harves ted seed produced in 2010, whic h represented 58% of world oilseed production that year (ASA, 2011). Soybean is gr own as a commercial crop in over 35 countries. The major producers of soybean are th e U.S., Brazil, Argentina, China, India, Paraguay and Canada, whic h 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 pro duced in the U.S. (ASA, 2011). For the fifth consecutive year, U.S. whole soybean expo rts 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 fo r 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 wa s planted on approximately 76.6 million acres in the U.S. in 2010, producing 3.33 billion bushels of soybean w ith 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, cooki ng, 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. Noneth eless, soybean oil accounted for approximately 29% of all the vegetable oils consumed globally, and was the secondMonsanto 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 s upplement in feed rations for liv estock. 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 y easts via fermentation to the manufacture of soaps, inks, paints, disinfectants, and biodies el. 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 ha ve taken place in Ch ina 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 Ko rea. The movement of soybean germplasms was probably associated with the developmen t and consolidation of territories and the disintegration of Chinese dynastie s (Hymowitz and Newell, 1981).

From the first century A.D. to approximat ely the 15th and 16th centuries, soybean was

introduced into several count ries, with land races eventu ally 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 cen tury and throughout the 17th cent ury, 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 subs equently 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 extensivel y 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 diploidize d 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 indige nous to Australia, West, Central and South Pacific Islands, China, Russia, Japan, I ndonesia, Korea, Papua New Guinea, the Philippines, and Taiwan(Hymowitz, 2004). The subgenus Soja includes the cultivated soybean, G. max (L.) Merr. and its wild a nnual relatives from Asia, G. soja Sieb. and Zucc. The list of species in the genus Glycine Willd. is presented in Table II-2.Monsanto Company 11-SY-217U 33 of 467 

### Table II-2. List of Species in the Genus Glycine Willd., 2n Chromosome Number, Genome Symbol, and Distribution

Genus 2n Genome 1 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 Complex 3 Australia, West Central and

South Pacific Islands

22. G. tomentella Hayata 38 EE Australia

40 DD Australia, Papua New Guinea

78 Complex 4 Australia, Papua New Guinea

80 Complex 5 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).Monsanto Company 11-SY-217U 34 of 467 

### Glycine soja grows wild in China, Ja pan, 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 wi th narrow trifoliolate leaves . The purple or very rarely

white flowers are inserted on s hort, 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 hab it 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 no rmally and subsequently produce fertile pollen and seed (Singh and Hy mowitz, 1989). The taxonomic position of G. gracilis has been an area of debate, and neith er 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 out crossing (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 under gone aneuploid reduction (loss of a specific chromosome), which is prevalent throughout the Papilionoide ae, to a base number of 10 chromosomes (Lackey, 1981). Te traploidization (2 n = 2 x = 40) through autopolyploidy or allopolyploidy of the pr ogenitor species occurred either prior to or after dissemination from the ancestral region. The path of mi gration from a common progenitor is assumed by Singh et al., (2001) as: wild perennial (2 n = 4 x = 40, unknown or ex tinct) to wild annual (2 n = 4 x = 40; G. soja ) to soybean (2 n = 4 x = 40; G. max ). Soybean should be regarded as a stable tetraploid with diploi dized 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 caly x of five sepals, a corolla of five petals, one pistil, and nine fused stamens with a sing le 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 ar ound the stigma (OECD, 2000). The soybean flower stigma is receptive to pollen approxi mately 24 hours before anthesis and remainsMonsanto Company 11-SY-217U 35 of 467 

### receptive for 48 hours after anthesis. The anther s mature in the bud and directly pollinate the stigma of the same flower. As a result , soybean is consideredto 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 la te 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 typi cally not a concern in most environments where soybean is cultivated (CFIA, 1996; OECD, 2000). Soybean seed rarely exhibit any dormancy characteristics, and seed rema ining in the field after harvest likely will readily imbibe water (Lersten and Carlson, 20 04), 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 controlle d 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, wa s 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 li ne of MON 87712 and was used as the conventional soybean comparator (hereafter referred to as the conventiona l 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 th erefore be assessed in an unbiased manner in the comparative safety a ssessment. In addition, reference materials were used to establish ranges of natural va riability 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 commer cial soybean varieties that were derived through conventional methods and/or Roundup R eady soybean varieties. The commercial

reference varieties used at each location were selected based on their availability and agronomic fit for the resp ective geographic region.Monsanto Company 11-SY-217U 36 of 467 

### III. DESCRIPTION OF TH E 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 co mprised of sequences to be transcribed and the regulatory elements necessary fo r the expression of those sequences.

III.A. The Plasmid Vector PV-GMAP5779

PV-GMAP5779 was used for the transformati on 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, de signated 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-1 α promoter, EF-1 α leader, EF-1 α intron, CTP2 targeting sequence, and the E9 3 ′ untranslated region. During tr ansformation, both T-DNAs were inserted into the soybean genome (Secti on III.B) where T-DNA II, containing the cp4 epsps expression cassette, fu nctioned as a marker gene for the selection of transformed plantlets. Subsequently, conven tional self-pollinated breeding methods and segregation, along with a combin ation 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 bacteria l 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 gene tic elements and their prefix es (e.g., P-, L-, I-, TS-, OR-, B-, CS-, and T-) in PV-GMA P5779 is provided in Table III-1.

III.B. Description of the Transformation System

MON 87712 was developed through Agrobacterium -mediated transfor mation 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. Br iefly, meristem tissues were excised from the embryos of germinated conv entional 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 R 0 

### plants generated through th is transformation were se lf-pollinated to produce R 1 

### plants, and the unlinked inse rtions of T-DNA I and T-DNA II were segregated. A non-lethal dose of glyphosate was applied to R 1 

### 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. Subs equently, 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 th e 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) (Ho ltan et al., 2011; Khanna et al., 2009; Putterill et al., 1995). The pr esence of BBX32 modulates asp ects 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 th e 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 prot ein 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 cassett e were isolated through conventional self-pollinated breedin g 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 incl uded in the T-DNA II probes. 11428 bp CS- BBX32

T- E6

B-Right Border Region

B-Right Border Region

P- FMV/EF-1 α

L- EF-1 α

I- EF-1 α

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 IIMonsanto Company 11-SY-217U 39 of 467 

### Figure III-2. Schematic of the Development of MON 87712 Transformed meristem tissue fr om 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 pr ogeny generations in laboratory and

field assessments Assembled Agrobacterium binary plasmid vector PV-GMAP5779 and

transferred to Agrobacterium tumefaciens strain ABIMonsanto Company 11-SY-217U 40 of 467 

### 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), whic h functions to direct transc ription 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 polya denylation of the RNA transcripts.

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

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

E9 3 ′ untranslated region. The FMV/EF-1 α promoter is the chimer ic 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-1 α gene from Arabidopsis

thaliana encoding elongation factor EF-1 α (Axelos et al., 1989), which functions to direct transcription in plant cells. The EF-1 α leader is the 5 ′ leader (exon 1) sequence of the

EF-1 α gene from Arabidopsis thaliana that encodes elongation factor EF-1 α (Axelos et al., 1989), which is involved in re gulating gene expression. The EF-1 α intron is the intron with flanking splice sites of the EF-1 α gene from Arabidopsis thaliana encoding elongation factor EF-1 α (Axelos et al., 1989), which is involved in regulating gene expression (Curie et al., 1991). The CTP2 targeting sequence is th e targeting sequence of

the ShkG gene from Arabidopsis thaliana encoding the EPSPS transit peptide region (Herrmann, 1995; Klee et al., 1987), which func tions to direct tr ansport 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 protei n (Coruzzi et al., 1984), wh ich functions to direct polyadenylation of the mRNA.

III.F. T-DNA Borders

PV-GMAP5779 contains Left a nd Right Border regions (Fi gure 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 regi ons each contain a 24-25 bp nick site that is the site of DNA exchange during tran sformation. 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 Ri ght Border regions, where one border region

set flanks T-DNA I and the other bo rder region set flanks T-DNA II.

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

Genetic elements that exis t 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 ofMonsanto 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 Tn 7 that confers spectinomycin and stre ptomycin resistance (Fling et al., 1985) in E. coli and Agrobacterium during molecular cloning. Because these elements are outside the border regions, they are not e xpected to be transfe rred 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 Geneti c Elements in the PV-GMAP5779

Genetic Element Location in

Plasmid Vector Function (Reference)

T-DNA I

B 1 -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

P 2

- 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

CS 3

- 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

T 4

- 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 seque nce 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 seque nce 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 Elem ents in the PV-G MAP5779 (continued)

Intervening Sequence 3078-3099 Sequence used in DNA cloning

P -FMV/EF-1 α 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-1 α gene from Arabidopsis thaliana encoding elongation factor EF-1 α (Axelos et al., 1989) that directs transcription in plant cells

L 5

- EF-1 α 4140-4185 Leader (exon 1) sequence of the EF-1 α gene from Arabidopsis thaliana that encodes elongation factor EF-1 α (Axelos et al., 1989) that is involved in regulating gene expression

I 6

- EF-1 α 4186-4807 Intron with flanking splice sites of the EF-1 α gene from Arabidopsis thaliana encoding elongation factor EF-1 α (Axelos et al., 1989) that involved in regulating gene expression (Curie et al., 1991) Intervening Sequence 4808-4816 Sequence used in DNA cloning

TS 7

- 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 cloningMonsanto Company 11-SY-217U 44 of 467 

### Table III-1. Summary of Genetic Elem ents in the PV-G MAP5779 (continued)

aadA 7582-8470 Bacterial promoter, coding sequence, and 3' UTR for an aminoglycoside-modifying enzyme, 3''(9)- O

nucleotidyltransferase from the transposon Tn 7 (Fling et al., 1985) that confers spectinomycin and streptomycin

resistance Intervening Sequence 8471-9000 Sequence used in DNA cloning

OR 8

- 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 maintenan ce of plasmid copy number

in E. coli (Giza and Huang, 1989) Intervening Sequence 10209-10945 Sequence us ed 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 us ed 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 stab ly 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 analys es 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-GMAP57 79, and to confirm that a single copy of T-DNA I was inserted at a singl e site and is stably inheri ted; 2) DNA sequence analyses to determine the exact seque nce of the inserted DNA a nd the DNA sequences flanking

the 5' and 3' ends of the insert; 3) DNA sequence compar ison of the inserted DNA sequence to the T-DNA I sequence in PV-GMA P5779 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 characte rization of the genetic modi fication 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 dete rmine 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-D NA 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 copi es 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 v ector 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 fla nking 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 se quence. 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 anot her predictable size segment containing a portion of the insert wi th 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 det ecting 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 vect or 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 mo lecular 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 Southe rn blots, and the molecular we ight 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 M ON 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 sequenc es, 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 se quences, 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 addi tion, DNA sequencing analyses confirmed each genetic element in the insert and the sequenc e of the insert matches the corresponding sequence in PV-GMAP5779. Fu rthermore, genomic orga nization at the MON 87712 insertion site was determined by comparing th e 5' and 3' flanking sequences of the insert to the sequence of the inserti on site in conventional soybean.

The stability of T-DNA I present in MON 87712 across multiple generations (R 3 

### , R 4 

### , R 5 

### , R 6 

### , and R 7 

### ) was demonstrated by Southern blot fi ngerprint analysis. Genomic DNA from five generations of MON 87712 (F igure IV-11) was digested w ith 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 en tire T-DNA I insert (Figure IV-12). This fingerprint strategy consists of two border se gments that assess not only the stability of T-DNA I, but also the stability of genomi c DNA directly adjacent to T-DNA I. Generational stability analysis demonstrated th at the expected Southern blot fingerprint of MON 87712 was maintained through five ge nerations of the breed ing history, thereby confirming the stability of T-DNA I in MON 87712.

Segregation analysis was employed to exam ine the genetic behavior of the T-DNA I

insert in MON 87712. The results from this anal ysis showed heritability and stability of the insert occurred as expected across mu ltiple generations (Fi gure IV-13, Table IV-3)

which corroborates the molecular insert stabil ity 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 summ arized in Table IV- 2. The results fromMonsanto 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 c onventional control for DNA sequence analysis are shown in Figure IV -9 and Figure IV-10, respectively. The generations used in the genera tional stability analysis are de picted 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 fo r the segregation analysis ar e presented in Table IV-3.

Materials and methods used for the charac terization of the insert in MON 87712 are found in Appendix C.Monsanto Company 11-SY-217U 49 of 467Figure IV-1. Schematic Representation of theInsert and Flanking DNA Sequences in MON 87712DNA derived from the T-DNA I of PV-GMAP5779 integrated in MON 87712. Right-angled arrowsindicate the ends of theintegrated T-DNA I and the beginning of theflanking sequence. Identified on the map are the genetic elements within the insert, aswell as restriction sites for enzymes withpositions relative to the size of the DNAsequence (flanks and insert) used in the Southernanalyses. The relative locations of the T-DNA I probes and the expected sizes of restriction segments are indicated. This schematicdiagram is not drawn to scale. Locations of T-DNA I probes are approximate.Probes are described in Figure III-1.NcoI 148EcoRI 289EcoRI 3750NcoI 4418NcoI 6608SpeI 5905

B-Left Border Region r1 P- e35S CS- BBX32 T- E6 B-Right Border Region r15' Flank 3' FlankProbe 1Probe 217472NcoIEcoRI andSpeI~ 4.3 kb~ 2.2 kb~ 3.5 kb ~ 2.2 kbMonsanto Company 11-SY-217U 50 of 467Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used inMON 87712 AnalysisSouthern Blot Analysis T-DNA I T-DNA II BackboneInsertStabilitySouthern Blot FigureIV-2IV-3IV-4IV-5IV-6IV-7IV-8IV-12Probe Used123, 5467, 981Probing TargetDigestionenzyme Expected Band Sizes on each Southern BlotPV-GMAP5779NcoI~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 kb~7.8 kb~3.7 kbProbe Templates1N/A~~2~~2~1.3 kb~1.1 kb~~2~~2~1.8 kb~0.9 kb~~2~~2MON 87712NcoI~4.3 kb~2.2 kb~2.2 kbNoneNoneNoneNoneNone~4.3 kb~2.2 kbEcoRI andSpeI~3.5 kb~2.2 kb~2.2 kbNoneNoneNoneNoneNone--3*these bands were detected by the probes due to sequence homology to the other digested segment of PV-GMAP5779.1probe template spikes were used as positive hybridization controlsin Southern blot analyseswhen multiple probes were hybridized tothe 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 analysisMonsanto Company 11-SY-217U 51 of 467 

### Table IV-2. Summary of Ge netic 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

B 1 -Left Border Region r1 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

P 2

- 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

CS 3

- 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

T 4

- 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 Region r1 5465-5507 DNA region from Agrobacterium tumefaciens containing the right border seque nce 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.

r1 Superscripts in Left and Right Borders indicate that the border sequences in MON 87712 were truncated compared to the border se quences in PV-GMAP5779.Monsanto Company 11-SY-217U 52 of 467 

### IV.A. Southern Blot Analysis to Determ ine Insert and Copy Number of T-DNA I in

MON 87712

The copy number and inserti on sites of T-DNA I sequences in the soybean genome were evaluated by digesting the R 3 

### 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 addi tional copies and/or in tegration 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 c opy at a single integration si te 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 Tabl e 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 Sout hern 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 contro l 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 geno mic 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 a nd lane 9) and hybridized with Probe 1 (Figure III-1) showed no detect able 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). Det ection of the spiked co ntrols 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 indica te 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 hybridi zation 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 geno mic 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 a nd lane 9) and hybridized with Probe 2 (Figure III-1) showed no detect able 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 th at 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 (Figur e 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 indica te that the sequence covered by Probe 2 resides at a single detect able 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 32 

### P-labeled probe that spanned a portion of the T-DNA I sequence (Figure III-1, Probe 1). E ach 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

0.5Monsanto 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 32 

### P-labeled probe that spanned a portion of the T-DNA I sequence (Figure III-1, Probe 2). E ach 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, obt ained 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

0.5Monsanto Company 11-SY-217U 56 of 467 

### IV.B. Southern Blot Analysis to Det ermine 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 R 3 

### 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 pres ent in MON 87712, then probing with the T-DNA II sequences would result in the detecti on 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 geno mic 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) show ed no detectable hybridization bands, as expected. Conventional contro l 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 (Fig ure III-1, Probe 3 and Probe 5) generated from PV-GMAP5779 produced the expected ba nds 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 expect ed. The results presented in Figure IV-4 indicate that MON 87712 cont ains no detectable T-DNA II elements from

Probe 3 and Probe 5 of PV-GMAP5779.

IV.B.2. Probe 4

Conventional control geno mic 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 a nd 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 la ne 5 through lane 8) and ~1.0 kb ( EcoR I and Spe I digest, lane 3, lane 4, lane 9, a nd lane 10) in a darker exposure (data not shown). These hybridization signals most likely result from Probe 4 hybridizing to homologous sequences residi ng 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 hybridizationMonsanto Company 11-SY-217U 57 of 467 

### bands discussed above. Detection of the sp iked 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 de tectable bands other than the endogenous hybridization bands discussed above. The results presente d 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 De tect the Presence or Absence of T-DNA II Sequences in MON 87712: Probe 3 and Probe 5 The blot was hybridized with two 32 

### P-labeled probes that sp anned 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 tissu e. 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

0.5Monsanto Company 11-SY-217U 59 of 467 

### Figure IV-5. Southern Blot Analysis to De tect the Presence or Absence of T-DNA II Sequences in MON 87712: Probe 4 The blot was hybridized with one 32 

### P-labeled probe that spanned a portion of the T-DNA II sequence (Figure III-1, Probe 4). E ach 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

0.5Monsanto Company 11-SY-217U 60 of 467 

### IV.C. Southern Blot Analysis to De termine the Presence or Absence of PV-GMAP57779 Backbone Sequences in MON 87712

The presence or absence of PV-GMAP5779 b ackbone sequences in the soybean genome was evaluated by digesting the R 3 

### generation of MON 87712 and the conventional control genomic DNA samples with tw o 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, th en hybridizing with overlapping probes corresponding to the backbone sequence should result in the detec tion of hybridization

bands on the Southern blot. The results of th is analysis are shown in Figure IV-6 through Figure IV-8.

IV.C.1. Plasmid Vector Backbone Probe 6

Conventional control geno mic 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 a nd lane 9) and hybridized with Probe 6 (Figure III-1) showed no detect able 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 cont ained 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 ba nds, as expected. The data indicate that

MON 87712 contains no detectab le backbone elements from Probe 6 of PV-GMAP5779.

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

Conventional control geno mic 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) show ed no detectable hybridization bands, as expected. Conventional contro l 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 expo sure, data not shown) was al so detected because a small region of the intervening seque nce 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) ge nerated from PV-GMAP5779 produced the expected bands at ~1.8 kb and ~0.9 kb, respectiv ely (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 cont ains no detectable backbone elements from Probe 7 and

Probe 9 of PV-GMAP5779.

IV.C.3. Plasmid Vector Backbone Probe 8

Conventional control geno mic 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 a nd lane 9) and hybridized with Probe 8 (Figure III-1) showed no detect able 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 contro ls 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 ba nds, as expected. The data indicate that

MON 87712 contains no detectab le 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 Presen ce or Absence of PV-GMAP5779 Backbone Sequences in MON 87712: Probe 6 The blot was hybridized with one 32 

### P-labeled probe that spa nned a portion of the PVGMAP5779 backbone sequence (Figure III-1, Probe 6). Each lane contains ~10 μg of digested genomic DNA isolated from leaf tissu e. 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/ Hin d 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 RunMonsanto Company 11-SY-217U 63 of 467 

### Figure IV-7. Southern Blot Analysis to Determine the Presen ce or Absence of PV-GMAP5779 Backbone Sequences in MON 87712: Probe 7 and Probe 9 The blot was hybridized with two 32 

### P-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 isol ated 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/ Hin d 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 RunMonsanto 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 32 

### P-labeled probe that spanned a portion of the PV-GMAP5779 backbone sequence (Figure III-1, Pr obe 8). Each lane contains ~10 μg of digested genomic DNA isolat ed 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/ Hin d 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 RunMonsanto 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 el ements within the MON 87712 insert was confirmed by DNA sequence analyses. PCR pr imers were designed w ith the intent to amplify three overlapping DNA regions that sp an 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 seque nce of PV-GMAP5779 as described in Table III-1. From the sequence analyses, 3493 base pair s flanking the 5' end of the MON 87712 insert and 1965 base pair s 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 Analys is Across the Insert in MON 87712 PCR was performed on both conventiona l 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 veri fy 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 generate d 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-e35SCS-BBX32T-E6B-Right Border Regionr1 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 4.1B-Left Border Regionr1Monsanto 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 perf ormed on genomic DNA extracted from

MON 87712 and the conventional control to ex amine 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 th e 3' end of the inse rt (Figure IV-10). The amplified PCR product from the conve ntional control was subjected to DNA sequence analysis. Sequence alignments were performed between the conventional control sequence and the sequences flanki ng the 5' and 3' end of the MON 87712 T-DNA I insert. The alignment analyses i ndicated 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 strand ed break repair mechanisms in the plant during the Agrobacterium -mediated transformation proce ss (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 us ing 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 th e 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 ethi dium 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 BB-Left Border Regionr1P-e35SCS-BBX32T-E6B-Right Border Regionr1 5'

Flank 3'

Flank Primer A Primer B 2 1 4 3 5Monsanto Company 11-SY-217U 69 of 467 

### IV.F. Southern Blot Analysis to Examine In sert Stability in Multiple Generations of

MON 87712

In order to demonstrate the st ability of the insert in M ON 87712, Southern blot analysis was performed using genomic DNA obtained from fi ve 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 R 3 

### generation was used for the molecular characterization analyses as show n in Figure IV-2 through Figure IV-8. To analyze insert stability, four additional generations, R 4 

### , R 5 

### , R 6 

### , and R 7 

### , 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 wa s 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 fingerp rint 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). Det ection of the spiked controls indicates that the probe hybridized to its target sequences.

MON 87712 DNA extracted from generations R 3 

### , R 4 

### , R 5 

### , R 6 

### , and R 7 

### 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 R 3 

### 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 R 3 

### generation was used for the molecu lar characterization and commercial development of MON 87712. R0 corresponds to the transformed plant. All generations were self pollinated (  ). Generations used for in sert stability analysis (R 3 

### , R 4 

### , R 5 

### , R 6 

### , and R 7 

### ) 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 32 

### P-labeled probe that spanned a portion of the T-DNA I sequence (Figure III-1, Probe 1). E ach 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 (R 3 ) ( Nco I)

5 MON 87712 (R 4 ) ( Nco I)

6 MON 87712 (R 5 ) ( Nco I)

7 MON 87712 (R 6 ) ( Nco I)

8 MON 87712 (R 7 ) ( 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 8Monsanto 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 ( χ 2) 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 segregati on ratio according to Mendelian principles.

The MON 87712 breeding path for generating segreg ation data is described in Figure IV13. The transformed R 0 

### plant was self-pollinated to produce R 1 

### seed. From the R 1 

### 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 R 1 

### MON 87712 plant was self-pollinated to give rise to a population of R 2 

### plants, which were in turn se lf-pollinated to obtain the R 3 

### generation. At each generation, the fixed homozygous plants were tested fo r the expected segreg ation pattern of 1:0 (positive:negative) for the BBX32 gene using the Invader analysis.

Homozygous R3 MON 87712 plants were crosse d to a Monsanto proprietary soybean line (EXP0224AAC) that did not contain the BBX32 gene to produce F 1 

### hemizygous seed. The resulting F 1 

### plants were tested fo r the copy number of the BBX32 gene by Endpoint Taqman zygosity analysis. A hemizygous F 1 

### plant was selected and then selfpollinated to produce F 2 

### seed. This process of self-pollination and Endpoint Taqman zygosity analysis was repeated for the F 2 

### , F 3 

### , and F 4 

### 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 Mendeli an inheritance principles. The χ 2 was calculated as:

χ

2 

### = ∑ [( | o - e | )2 / e]

where o = observed frequency of the phenot ype and e = expected frequency of the phenotype. The level of statistical signi ficance was predetermined to be 5% ( α = 0.05).

The results of the χ 2 

### analysis of the segregating proge ny of MON 87712 are presented in

Table IV-3. The χ 2 

### value in the F2, F3, and F4 gene rations 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 ar e also consistent with the molecular

characterization data that indicate MON 87712 contains a single, intact copy of the

BBX32 expression cassette that was inserted in to the soybean genome at a single locus.Monsanto Company 11-SY-217U 73 of 467Figure 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 F4generations.Note: An individual hemizygous plant from each of the F1, F2, and F3populations was self-pollinated to produce the population of the next generation.Transformed and regenerated R0plantR1Plant #2 (MON 87712) selectedR2(homozygous positive)R3(homozygous positive)Breeding path continuedCross with a Monsantoproprietary variety that didnot contain theBBX32geneF1(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 negativeMonsanto Company 11-SY-217U 74 of 467Table IV-3. Segregation of the ExpressionCassette During the Development of MON 877121:2:1 SegregationGenerationTotalPlants1Observed #PlantsHomozygousPositiveObserved #PlantsHemizygousObserved #PlantsHomozygousNegativeExpected #PlantsHomozygousPositiveExpected #PlantsHemizygousExpected #PlantsHomozygousNegativeχ2ProbabilityF221859986154.50109.0054.502.260.3235F318448944246.0092.0046.000.480.7873F418244875145.5091.0045.500.890.64081Plants were evaluated for the presence of theBBX32gene by Endpoint Taqman zygosity analysis. “Total plants” refers to the total number ofplants 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 integrat ed 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 performe d on MON 87712 and conventional control determined the following: the complete DNA sequence of the in sert and adjacent DNA sequences in MON 87712; the or ganization of the genetic elem ents within the insert; the expected sequence of each elemen t in the inserted DNA; and the 5 ′ and 3 ′ insert to genomic DNA junctions. The PCR and DNA seque nce analysis identified a 42 base pair

deletion that occurred at th e insertion site in MON 87712.

Southern blot analysis of MON 87712 dem onstrated that the inserted DNA has been maintained through five generations of breedi ng, thereby, confirming the stability of the insert. Results from segregation analyses sh ow heritability and stab ility of the insert occurred as expected across multiple generati ons, which corroborates the molecular insert stability analysis and establishes the geneti c 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 SAF ETY ASSESSMENT OF THE BBX32

PROTEIN IN MON 87712

Characterization of the intr oduced protein in a biotechno logy-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 eviden ce, 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 stab ilized by binding zinc ions. The B-box domain is predicted to be involved in protein-prot ein interactions (Khanna et al., 2009). BBX32 contains a single annotated pr otein domain, the B-Box B1 dom ain (Khanna et al., 2009). The B-box zinc finger family is found in many plant species; for ex ample, 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 accesso ry protein. As descri bed in Appendix B, BBX32 in MON 87712 plays a role in the tr anscriptional regulation of the plant’s day/night processes, and results in increased av ailability of assimilates in the plant. Plant

nutrient assimilation and utilization are crit ical processes to drive yield improvement.

V.B. Characterization and Equiva lence of BBX32 Protein from MON 87712

The safety assessment of crops derived th rough biotechnology incl udes characterization of the functional and physicoche mical properties of, and confir mation 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 expr ession 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 purit y, but instead was enriched in leaf extract to a purity of approximately 0.0001%. This low purity limite d the physicochemical characterization of MON 87712-produced BBX32 and the overall equiva lence analysis to ch aracteristics that could be evaluated by western blot. E. coli -produced BBX32 was characterized using a panel of analytic techniques including west ern blot analysis, N-terminal sequence analysis, and matrix-assisted laser desorpti on/ionization time-of-fli ght mass spectrometry (MALDI-TOF MS) to confirm identity, s odium 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 demonstr ate protei n-protein binding functionality

.

MON 87712-produced BBX32 prot ein was characterized by we stern 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 prot eins on SDS-PAGE, as detected by western blot analysis, and through bioinformatic co mparison of the BBX32 coding sequence from

the E. coli expression vector and MON 87712. The de tails of the materials, methods, and results are described in Appendix D, while the conclusions are summarized as below.

Western blot analysis with a monoclonal an tibody developed against BBX32 showed that the MON 87712-produced BBX32 had e quivalent mobility to the E. coli -produced BBX32 protein on the blot, indicating th e 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 BBX 32 coding sequence from the vector used to transform conventional soybean to pr oduce MON 87712, PV-GMAP5779, and from the

E. coli BBX32 expression vector, pMON102114, re sulted 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 th e corresponding T-DNA I sequence of PVGMAP5779. Thus, the pMON102114 BBX32 coding sequence is identi cal to the BBX32 coding sequence in MON 87712.

Direct assessments of BBX32 partner protei n binding activity, glycos ylation status, and N-terminal sequence , were not conduc ted for MON 87712-produced BBX32 due to the low purity of the plant-produced protein. In stead, these characteristics of BBX32 in

MON 87712 were assessed indir ectly. With respect to functional activity, the phenotypes observed for MON 87712 compared to convent ional 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-term inus predicted by the BBX32 coding sequenceMonsanto Company 11-SY-217U 78 of 467 

### 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, starti ng at position 172), but lacks the N-terminal signal sequence required for tr ansport to the endoplasmic re ticulum 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, wh ich contains potential N-glycosylation sites but has been shown to not be gl ycosylated (Harrison et al., 1 996). 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 rele vant to the risk assessment were determined by western blot anal ysis. For western blot s, protein extracts are separated by gel electrophores is 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 pres ent in MON 87712 tissue extracts from other proteins present in the tissue matrix, whic h allowed for the detection of this low expressing protein in the presen ce of other matrix proteins. The levels of the BBX32 protein in various tissues ofMON 87712 were estimated by densitometric analysis of Xray films exposed to immunoblots probed w ith BBX32 antibodies and visualized using chemiluminescent detection reagents (refer to Appendix E).

Tissue samples of MON 87712 and the pare ntal conventional control A3525 were collected during the 200 9 growing season from eight geogra phically diverse field sites in the U.S.: Jackson County, Arkansas; Parke County, Indiana; Clinton County, Illinois; Madison County, Illinois; Stark County, Illinoi s; York County, Nebraska; Boone County, Indiana; and Pawnee County, Kansas. These fi eld sites were representative of soybean producing regions suitable for co mmercial production. At each si te, four replicated plots

of MON 87712 and the conventional contro l 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 colle cted from each replicated plot at all field

sites.

Analyses of BBX32 protein levels were carried out in all se ven tissue types collected as

described in Table V-1. For leaf and root sa mples, densitometric analysis of the BBX32- specific immunoblots yielded the reported quan titative values of BBX32 protein by interpolation from standard curves prepared using purified BBX32 pr otein standard. The results obtained from western blot analysis are summarized in Table V-1and the details of the materials, methods, and sample colle ction are described in Appendix E.

The mean BBX32 protein levels were determ ined across eight sites and seven tissue types. BBX32 protein levels in MON 87712 across tissue types ra nged from <LOD to 110 ng/g dwt. The western blot method deve loped 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 develope d, the BBX32 protein was not detected in several samples including all harvested seed, forage and OSL-4 samples. The mean protein level in rootMonsanto Company 11-SY-217U 79 of 467 

### was 3.9 ng/g dwt, though most samples were <LOD. Expression was highest in leaf tissue, specifically OSL-3 (mean protei n level of 35 ng/g dwt) where BBX32 was determined in all samples. The levels in OSL-1 and OSL-2 were s lightly below those of OSL-3. These results indicate that the expr ession 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 ti ssue extracts. The LOD of BBX32 on the western blots was defined by the lowest amount of BBX32 protei n that resulted in a visible band on the

western blots. The LOD was determined to be 0.5 pg in forage extrac ts 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. (20 04) 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 m echanisms that regulate protein abundance.Monsanto Company 11-SY-217U 80 of 467 

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

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

Tissue 1 

### Development Stage 2 

### Days

After Planting

(DAP) BBX32 Mean (SD) Range (ng/g fwt) 3 

### BBX32 Mean (SD) Range (ng/g dwt) 4 

### LOQ/LOD 5



### (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 NA 6 

### 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 ND 7 

### 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 wa s collected. Soybean plant growth stages described in Soybean Growth and Development (Pedersen, 2004).

3 Protein levels are expressed as th e 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 a ll 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 th e 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 blot s 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 detectedMonsanto Company 11-SY-217U 81 of 467 

### V.D. Assessment of Potential A llergenicity 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 pr otein is not likely to be associated with allergenicity if: 1) the protein is from a non-allergenic s ource, 2) the protein represents a very small portion of the total plant protein, 3) the protein does not share structural similarities to known allergens ba sed on the amino acid sequence, and 4) the protein is rapidly di gested in mammalian gastrointest inal systems. BBX32 has been assessed for its potential allergenicity accord ing 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, th erefore, is highly unlikely to contain immunologically cross-reactive allergenic epitopes.

4) In vitro digestive fate experiments conducted w ith BBX32 demonstrate that the protein is rapidly digested in simulated gastric fluid (SGF) and in simula ted intestinal fluid (SIF).

Taken together, these data support the conclu sion 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 prot eins 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 expos ure 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 su ch 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). CamelinaMonsanto Company 11-SY-217U 82 of 467 

### 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 fi nger family is found in many plant species including soybean, where the B-box family cont ains 61 genes (Preuss et al., submitted for publication). Bioinformatic searches usi ng the BBX32 amino acid sequence as the query identify homologous sequences from several di fferent plant species, including the food crops: citrus, grape, apple, s oybean, rice, lettuce, and corn. Overall the protein sequence identity of BBX32 to homologs in these sp ecies 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 c onsumed 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 alle rgens, gliadins, glutenins, or protein

toxins which could have adverse eff ects 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 adve rse 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, w ith a No Observable Adverse Effect Level (NOAEL) of 29 mg/kg body weight (BW) , the highest dose level tested.

Potential human health risks from consump tion of foods derived from MON 87712 were evaluated using a Margin of Exposure (MOE) a pproach. 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 anMonsanto Company 11-SY-217U 83 of 467 

### 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 :



### ratio) tissue (buffer to l) ( lane per loaded extract the of volume (pg) band visible a with standard BBX32 spiked lowest the fwt ng/g

   3



### The corresponding expression level calculated fo r 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 corres ponds to an expressi on 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 95 th 

### percentile “eater-only” estimates of acute dietary exposure determined using the Dietar y 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 F ood Intakes by Individuals (CSFII), and assume that 100% of soybean products c onsumed, excluding oil, are derived from MON 87712. The MOEs for acute dietary in take 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 fo r BBX32, support the conclu sion that there is no meaningful risk to human health from dietary exposure to BBX32 in MON 87712.

Potential health risks to animals from th e 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 calc ulated 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, swin e, 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 leve l of exposure of animals to BBX32 in their feed, in addition to the above mentioned sa fety 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 conclusi on 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 M ON 87712 are as safe as soyb ean currently on the market for human and animal consumption.

V.F. BBX32 Protein Characteri zation and Safety Conclusion

3 Buffer to tissue ratio includes the dilutions factor. Fo r 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 safe ty studies. Expression studies using western blot analysis demonstrated that BBX32 was de tected in leaf and root of MON 87712, and was expressed at levels below the limit of de tection 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 expressi on values of 1 ng/g fwt and 10 ng/g fwt respectively, representing a low percenta ge of the total protein in soybean. Bioinformatics analysis de termined that BBX32 does not share amino acid sequence similarities with known allergens, gliadins, glutenins, or protei n toxins. BBX32 was rapidly digested in in vitro assays using simulated gastric an d 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 AS SESSMENT OF MON 87712

Safety assessments of biotechnology-derive d crops follow the comparative safety assessment process (Codex Alimentarius, 2009) in which the composition of grain and/or other raw agricultural commodities of the bi otechnology-derived crop is compared to the appropriate conventional counter part that has a history of safe use. Compositional assessments are performed using the princi ples 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 cr op varieties, nine countries, and eleven growing seasons concluded th at incorporation of biotechnology-derived agronomic traits has had little impact on na tural variation in crop composition; most compositional variation is attributable to growing region, agr onomic 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 breedi ng efforts to improve nutri tion, 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 gene tically modified plants” (OECD, 1998). The OECD consensus documents emphasize quantita tive 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. Leve ls of the components in harvested seed and forage of the biotechnology-derived crop are co mpared 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 in cluded at each site of the field , with a total of sixteen unique refere nces included in this study, production to provide data on natural variability of each compositional component analyzed. The field production was conducted at eight geographi cally diverse sites: Jackson County, Arkansas (ARNE); Parke County, Indiana (INRC); Clinton County, Illinois (ILCY); Madison County, Illinois (ILHI); Stark Count y, Illinois (ILWY); Boone County, Indiana (INSH); Pawnee County, Kansas (KSLA); and York County, Nebras ka (NEYO). All soybean plants including MON 87712, the c onventional control A3525, and conventionalMonsanto 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 c onventional control A3525 and the composition of the conventional commerci al 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 proxima tes and fiber in forage. Th e anti-nutrients assessed in harvested seed included raffinose, stachyose, lectin, phytic acid, tryp sin inhibitors, and

isoflavones.

In all, 63 different analytical components we re measured (seven in forage and 56 in harvested seed). Due to statistical constraint s, 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 a ssay 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 an alysis of variance method. Values for all assessed components were reported on a dry wei ght basis with the exception of moisture, which was reported as % fresh we ight and fatty acids (FA), wh ich were reported as % of

total FA.

For MON 87712, nine sets of statistical comp arisons to the conventional control A3525 were conducted. One comparison was based on compositional data combined across all eight field sites (the combin ed-site analysis) and eight comparisons were conducted on data from each of the eight individual field site s. 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 consum ption and that were grown concurrently with

MON 87712 and the conventional cont rol A3525 in the same trial.

For the combined-site analysis, significant differences in nutrient and anti-nutrient components were evaluated further using cons iderations relevant to the safety and nutritional quality of MON 87712 when compar ed to the conventional control A3525. The evaluation included: 1) the relative magn itude of the significant difference in the mean values (mean difference as % of the conventional control A 3525) of nutrient and anti-nutrient components of MON 87712 compared to the conventional control A3525,

relative to natural variabil ity 2) whether the MON 8771 2 component mean value is within the range of natural variability of that component as represented by the 99%

tolerance interval of conventi onal commercial reference varie ties grown concurrently in the same trial, 3) analyses of the repr oducibility of the significant combined-site component differences at indivi dual sites, and 4) assessing the combined-site significant differences and reproducible indi vidual site significant differences within the context of natural variability of comme rcial soybean composition published in the scientificMonsanto 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 comp arative assessment of the levels of key

nutrients and anti-nutrients in harvested s eed, 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 fora ge of MON 87712 is equi valent to that of the conventional control A3525, and within the natural variabi lity 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 differe nt 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 (a sh, carbohydrates by calculation, moisture, and total fat), two types of fiber (ADF and NDF), nine amino acids (histidine, leucine, lysine, methionine, proline, serine, th reonine, tryptophan, and tyrosine), six fatty acids (18:1 oleic acid, 18:2 linoleic acid, 18:3 lin olenic 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 pr oximate (protein), nine amino acids (alanine, arginine, aspartic acid, cystine, glutam ic acid, glycine, isoleucine, phenylalanine, and valine), and two fatty acid s (16:0 palmitic acid and 18:0 stearic acid) (Table VI-1 and VI-2).

The significant differences in nutrients we re further evaluated using considerations relevant to the safety and nutritional qua lity of MON 87712 when compared to the

conventional control A3525:

1) All nutrient component significant di fferences observed in the combined-site statistical analysis, whether reflecting in creased or decreased mean values for MON 87712 with respect to the convent ional control A3525, had small relative magnitudes when compared to natural variab ility, 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% to lerance interval

established from the conventional commercial reference varieties gr own concurrently in the same trial, and were, therefore, within th e range of natural vari ability in commercial soybean varieties with a hist ory of safe consumption.Monsanto Company 11-SY-217U 88 of 467 

### 3) Assessment of the reproducibility of th e combined-site significant differences at the eight individual sites showed significant diff erences for: protein at one site; alanine, aspartic acid, isoleucine, and valine at two sites; argini ne, glutamic acid, glycine, phenylalanine, and 18:0 stearic acid at three site s; and 16:0 palmitic acid at four sites. Cystine was not significantly diffe rent at any of the individual sites. Individual site mean values of MON 87712 for all nutrient compon ents with significant differences were

within the 99% tolerance interval establ ished 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 na tural variability of commer cial 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 nutritiona l perspective. Ten of the 12 significant

differences in the harvested seed nutrient levels between MON 87712 and the

conventional control A3525 observed in the combin ed-site data analysis were attributable to small differences in protein and nine am ino acids (all expressed as % dw). The relative magnitude of the difference betw een the mean protein values for MON 87712 and the conventional control A3525 was small (a n 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 relativ e 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 signi ficant differences at all individual sites. Eight of the nine amino acids ob served to be different in the combined-site analysis were increased (1.22 – 2.26%) consistent with an in crease in protein, one amino acid (cystine) was decreased (3.07%) relative to the convent ional control A3525 and, as with protein, significant differences between MON 87712 a nd the conventional control A3525 were not consistently observed at all sites. Cystine, the amino aci d observed to be significantly decreased (3.07%) in MON 87712 when compared to the c onventional cont rol A3525 in the combined-site analysis, was not significantly different at any of th e 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 pers pective because they were not consistently reproduced at the individual si tes, and the mean MON 87712 values were within the 99%

tolerance interval es tablished by conventional commerci al 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 consid ered to be meaningful from a food and feed safety and nutritional pers pective. Two of the combined -site significant differences

between MON 87712 and the conventional control A3525 were attributable to fatty acidsMonsanto 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 M ON 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 ac id. Neither of thes e fatty acids were significantly different between MON 87712 and the conventional control A3525 at more than four of the eight individua l sites. Thus, observed signifi cant differences in fatty acid

values between MON 87712 and the conventiona l control A3525 were not considered to be meaningful from a food and feed safety and nutritional perspectiv e because they were not consistently reproduced at the individual sites, and th e mean MON 87712 values were

within the 99% tolera nce interval established by conve ntional commercial reference varieties grown concurrently in the same trial.

In summary, statistical anal yses found no consistent signif icant differences between the levels of nutrient components in harves ted 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, genistei n, and glycitein) between MON 87712 and the conventional control A3525. Thus, the eval uation of anti-nutrient components in harvested seed supported the conclusion th at 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 conventiona l control A3525 for six of the seven nutrients. No significant differences were observed for ash, carbohydrates, moistu re, protein, ADF or NDF (Table VI-4). One significant diffe rence for total fat was observed between

MON 87712 and the conventional control. The re lative magnitude of the difference, with respect to the convent ional control, was only 11.34%, a nd the value was within the 99%

tolerance interval es tablished by conventional commerci al 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 ener gy. If the gross energy of the soybean forage is calculated using Atwate r Values (Merrill and Watt, 1973) and the composition observed in the combined-site analys is, 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 significan t difference between MON 87712 and the

conventional control A3525 observed in the co mbined-site analysis was evaluated for reproducibility at the individua l sites. Significant differences were observed between

MON 87712 and the conventional control A3525 in to tal fat values at only two of the eight individual sites. The relative magnit udes of the significant differences were 12.96Monsanto Company 11-SY-217U 90 of 467 

### and 39.10% lower, but all mean values at th e individual sites that were significantly

different were within the 99% tolerance inte rval established by conventional commercial reference varieties grown concur rently in the same trial.

Thus, an evaluation of nutrient components in forage supported the conclusion of compositional equivalence of M ON 87712 to conventional soybean.Monsanto Company 11-SY-217U 91 of 467Table VI-1. Summary of Differences (p<0.05) for theComparison of Soybean Component Levels for MON 87712 vs.Conventional ControlMean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin Combined-Site AnalysisSeed Proximate (% dwt)Protein40.9340.491.090.00538.87 - 42.7935.06, 43.58Seed Amino Acid (% dwt)Alanine1.771.751.300.0141.69 - 1.851.54, 1.88Arginine3.143.072.260.0272.93 - 3.382.51, 3.33Aspartic Acid4.734.671.300.0294.44 - 4.924.04, 5.07Cystine0.590.61-3.07<0.0010.55 - 0.630.51, 0.67Glutamic Acid7.607.491.490.0387.18 - 7.966.28, 8.18Glycine1.791.771.220.0201.70 - 1.851.52, 1.90Monsanto Company 11-SY-217U 92 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin Combined-Site AnalysisSeed Amino Acid (% dwt)Isoleucine1.901.871.570.0321.78 - 1.981.62, 2.03Phenylalanine2.152.121.590.0161.98 - 2.261.81, 2.33Valine1.991.952.020.0131.88 - 2.081.71, 2.13Seed Fatty Acid (% Total FA)16:0 Palmitic11.4811.64-1.420.00210.78 - 11.947.76, 13.1418:0 Stearic4.054.17-3.040.0043.54 - 4.723.06, 5.10Forage Proximate (% dwt)Total Fat6.237.03-11.340.0053.41 - 8.620.54, 13.11Monsanto Company 11-SY-217U 93 of 467Table VI-1 . Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed AntinutrientRaffinose (% dwt) Site ARNE0.820.7313.42<0.0010.77 - 0.860.39, 1.01Raffinose (% dwt) Site ILCY0.780.7010.760.0060.72 - 0.830.39, 1.01Raffinose (% dwt) Site ILHI0.780.90-13.270.0020.76 - 0.800.39, 1.01Raffinose (% dwt) Site KSLA0.870.825.250.0110.86 - 0.880.39, 1.01Raffinose (% dwt) Site NEYO0.660.71-6.570.0230.65 - 0.660.39, 1.01Seed Fatty Acid (% Total FA)16:0 Palmitic Site ARNE11.6912.01-2.660.00111.54 - 11.807.76, 13.1416:0 Palmitic Site ILHI11.3711.55-1.560.00211.30 - 11.497.76, 13.14Monsanto Company 11-SY-217U 94 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed Fatty Acid (% Total FA)16:0 Palmitic Site KSLA11.8912.12-1.880.00211.84 - 11.947.76, 13.1416:0 Palmitic Site NEYO11.6311.89-2.170.01211.59 - 11.717.76, 13.14Seed Amino Acid (% dwt)Arginine Site ILCY3.283.115.760.0073.23 - 3.382.51, 3.33Arginine Site KSLA3.052.905.220.0013.03 - 3.082.51, 3.33Arginine Site NEYO3.193.025.51<0.0013.16 - 3.242.51, 3.33Glutamic Acid Site ILCY7.787.572.760.0427.63 - 7.876.28, 8.18Glutamic Acid Site KSLA7.247.032.960.0207.19 - 7.366.28, 8.18Monsanto Company 11-SY-217U 95 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed Amino Acid (% dwt)Glutamic Acid Site NEYO7.687.413.660.0077.60 - 7.766.28, 8.18Glycine Site ILCY1.841.792.440.0351.80 - 1.851.52, 1.90Glycine Site KSLA1.721.691.860.0341.70 - 1.751.52, 1.90Glycine Site NEYO1.791.742.990.0011.78 - 1.801.52, 1.90Leucine Site ILCY3.233.152.300.0413.18 - 3.272.71, 3.38Leucine Site KSLA3.022.962.120.0433.00 - 3.052.71, 3.38Leucine Site NEYO3.183.093.200.0023.17 - 3.212.71, 3.38Phenylalanine Site ILCY2.222.153.240.0222.17 - 2.261.81, 2.33Monsanto Company 11-SY-217U 96 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 877712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed Amino Acid (% dwt)Phenylalanine Site KSLA2.041.982.800.0491.98 - 2.071.81, 2.33Phenylalanine Site NEYO2.172.084.580.0032.15 - 2.191.81, 2.33Seed Fatty Acid (% Total FA)18:0 Stearic Site ARNE3.854.16-7.37<0.0013.78 - 3.893.06, 5.1018:0 Stearic Site ILCY3.774.00-5.540.0013.71 - 3.913.06, 5.1018:0 Stearic Site INRC3.673.96-7.250.0013.54 - 3.843.06, 5.1018:2 Linoleic Site ARNE51.5053.62-3.960.00350.26 - 52.2750.14, 57.8118:2 Linoleic Site ILCY54.9354.341.090.04554.84 - 55.0350.14, 57.81Monsanto Company 11-SY-217U 97 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed Fatty Acid (% Total FA)18:2 Linoleic Site INSH52.7253.25-0.980.03452.33 - 53.2550.14, 57.81Seed Amino Acid (% dwt)Alanine Site KSLA1.701.672.200.0311.69 - 1.721.54, 1.88Alanine Site NEYO1.781.733.010.0071.76 - 1.801.54, 1.88Aspartic Acid Site KSLA4.564.452.300.0374.51 - 4.624.04, 5.07Aspartic Acid Site NEYO4.774.642.750.0054.74 - 4.814.04, 5.07Histidine Site KSLA1.041.013.050.0211.03 - 1.060.91, 1.17Histidine Site NEYO1.091.062.340.0011.08 - 1.090.91, 1.17Monsanto Company 11-SY-217U 98 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed Amino Acid (% dwt)Isoleucine Site ILCY1.941.873.820.0291.87 - 1.971.62, 2.03Isoleucine Site NEYO1.921.853.590.0091.90 - 1.931.62, 2.03Valine Site ILCY2.051.964.200.0401.95 - 2.081.71, 2.13Valine Site NEYO2.031.954.290.0132.00 - 2.051.71, 2.13Seed Fatty Acid (% Total FA)18:1 Oleic Site ARNE24.1821.0314.990.00123.08 - 25.7117.37, 26.8618:1 Oleic Site INSH22.2621.254.760.00122.07 - 22.5017.37, 26.8620:0 Arachidic Site ARNE0.290.32-9.780.0400.26 - 0.300.22, 0.39Monsanto Company 11-SY-217U 99 of 467Table VI-1. Summary of Differences(p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteSeed Fatty Acid (% Total FA)20:0 Arachidic Site KSLA0.300.2612.230.0060.29 - 0.300.22, 0.39Seed Vitamin (mg/100g dwt)Vitamin E Site ARNE1.782.16-17.890.0231.58 - 1.880.10, 2.85Vitamin E Site NEYO0.961.13-15.030.0270.94 - 0.990.10, 2.85Seed Secondary Metabolite (% dwt)Stachyose Site ARNE4.133.808.770.0213.94 - 4.332.45, 5.34Stachyose Site NEYO4.274.113.890.0094.24 - 4.292.45, 5.34Forage Proximate (% dwt)Moisture (% fwt) Site ARNE75.5372.633.990.00474.60 - 77.5065.61, 80.67Monsanto Company 11-SY-217U 100 of 467Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observedin More than One Individual SiteForage Proximate (% dwt)Moisture (% fwt) Site ILCY72.8574.70-2.480.00671.80 - 74.5065.61, 80.67Total Fat Site ILHI8.159.36-12.960.0087.44 - 8.620.54, 13.11Total Fat Site KSLA3.746.14-39.100.0013.41 - 4.030.54, 13.11Statistical Differences Observed in One SiteSeed Proximate (% dwt)Carbohydrates Site ILWY40.4639.223.170.02039.98 - 41.5032.36, 41.63Moisture (% fwt) Site INRC8.047.457.860.0217.91 - 8.115.41, 10.36Protein Site NEYO41.0540.142.27<0.00140.85 - 41.3635.06, 43.58Total Fat Site ILWY14.3515.35-6.520.03913.91 - 14.6013.15, 23.90Monsanto Company 11-SY-217U 101 of 467Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observed in One SiteSeed Fiber (% dwt)Acid Detergent Fiber Site ILCY15.3013.4613.690.00413.99 - 16.409.99, 22.21Neutral Detergent Fiber Site ILCY16.4214.5812.650.01415.64 - 17.2711.03, 23.27Seed Amino Acid (% dwt)Lysine Site NEYO2.682.622.200.0042.66 - 2.682.33, 2.81Proline Site NEYO2.021.972.570.0462.00 - 2.041.70, 2.13Serine Site NEYO2.192.123.470.0442.17 - 2.241.86, 2.33Tyrosine Site KSLA1.451.403.390.0351.41 - 1.481.28, 1.57Seed Fatty Acid (% Total FA)18:3 Linolenic Site ARNE8.068.39-3.860.0317.87 - 8.295.60, 11.61Monsanto Company 11-SY-217U 102 of 467Table VI-1. Summary of Differences (p<0.05) for the Comparison of Soybean Component Levels for MON 87712 vs.Conventional Control (continued)Mean Difference(Test minus Control)Analytical Component (Units)1MON 87712Mean2Control3MeanMeanDifference(% of Control)Significance(p-Value)TestRangeCommercialToleranceInterval4Statistical Differences Observed in One SiteSeed Fatty Acid (% Total FA)22:0 Behenic Site KSLA0.290.2322.350.0170.28 - 0.300.18, 0.43Seed AntinutrientTrypsin Inhibitor (TIU/mg dwt) SiteARNE28.4736.40-21.780.02924.54 - 34.9220.97, 50.01Seed Isoflavone (μg/g dwt)Glycitein Site ILWY117.8896.6821.920.005112.20 - 122.628.13, 299.671dwt = dry weight; fwt = fresh weight; FA = fatty acid; TIU = Trypsin Inhibitor Units.2Mean = least-square mean.3Control refers to the conventional control, A35254With 95% confidence,the interval contains 99% of the values expressed in the population of commercial substances. Negative limits were set to zero.Monsanto Company 11-SY-217U 103 of 467Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Proximate (% dwt)Ash4.99 (0.11)5.07 (0.11)-0.081 (0.069)-0.22, 0.0550.2384.43, 5.89(4.48 - 6.36)(4.46 - 5.71)(-0.78 - 1.42)(4.43 - 6.14)Carbohydrates37.83 (0.57)37.96 (0.57)-0.14 (0.25)-0.63, 0.350.58232.36, 41.63(34.64 - 41.50)(34.72 - 40.86)(-3.07 - 2.47)(33.43 - 40.39)Moisture (% fwt)8.01 (0.20)7.89 (0.20)0.12 (0.13)-0.13, 0.380.3455.41, 10.36(5.72 - 9.13)(6.44 - 10.30)(-1.69 - 1.84)(5.43 - 9.86)Protein40.93 (0.35)40.49 (0.35)0.44 (0.15)0.14, 0.750.00535.06, 43.58(38.87 - 42.79)(37.73 - 42.14)(-1.65 - 2.56)(35.11 - 42.16)Total Fat16.27 (0.44)16.48 (0.44)-0.21 (0.18)-0.58, 0.150.25013.15, 23.90(13.91 - 18.78)(13.91 - 19.05)(-2.38 - 1.47)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber15.54 (0.50)15.59 (0.49)-0.048 (0.45)-1.01, 0.910.9169.99, 22.21(12.94 - 19.11)(12.41 - 20.81)(-4.82 - 2.81)(11.74 - 22.13)Monsanto Company 11-SY-217U 104 of 467Table VI-2. Summary of Combined-site Soybean SeedNutrients for MON 87712 vs. Conventional Control(continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Fiber (% dwt)Neutral Detergent Fiber16.54 (0.42)16.76 (0.42)-0.23 (0.40)-1.09, 0.630.57911.03, 23.27(13.68 - 20.74)(13.40 - 20.37)(-2.65 - 2.76)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.77 (0.014)1.75 (0.014)0.023 (0.0081)0.0053, 0.0400.0141.54, 1.88(1.69 - 1.85)(1.65 - 1.83)(-0.055 - 0.073)(1.58 - 1.84)Arginine3.14 (0.036)3.07 (0.036)0.069 (0.028)0.0089, 0.130.0272.51, 3.33(2.93 - 3.38)(2.85 - 3.31)(-0.20 - 0.23)(2.57 - 3.24)Aspartic Acid4.73 (0.043)4.67 (0.043)0.061 (0.025)0.0069, 0.110.0294.04, 5.07(4.44 - 4.92)(4.36 - 4.89)(-0.16 - 0.24)(4.06 - 4.89)Cystine0.59 (0.0082)0.61 (0.0082)-0.019 (0.0043)-0.027, -0.010<0.0010.51, 0.67(0.55 - 0.63)(0.56 - 0.66)(-0.072 - 0.030)(0.54 - 0.69)Glutamic Acid7.60 (0.081)7.49 (0.080)0.11 (0.049)0.0068, 0.220.0386.28, 8.18(7.18 - 7.96)(6.86 - 7.97)(-0.37 - 0.38)(6.40 - 7.94)Monsanto Company 11-SY-217U 105 of 467Table VI-2 . Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Amino Acid (% dwt)Glycine1.79 (0.015)1.77 (0.015)0.021 (0.0082)0.0038, 0.0390.0201.52, 1.90(1.70 - 1.85)(1.66 - 1.84)(-0.041 - 0.081)(1.54 - 1.85)Histidine1.07 (0.0080)1.06 (0.0079)0.0089 (0.0057)-0.0033, 0.0210.1400.91, 1.17(1.03 - 1.12)(1.00 - 1.12)(-0.046 - 0.051)(0.93 - 1.16)Isoleucine1.90 (0.019)1.87 (0.019)0.029 (0.012)0.0028, 0.0560.0321.62, 2.03(1.78 - 1.98)(1.70 - 1.97)(-0.073 - 0.13)(1.60 - 2.00)Leucine3.15 (0.029)3.11 (0.029)0.035 (0.017)-0.00077, 0.0710.0542.71, 3.38(3.00 - 3.30)(2.90 - 3.26)(-0.12 - 0.13)(2.77 - 3.29)Lysine2.66 (0.019)2.64 (0.019)0.013 (0.011)-0.011, 0.0380.2632.33, 2.81(2.54 - 2.74)(2.49 - 2.75)(-0.11 - 0.074)(2.36 - 2.74)Methionine0.57 (0.0037)0.57 (0.0036)-0.0034 (0.0039)-0.012, 0.00500.4030.51, 0.59(0.52 - 0.60)(0.53 - 0.60)(-0.054 - 0.036)(0.51 - 0.60)Monsanto Company 11-SY-217U 106 of 467Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control(continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Amino Acid (% dwt)Phenylalanine2.15 (0.023)2.12 (0.023)0.034 (0.012)0.0071, 0.0600.0161.81, 2.33(1.98 - 2.26)(1.95 - 2.24)(-0.077 - 0.15)(1.81 - 2.25)Proline2.00 (0.018)1.99 (0.018)0.0060 (0.014)-0.022, 0.0340.6671.70, 2.13(1.83 - 2.14)(1.85 - 2.11)(-0.20 - 0.14)(1.69 - 2.09)Serine2.16 (0.019)2.15 (0.019)0.0080 (0.015)-0.023, 0.0390.5931.86, 2.33(2.01 - 2.25)(2.01 - 2.34)(-0.16 - 0.14)(1.90 - 2.30)Threonine1.59 (0.011)1.58 (0.011)0.015 (0.0098)-0.0056, 0.0360.1371.40, 1.69(1.51 - 1.66)(1.50 - 1.66)(-0.089 - 0.13)(1.36 - 1.68)Tryptophan0.44 (0.0052)0.45 (0.0051)-0.0074 (0.0062)-0.021, 0.00600.2560.36, 0.50(0.36 - 0.49)(0.38 - 0.49)(-0.094 - 0.10)(0.38 - 0.48)Tyrosine1.48 (0.014)1.46 (0.014)0.013 (0.012)-0.010, 0.0370.2671.28, 1.57(1.40 - 1.57)(1.33 - 1.56)(-0.095 - 0.18)(1.28 - 1.55)Monsanto Company 11-SY-217U 107 of 467Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control(continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Amino Acid (% dwt)Valine1.99 (0.017)1.95 (0.017)0.039 (0.014)0.0095, 0.0690.0131.71, 2.13(1.88 - 2.08)(1.78 - 2.07)(-0.072 - 0.15)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.48 (0.13)11.64 (0.13)-0.16 (0.045)-0.26, -0.0670.0027.76, 13.14(10.78 - 11.94)(11.13 - 12.23)(-0.44 - 0.091)(9.00 - 12.03)18:0 Stearic4.05 (0.099)4.17 (0.099)-0.13 (0.037)-0.21, -0.0460.0043.06, 5.10(3.54 - 4.72)(3.78 - 4.65)(-0.37 - 0.25)(3.49 - 4.97)18:1 Oleic20.82 (0.46)20.21 (0.46)0.61 (0.33)-0.093, 1.310.08317.37, 26.86(18.72 - 25.71)(18.72 - 21.54)(-1.57 - 4.66)(18.93 - 25.33)18:2 Linoleic54.01 (0.32)54.18 (0.32)-0.17 (0.25)-0.71, 0.370.51450.14, 57.81(50.26 - 55.60)(53.21 - 55.02)(-3.63 - 1.50)(51.57 - 56.25)18:3 Linolenic8.95 (0.27)9.07 (0.27)-0.12 (0.057)-0.24, 0.00160.0525.60, 11.61(7.46 - 10.22)(7.77 - 10.31)(-0.68 - 0.52)(5.89 - 10.16)Monsanto Company 11-SY-217U 108 of 467Table VI-2. Summary of Combined-site Soybean Seed Nutrients for MON 87712 vs. Conventional Control(continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.29 (0.0072)0.30 (0.0072)-0.010 (0.0060)-0.023, 0.00280.1170.22, 0.39(0.25 - 0.34)(0.25 - 0.34)(-0.077 - 0.049)(0.23 - 0.38)20:1 Eicosenoic0.14 (0.012)0.14 (0.012)-0.0080 (0.0057)-0.019, 0.00330.1630.094, 0.23(0.075 - 0.19)(0.073 - 0.19)(-0.082 - 0.071)(0.072 - 0.21)22:0 Behenic0.26 (0.0099)0.28 (0.0098)-0.014 (0.012)-0.039, 0.0110.2580.18, 0.43(0.18 - 0.33)(0.18 - 0.32)(-0.11 - 0.096)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E1.24 (0.15)1.37 (0.15)-0.13 (0.064)-0.27, 0.00250.0530.10, 2.85(0.81 - 2.05)(0.79 - 2.23)(-0.76 - 0.44)(0.86 - 2.73)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, A35255With 95% confidence, the interval contains 99% of the valuesexpressed in the population of commercial substances. Negative limits set wereto zero.Monsanto Company 11-SY-217U 109 of 467Table VI-3. Summary of Combined-site Soybean SeedAnti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)AntinutrientLectin (H.U./mg dwt)2.06 (0.14)2.22 (0.13)-0.17 (0.18)-0.54, 0.210.3630, 6.11(1.10 - 3.41)(0.58 - 4.15)(-1.84 - 1.63)(0.60 - 6.99)Phytic Acid (% dwt)1.18 (0.077)1.20 (0.077)-0.029 (0.043)-0.12, 0.0630.5130.50, 1.92(0.65 - 1.81)(0.86 - 1.77)(-0.58 - 0.42)(0.66 - 1.74)Raffinose (% dwt)0.73 (0.030)0.72 (0.030)0.0095 (0.022)-0.038, 0.0570.6740.39, 1.01(0.54 - 0.88)(0.52 - 0.93)(-0.17 - 0.14)(0.45 - 0.93)Stachyose (% dwt)4.28 (0.081)4.19 (0.081)0.092 (0.060)-0.037, 0.220.1472.45, 5.34(3.77 - 4.76)(3.52 - 4.88)(-0.41 - 0.90)(2.57 - 4.68)Trypsin Inhibitor (TIU/mgdwt)32.94 (1.09)34.14 (1.07)-1.20 (1.53)-4.23, 1.840.43520.97, 50.01(24.54 - 40.86)(20.34 - 54.88)(-23.03 - 14.32)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein1089.62 (85.86)1054.04 (85.80)35.58 (30.54)-29.88, 101.040.2630, 1756.99(549.34 -1609.37)(537.10 -1533.78)(-187.49 -384.84)(138.15 -1548.98)Monsanto Company 11-SY-217U 110 of 467Table VI-3. Summary of Combined-site Soybean SeedAnti-nutrients for MON 87712 vs. Conventional Control(continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Isoflavone (μg/g dwt)Genistein867.61 (58.80)832.31 (58.73)35.30 (26.14)-20.80, 91.410.19887.22, 1792.07(503.75 -1204.29)(508.77 -1175.72)(-219.09 -391.63)(335.67 -1409.07)Glycitein102.13 (4.02)100.86 (3.98)1.27 (5.47)-10.44, 12.990.8198.13, 299.67(75.71 - 131.79)(77.28 - 162.85)(-46.46 - 33.87)(66.83 - 280.71)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, A35255With 95% confidence, the interval contains 99% of the valuesexpressed in the population of commercial substances. Negative limits wereset to zero.Monsanto Company 11-SY-217U 111 of 467Table VI-4. Summary of Combined-site Soybean Forage Nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Proximate (% dwt)Ash6.56 (0.31)6.47 (0.31)0.089 (0.17)-0.27, 0.450.6064.29, 8.65(4.55 - 8.75)(4.79 - 8.74)(-2.60 - 1.62)(4.82 - 8.98)Carbohydrates65.10 (0.78)64.76 (0.78)0.33 (0.34)-0.35, 1.020.33255.73, 77.45(59.41 - 70.88)(58.14 - 70.63)(-3.97 - 5.67)(54.40 - 72.96)Moisture (% fwt)72.80 (0.92)72.01 (0.92)0.79 (0.42)-0.099, 1.680.07765.61, 80.67(67.60 - 77.50)(66.60 - 75.50)(-3.50 - 4.70)(64.50 - 79.80)Protein22.13 (0.51)21.75 (0.51)0.39 (0.22)-0.058, 0.830.08613.77, 26.51(18.45 - 26.20)(17.88 - 26.12)(-2.05 - 3.29)(16.56 - 27.76)Total Fat6.23 (0.56)7.03 (0.56)-0.80 (0.25)-1.32, -0.270.0050.54, 13.11(3.41 - 8.62)(4.57 - 9.80)(-3.15 - 1.00)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber29.72 (0.93)29.05 (0.93)0.67 (0.62)-0.67, 2.010.30123.12, 38.15(24.36 - 38.10)(24.46 - 35.21)(-5.70 - 11.81)(22.60 - 41.29)Monsanto Company 11-SY-217U 112 of 467Table VI-4. Summary of Combined-site Soybean Forage Nutrients for MON 87712 vs. Conventional Control(continued)Difference (Test minus Control)Analytical Component(Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceIntervalSignificance(p-Value)CommercialToleranceInterval5(Range)Fiber (% dwt)Neutral Detergent Fiber33.30 (1.16)32.58 (1.15)0.72 (0.77)-0.94, 2.370.36824.96, 43.33(26.56 - 41.73)(24.92 - 41.41)(-6.68 - 8.04)(25.78 - 44.41)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, A35255With 95% confidence, the interval contains 99% of the valuesexpressed in the population of commercial substances. Negative limits wereset to zero.Monsanto Company 11-SY-217U 113 of 467 

### Table VI-5. Literature and ILSI Databa se Ranges for Components in Soybean Forage and Seed

Seed Tissue Components 1 Literature Range 2 ILSI Range 3

Seed Nutrients Proximates (% dwt)

Ash 4.61 – 6.32 a ; 4.32 – 5.88 b 3.89 – 6.99 Carbohydrates by calculation 32.75 – 40.98 a ; 29.88 – 43.48 b 29.6 – 50.2 Moisture (% fwt) 6.24 – 12.10 a ; 5.44 – 11.70 b 4.7 – 34.4

Protein 34.78 – 43.35 a ; 32.29 – 42.66 b 33.19 – 45.48

Total Fat 14.40 – 20.91 a ; 15.10 – 23.56 b ; 15.5 – 24.7 4 8.10 – 23.56

Fiber (% dwt) Acid Detergent Fiber 9.22 – 26.26 a ; 11.81 – 19.45 b 7.81 – 18.61 Neutral Detergent Fiber 10.79 – 23.90 a ; 13.32 – 23.57 b 8.53 – 21.25

Amino Acids (% dwt)

Alanine 1.62 – 1.89 a ; 1.43 – 1.93 b 1.51 – 2.10 Arginine 2.57 – 3.34 a ; 2.15 – 3.05 b 2.29 – 3.40 Aspartic acid 4.16 – 5.02 a ; 4.01 – 5.72 b 3.81 – 5.12 Cystine/Cysteine 0.52 – 0.69 a ; 0.41 – 0.71 b 0.37 – 0.81

Glutamic acid 6.52 – 8.19 a ; 5.49 – 8.72 b 5.84 – 8.20 Glycine 1.59 – 1.90 a ; 1.41 – 1.99 b 1.46 – 2.00

Histidine 0.96 – 1.13 a ; 0.86 – 1.24 b 0.88 – 1.18

Isoleucine 1.59 – 2.00 a ; 1.41 – 2.02 b 1.54 – 2.08

Leucine 2.79 – 3.42 a ; 2.39 – 3.32 b 2.59 – 3.62 Lysine 2.36 – 2.77 a ; 2.19 – 3.15 b 2.29 – 2.84

Methionine 0.45 – 0.63 a ; 0.39 – 0.65 b 0.43 – 0.68 Phenylalanine 1.82 – 2.29 a ; 1.62 – 2.44 b 1.63 – 2.35

Proline 1.83 – 2.23 a ; 1.63 – 2.25 b 1.69 – 2.28

Serine 1.95 – 2.42 a ; 1.51 – 2.30 b 1.11 – 2.48

Threonine 1.44 – 1.71 a ; 1.23 – 1.74 b 1.14 – 1.86 Tryptophan 0.30 – 0.48 a ; 0.41 – 0.56 b 0.36 – 0.50 Tyrosine 1.27 – 1.53 a ; 0.74 – 1.31 b 1.02 – 1.61

Valine 1.68 – 2.11 a ; 1.50 – 2.13 b 1.60 – 2.20

Fatty Acids (% total FA) 8:0 Caprylic not available 0.148 – 0.148 10:0 Capric 0.15 – 0.27 b not available

12:0 Lauric not available 0.082 – 0.132 14:0 Myristic 0.063 – 0.11 b 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 – 12.63 b 9.55 – 15.77

16:1 Palmitoleic 0.055 – 0.14 b 0.086 – 0.194 17:0 Heptadecanoic 0.076 – 0.13 b 0.085 – 0.146 17:1 Heptadecenoic 0.019 – 0.064 b 0.073 – 0.087

18:0 Stearic 3.21 – 5.63 b 2.70 – 5.88

18:1 Oleic 16.69 – 35.16 b 14.3 – 32.2

18:2 Linoleic 44.17 – 57.72 b 42.3 – 58.8Monsanto 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 Components 1 Literature Range 2 ILSI Range 3

18:3 Gamma Linolenic not available not available

18:3 Linolenic 4.27 – 9.90 b 3.00 – 12.52

20:0 Arachidic 0.35 – 0.57 b 0.163 – 0.482

20:1 Eicosenoic 0.13 – 0.30 b 0.140 – 0.350

20:2 Eicosadienoic 0.016 – 0.071 b 0.077 – 0.245

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.35 – 0.59 b 0.277 – 0.595

Vitamins (mg/100g dwt)

Vitamin E 1.29 – 4.80 a ; 1.12 – 8.08 b 0.19 – 6.17

Seed Anti-Nutrients Lectin (H.U./mg fwt) 0.45 – 10.87 a ; 0.090 – 11.18 b 0.09 – 8.46 Trypsin Inhibitor (TIU/mg dwt) 20.79 – 59.03 a ; 18.14 – 42.51 b 18.00 – 108.00

Phytic Acid (% dwt) 0.41 – 1.92 a ; 0.81 – 2.66 b 0.63 – 1.96 Raffinose (% dwt) 0.26 – 0.84 a ; 0.43 – 1.85 b 0.21 – 0.66 Stachyose (% dwt) 1.53 – 3.04 a ; 1.97 – 6.65 b 1.21 – 3.50

Isoflavones ( μ g/g dwt) (mg/kg dwt)

Daidzein 224.03 – 1571.91 a ; 198.95 – 1458.24 b 60.0 – 2453.5

Genistein 338.24 – 1488.89 a ; 148.06 – 1095.57 b 144.3 – 2837.2 Glycitein 52.72 – 298.57 a ; 32.42 – 255.94 b 15.3 – 310.0

Forage Tissue Components 1 Literature Range 2 ILSI Range 3

Forage Nutrients

Proximate (% dwt)

Ash 5.28 – 9.24 a ; 4.77 – 8.54 b 6.72 – 10.78 Carbohydrates by calculation 62.25 – 72.30 a ; 60.61 – 77.26 b 59.8 – 74.7 Moisture (% fwt) 68.50 – 78.40 a ; 62.76 – 80.20 b 73.5 – 81.6

Protein 16.48 – 24.29 a ; 12.68 – 23.76 b 14.38 – 24.71

Total Fat 2.65 – 9.87 a ; 2.96 – 7.88 b 1.30 – 5.13

Fiber (% dwt) Acid Detergent Fiber 23.86 – 50.89 a ; 25.49 – 47.33 b not available Neutral Detergent Fiber 19.61 – 43.70 a ; 30.96 – 54.55 b not available

1 fw=fresh weight; dwt=dry weight; H.U. = hemaggl utinating 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 c onducted 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, c onducted in accordance with OECD guidelines,

also included measurement of these OECD de fined soybean nutrients and anti-nutrients in the conventional commercial soybean variet ies, 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 fo rage 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 we re 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 inthe combined-site analysis for antinutrients. Seed nutrient component differenc es were observed for mean values for protein, amino acids (nine com ponents), and fatty acids (two components), and the forage nutrient component difference was in total fa t and were evaluated using considerations relevant to the safety and nutritional qua lity of MON 87712 when compared to the

conventional control:

1) All nutrient component differences obser ved in the combined -site statistical analysis, whether reflecting increased or decreased MON 87712 mean values with respect to the conventional control A 3525, had small relative magnitudes when compared to natural variability, and therefore were not meaningful from a food/feed nutrition or safety perspect ive. The relative magnitude of the significant difference for protein when MON 87712 was compared to A3525 was 1.09%, and relative magnitudes of the diffe rences ranged from 1.22 to 3.07% for amino acids, from 1.42 to 3.04% for fatty aci ds, and a relative magnitude of the difference of 11.34% was observed for forage total fat.

2) Mean values for all significantly di fferent nutrient components from the combined-site analysis of MON 87712 were within the 99% to lerance interval

established from the conventional co mmercial reference varieties grown concurrently in the same trial, and were, therefore, within the range of natural variability of that component in commerc ial soybean varieties with a history of safe consumption.

3) Assessment of the reproducibility of the combined-site significant differences at the eight individual sites s howed significant differences for: protein at one site; alanine, aspartic acid, isoleucine, and valin e at two sites; arginine, glutamic acid, glycine, phenylalanine, and 18:0 stearic ac id 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 va lues of MON 87712 for all nutrient components withMonsanto Company 11-SY-217U 116 of 467 

### statistically significant differences were within the 99% tolerance interval

established from the conventional co mmercial reference varieties grown concurrently in the same trial and were, therefore, within the range of natural variability of that compone nt in commercial soybean with a history of safe consumption..

4) All combined-site mean values of M ON 87712 for all nutrient components were

within the context of the natural vari ability 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 sign ificantly different ( α <0.05) from that of the parental conventi onal 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 co mpared to the conventional control A3525, none of the 49 components asse ssed were observed to be significantly different at all eight indi vidual sites. All MON 87712 m ean component values were

within the 99% tolerance interval establ ished from the conventional commercial references varieties grown concurrently at the same field sites. Additionally, the combined-site component values were within th e range of values reported in the scientific literature and/or in the ILSI Crop Compositio n Database. Based on these data, it is concluded that harvested soybean seed a nd soybean forage produced from MON 87712 are compositionally equivalent to that of th e conventional soybean and that the presence

of BBX32 in MON 87712 does not have a me aningful 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, AGRO NOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides an assessment of th e phenotypic, agronomic, and environmental interaction characteristics including plant-sy mbiont associations, and volunteer potential and persistence outside cult ivation. These assessments were comparative between MON 87712 and the parental conventional control A 3525, a conventional soybean variety that has background genetics sim ilar to MON 87712 but does not possess the

BBX32 gene. The data support a determina tion 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 characte ristics of MON 87712 were evaluated in a comparative manner to assess plant pest potential. These assessments included evaluation of seed germination char acteristics, plant growth and development characteristics, observations of plant responses to abiotic st ress, plant-disease and plantarthropod interactions, pollen characteristics, plant-symbiont interact ion associations, and volunteer potential, and persiten ce outside of cultivation characteristics. Results from the phenotypic, agronomic, and environmental in teractions assessment demonstrate that MON 87712 does not possess a) increased weed iness characteristic s; b) increased susceptibility or tolerance to specific abio tic 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. Characteristic s Measured for Assessment

In the phenotypic, agronomic, and envi ronmental interactions assessment of MON 87712, data were collected to evaluate specific aspects of altered plant pest potential. A detailed description of the regulat ed article phenotype is requested as part of the petition for determination of nonregul ated status in 7 CFR § 340. 6 including differences from the unmodified recipient or ganism that would “s ubstantiate 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 detail ed phenotypic, agronomic, a nd environmental interaction description of MON 87712. A su bset of these data were in cluded in an evaluation of specific characteristics related to altered pl ant pest potential and adverse environmental impact.

The MON 87712 plant characterization and asse ssment encompassed seven general data categories: 1) seed germination, dormancy, a nd emergence; 2) vegetative growth; 3) reproductive development (including pollen char acteristics); 4) seed retention on the plant and lodging; 5) plant response to abioti c stress and interactions with diseases and arthropods; 6) plant-symbiont interactions and 7) volunt eer 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 cont rol 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 pheno typic, agronomic, and environmental

interaction characteristic. Commercial re ference varieties are developed through a process of selecting and breeding for variou s 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, Agronomi c, 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) Percen tage of seed producing seedlings

that could not be cl assified 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

test 2 )

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 th at imbibed water and

were firm to the touch but did not germinate (viability determined by a

tetrazolium test 2 ) 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) Pubescen ce 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% senescenceMonsanto Company 11-SY-217U 120 of 467 

### Table VII-1. Phenotypic, Agronomi c 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 usin g descriptions and guidelines outlined in Soybean Growth and Development (Pedersen, 2004).

2 Viability of hard and firm-swollen seed were de termined 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 ar e comparative assessments. Familiarity provides a basis from which th e 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 inter action of these factors, and provides a basis for comparative environmental risk assessm ent between a biotechnology-derived plant and its conventional counterpart.

Expert knowledge and experience with conve ntionally bred soybean was the basis for selecting appropriate endpoints and estimatin g 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, ag ronomic, and environmental interaction

characteristics. An overview of the characte ristics 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 ch aracteristics, and lodging) was us ed to assess whether there was

an increase in weediness potential of MON 87712, an el ement of Animal and Plant Health Inspection Service’s (APHIS) plan t pest determination. Evaluation of environmental interaction characteristics ( e.g ., plant abiotic stress, plant-disease, plantarthropod, and plant-symbiont in teractions) was also consider ed 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 pl ant pest risk compared to conventional soybean. Prior to analysis, the overall datase t was evaluated for evidence of biologically relevant changes, and for possible eviden ce of an unexpected plant response. No unexpected observations or issues were identified.

VII.B.1. Interpretation of Detected Differences Criteria

Comparative plant characteriz ation data between a biotec hnology-derived crop and the conventional control are interpre ted in the context of contribu tions to increased plant pest potential as assessed by APHIS. Under the fr amework of familiarity, characteristics for which no differences are detected support a conclusion of no increased plant pest potential of the biotechnology-derived cr op compared to the conventional crop.

Characteristics for which differences are dete cted are considered in a step-wise method (Figure VII-1) or in a similar method. All de tected differences for a characteristic are

considered in the context of whether or not the difference would incr ease the plant pest potential of the biotechnology- derived crop. Ultimately, a we ight of evidence approach considering all characteristics and data is used for the overall risk assessment of differences and their significance. In deta il, 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 th e 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 St atistically Significant Differences

Data on each measured characteristic are sta tistically analyzed, where appropriate, within

each individual-site and in a combined-site an alysis, 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 poten tial. Differences detected in individual-site analyses that are not consistently observed across multiple en vironments in the combined-site analysis are considered not biologically meaningful in terms of plant pest po tential and, therefore, are not further considered in subsequent step s. 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 dete cted in the combined-site analysis across multiple environments, then that mean va lue 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 1Monsanto Company 11-SY-217U 123 of 467 

### Step 4 - Evaluate Differences in the Context of the Crop

If the mean value of the biotechnology-deri ved crop is outside the variation of the commercial reference varieties ( e.g ., reference range), the mean value of the biotechnology-derived crop for th e characteristic is assesse d relative to known values common for the crop ( e.g. , published values).

Step 5 - Plant Pest Potential

If the mean value of the biotechnology-deri ved crop is outside the range of values common for the crop, the detected difference fo r 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.Monsanto Company 11-SY-217U 124 of 467 

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

Environmental Interaction Characteristics of MON 87712

This section provides the results of compar ative assessments condu cted in re plicated laboratory, greenhouse, and/or multi-site field experime nts to provide a detailed phenotypic, agronomic, and environmental in teraction description of MON 87712. The

MON 87712 characteristics evaluated in thes e assessments included: seed dormancy and germination characteristics (Section VII. C.1), plant phenotypic, agronomic, and

environmental interaction obs ervations under field conditions (Section VII.C.2), pollen characteristics (Section VII.C.3), symbiont interactions (Section VII.C.4), volunteer potential characteristics (S ection VII.C.5), and persiste nce 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 weedin ess 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 ar e available and routinely used to measure the germination characteristics of soybean seed. The Association of Official Seed Analysts (AOSA), an internationally recogni zed seed testing organi zation, recommends a temperature range of 20/30°C as optimal for germination of soybean (AOSA, 2007).

Comparative assessments of seed dormanc y and germination characteristics were

conducted on MON 87712 and the conventiona l control A3525. In addition, ten

commercial reference varieties were included to provide a range of comparative values that are representative of existing commercia l soybean varieties. The seed lots for MON 87712, its conventional control A3525, a nd the commercial re ference varieties were produced in replicated field trials at three sites during 2009 in Illinois and Missouri, geographical areas which represent environm entally 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 additiona l 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 i ndividual 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 si gnificant 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 varian ce for percent viable firm swollen seed andMonsanto Company 11-SY-217U 125 of 467 

### viable hard seed because there was no variabil ity present in the data. For these data, the

values of MON 87712 and the conventional cont rol A3525 were all ze ro, indicating no biological differences.

The biological characterist ics 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 th is study, particularly th e 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 Characterist ics of MON 87712 and the Conventional

Control A3525

Temperature Regime Germination

Characteristic 1 Mean % (S.E.) 2 Reference Range 3

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-s ite 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 seed s. 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 characteri zation assessment of MO N 87712. These data were developed to provide USDA-APHIS w ith a detailed description of MON 87712 relative to the parental conventional contro l A3525 and commercial re ference varieties. According to 7 CFR § 340.6, as part of the pe tition to seek deregula tion, a petitioner must submit “a detailed description of the phenot ype of the regulated article.” This information is being provided to assess whet her there are phenotypi c differences between

MON 87712 and the conventional control A3525 that may imp act its pest potential. Although all data from the plant characterization is considered in th e 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 biotechnol ogy-derived trait (e.g. increased yield with a yield trait). Thus, the environmental assessment takes acco unt of this when evaluating weediness, pest potential, or environmental impact. Ce rtain 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 cons idered 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 charac teristics. 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 replicat ions. All plots of MON 87712, the parental conventional control A3525, and the commercial re ference varieties with in each site were uniformly managed in order to assess whet her the introduction of BBX32 altered the phenotypic and agronomic char acteristics or the environmental interactions of MON 87712 compared to the conventional control A3525. A description of the evaluated phenotypic and environmental inter action characteristics and the designated developmental stages when evaluations occurr ed 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 intr oduction of BBX32 prot ein did not alter MON 87712 compared to the conventional control A3525 in terms of weediness potential. The lack of differences in plan t response to abiotic st ress, disease damage, arthropod-related damage, and pest and bene ficial arthropod abunda nce further support

the conclusion that the introduc tion of BBX32 is not likely to result in increased plant pest potential or an adverse environmen tal impact from MON 87712 compared to conventional soybean.Monsanto Company 11-SY-217U 128 of 467 

### VII.C.2.1. Field Phenotypic and Agronomic Characteristics

A total of 13 phenotypic and agronomic charac teristics 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 vigo r, days to 50% flowering, days to 50% end of flowering, plant hei ght, lodging, pod shattering, gr ain moisture, or 100 seed weight (Table VII-4). Five statistically significant differences were detected between

MON 87712 and the conventional control A 3525 in the combined-site analysis. MON 87712 had higher early st and count (302.7 vs. 297.0 plan ts 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 c ount (296.8 vs. 286.4 plants per plot), and higher yield (52.6 vs. 49.0 bu/ac) . Although significantly diffe rent 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 w ithin 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 physio logical maturity, and yield were consistent with the mode of action. The increase in yiel d is agronomically desirable and would not contribute to increased weediness poten tial of MON 87712 without changes in a

combination of other characteristics a ssociated with weediness (Baker, 1974). Furthermore, each difference, regardless of whethe r it is associated with the trait or not is assessed for pest potential and environmen tal impact. For each, it is unlikely that a difference in these characteristics (50% sene scence, days of physiological maturity, and yield) would contribute to increased weedin ess potential of MON 87712 compared to the

conventional control A3525.

Flower color and plant growth stage data we re categorical and we re 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 obser ved between MON 87712 and the conventional

control A3525.

The plant phenotypic and agronomic character istics evaluated were used to provide a detailed description of MON 87712 compar ed to the conventional control A3525. A

subset of these characteristics was usef ul to assess the weediness potential of MON 87712. Based on the assessed phenotyp ic 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 envir onmental impact than conventional soybean.Monsanto Company 11-SY-217U 129 of 467 

### Table VII-3. Field Phenotypic E valuation 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 subs tantial 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 Range 1

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% flowering 2 209.0 (1.0) 208.9 (1.0) 190.5 215.9

Days to 50% end of flowering 3 231.7 (1.2) 231.5 (1.1) 215.3 239.0

Days to 50% senescence 4 267.7 (0.8)* 265.0 (0.8) 251.6 270.8

Days to physiological maturity 5 280.8 (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 A3 525 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 di fferences 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 in teraction of the biotechnology-derived crop compared to its conventional control to determine the potential for increased plant pest

characteristics. Evaluations of environmenta l 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 charac teristics 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 envir onmental consequences (Section IX) to assess plant pest potential an d 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 environmenta l interactions evaluation included data collected in the phenotypic studies (plant-ins ect, plant-disease, and plant-environmental interactions). Data was analyzed on an indi vidual 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 so ybean 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 prot ein is unlikely to result in an increased plant pest potential or cause an advers e 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, a nd arthropod abundance was as sessed quantitatively from collections performed four times during the growing season at three of the 19 sites ( i.e., ARNE, ILSE, and MOCB). The assessed stre ssors (abiotic, diseases, and arthropods)

were at natural levels as no artificial infest ation 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 numerical ly and then placed into one of the following cate gories: none, slight, moderate, or severe. Thes e categorical data were not subjected to statistical analysis. The response of MON 87712 and the

conventional control A3525 to an abiotic stress or disease we re considered different on a particular observation date at a site if th e range of injury seve rity 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 da mage. These numerical data along with the quantitative arthropod abundance data were subjected to statistical analysis.Monsanto Company 11-SY-217U 132 of 467 

### In an assessment of plant response to abiotic stressors and disease damage, no statistically significant differences were detected be tween MON 87712 and the conventional control A3525 for any of the 384 comparisons whic h included 186 abiotic stressors and 198 disease damage comparisons (Tables H-6 and H-7).

In an assessment of arthropod-related dama ge, no statistically si gnificant differences

were detected between MON 87712 and the conventional control A3525 for 129 out of 137 comparisons among the observations across a ll sites (Table H-8). In addition, no

numerical differences were observed for the 55 comparisons for whic h p-value could not be generated due to lack of variability in the data. The mean damage ratings were slightly higher than the respective refere nce ranges for eight detected differences. However, the differences detected were sma ll in magnitude and these differences were

not consistent across observations or sites. Thus, these differe nces were not indicative of a consistent plant response associated with the trait and are unlikely to be biologically meaningful in terms of an adverse envir onmental impact of MON 87712 compared to conventional soybean.

In an assessment of pest and beneficial ar thropod abundance, no stat istically 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 obs erved for the eight comparisons (including six pest arthropod comparisons and two bene ficial 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 respec tive 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 ra nge; however, these differences were not

consistent across collections or sites. Thus, these differenc es were not indicative of a consistent plant response associated with th e trait and are unlikely to be biologically meaningful in terms of an adverse envir onmental impact of MON 87712 compared to conventional soybean.

The results of the field evalua tions 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 plan t responses to abiotic stress, disease damage, arthropod-related damage, a nd 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 po tential or an adverse environmental impact compared to conventional soybean.Monsanto Company 11-SY-217U 133 of 467 

### VII.C.3. Pollen Characteristics

USDA-APHIS considers the potential for gene flow and introgression of the biotechnology-derived tr ait into other soybean varietie s and wild relatives. Pollen morphology and viability information are per tinent to this assessment and, therefore,

were assessed for MON 87712. In additi on, characterization of pollen produced by

MON 87712 and the conventional control A3525 is relevant to th e 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 as sess 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 conventio nal 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 complete d 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, a nd the commercial re ference 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 cont rol A3525. These results demonstrate

that the introduction of BBX32 did not alte r the overall morphology or viability of MON 87712 pollen compared to the conve ntional control A3525. The pollen

characterization data contribute to the detailed phenotypic desc ription 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 pl ant 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 Range 2



### 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 dete cted 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 maxi mum 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 pl ant pest potential and the e nvironment as well as on agricultural or cultiva tion practices compared to its conve ntional counterpart. Potential changes in the symbiotic relationship wi th 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 sy mbiotic 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-micr obe symbiosis results in the formation of root nodules, which provide an environment in which differentiate d bacteria called bacteroids are capable of re ducing or “fixing” atmospheri c nitrogen. The product of nitrogen fixation, ammonia, can then be utilized by the plant. As a result of this relationship, nitrogen inputs ar e typically not necessary fo r 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 an d nitrogen status ar e 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 contro l A3525, and six commercial reference varieties were produced from seeds planted in pots co ntaining nitrogen- deficient potting medium and grown in the greenhouse. S eeds were inoculated with a solution of B. japonicum . The pots were arranged in a randomi zed complete block design with eight replicates. At six weeks after emergence, pl ants 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 enumer ation and determination of dry weight. MON 87712 was compared to the conventiona l control A3525 for key characteristics related to their association with the soybean - B. japonicum symbiosis. Detailed

information on materials and methods used fo r 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 meas ured parameter, including nodule number, shoot percent total nitrogen, shoot total nitr ogen (g), and dry we ight of nodules, shoot material, and root material (Table VII-6).Monsanto Company 11-SY-217U 136 of 467 

### Based on the assessed characteristics, th e 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 conventi onal soybean. Thus, these data further support a conclusion of no change in plant pest potential and no e xpected impact to cultivation practices relative to nitr ogen inputs for MON 87712 compared to conventional soybean.Monsanto Company 11-SY-217U 137 of 467 

### Table VII-6. Symbiont Interaction A ssessment of MON 87712 and Conventional

Control A3525

Measurements Mean (S.E.) * Reference Range 1

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 replic ated blocks (n = 8) in a greenhouse using a randomized completed block design. S.E. = Standard Error.

* 

### No significant differences were detect ed between MON 87712 and the conventional control A3525 ( α =0.05).

1 

### Reference range is the minimum and ma ximum mean value observed among six

commercial reference varieties.Monsanto Company 11-SY-217U 138 of 467 

### 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-w inter and volunteer in the s ubsequent cropping season. The purpose of this study was to assess the volun teer potential of MON 87712 compared to the near isogenic conventional control A 3525. 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 cont rol A3525. In addition, 4 commercial

reference varieties were included as references at each site. It was determined that the seed were acceptable for evaluating voluntee r potential based on standard viability testing. Normal seed germination ra tes were confirmed for MON 87712, the conventional control A3525, and commercial re ference 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 ra ndomized complete block design with four replications. Each plot wa s 5-8 ft wide by 20 ft long a nd was hand-seeded by uniformly scattering approximately 400 seeds on the soil surface. Seeds were then mechanically incorporated to a maximum depth of appr oximately 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. Volunt eer plant counts were taken every two weeks after planting until the environmental conditions were no longer conducive for germination and emergence. Monitori ng 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 an alyzed on an individu al site basis; no combined site analysis was conducted.

No volunteer plants were observed at any si te or observation time during the fall of 2009. No volunteer plants were observed at any ob servation 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 cons istent across locations. Based on the assessed data, the results of this study s upport a conclusion that the introduction of BBX32 did not alter the ability of MON 87712 to volunteer compared to conventional soybean. Furthermore, these results dem onstrate 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 139 of 467Table VII-7. Observed Volunteer Soybean Plants of MON 87712 Compared to the Conventional Control A3525 andReferences in a 2009/2010 U.S. Field TrialSite1SeasonObservation Dates2Number of Volunteers Reference range3MON 87712ControlMin. Max.ARAU Fall 12/07/09, 12/21/090 0 0 0Spring4/22/10, 5/06/10, 5/20/10, 6/03/10,6/17/100 0 0 0ARNE Fall 12/07/09, 12/21/090 0 0 0Spring4/22/10, 5/06/10, 5/20/10, 6/03/10,6/17/100 0 0 0ILWY Fall 12/07/090 0 0 0Spring4/08/10, 4/22/10, 5/06/10, 5/20/10,6/03/10, 6/17/103 0 0 7MOAN Fall None- - - -Spring3/19/10, 4/02/10, 4/16/10, 4/28/10,5/12/10, 5/26/10, 6/09/100 1 0 1MeanAcross0.75 0.25 0.0 2.0Note: No statistical analyses were performed due to lack ofvariability. Numbers shown are total numbers of volunteers for each substance across allfour replications at each specific site (n=4).1ARAU = Woodruff County, AR; ARNE = Jackson County, AR; ILWY = Stark County, IL; MOAN = Shelby County, MO.2Observations were made approximately every two weeks.3Minimum 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 Excel2007.Monsanto Company 11-SY-217U 140 of 467 

### VII.C.6. Persistence Outside of Cultivation Assessment

Weediness or invasiveness may also be indicat ed if soybean exhibited an increased rate of persistence outside of cultivation. The pur pose 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 agricu ltural inputs a llowing 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 enco mpassing plant growth, development, and seed production were assessed for MON 87712, th e parental conventional control A3525, and the commercial reference varieties in unmanaged environments. The experiment was

established at each of four sites in a ra ndomized complete block design with three replications. Stand count, growth stage, an d plant vigor was evalua ted 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 calculat ed and used to evaluate the ability of MON 87712, the conventional control A3525, and the commercial reference varieties to persist outside of cultivation. Each replacemen t value is the ratio of number of seeds produced to the number of seeds sown. A re placement 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 replacem ent 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, invasi veness, or potential for soybean to persist outside of managed agricultural environments.Monsanto Company 11-SY-217U 141 of 467 

### VII.D. Conclusions for Phenotypic, Agro nomic, 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 cont rol 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 incl uded 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 poten tial and persistence out side of cultivation

characteristics. Results from the phenotypic, agronomic, a nd environmental interactions

assessment demonstrate that MON 87712 does not possess a) weedines s characteristics; b) increased susceptibility or to lerance to specific abiotic stress, disease, or arthropods; or c) characteristics that would confer a plant pe st risk or significant environmental impact compared to conventional soybean.Monsanto Company 11-SY-217U 142 of 467 

### VIII. U.S. AGRO NOMIC PRACTICES

VIII.A. Introduction

As part of the plant pest assessment re quired 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. fo r producing soybean and is included in this petition as a baseline to a ssess possible impacts to agricultural practices

due to the cultivation of MON 87712. Di scussions 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 pr esented in Section VIII.C.2 demonstrated that MON 87712 produced more yield per s oybean plant and has slightly delayed maturity compared to conventional soybean. However, MON 87712 is no more susceptible to diseases or pests than c onventional soybean and required no additional inputs to produce a crop from MON 87712, a nd no specific impacts were noted to agronomic practices employed for production of soybean. In the areas where there is potential for impact on agronomic practi ces 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 seedbe d 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 th e 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 pl ants with different genetic backgrounds and selected traits resulting in higher yields, compositiona l improvements, and desirable production traits. Biot echnological approaches complement traditional s oybean breeding efforts by targeting the same major char acteristics as traditional breeding.

Crop yield results from a sequ ential growth and developmen t 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 br eeding. The genetic changes that resulted in crop domestication and yield impr ovement in conventional vari eties have been shown by modern molecular biology analys is to have been typically ac hieved through the selection and safe use of plant genes encoding transcriptional regula tor proteins. Agricultural biotechnology provides the opportunity to fu rther 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 steadi ly 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 a nd oil contents, industria l 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 av erage 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 (M orrison 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 wa s conducted on data from the USDA National Agricultural

Statistics Service (USDA-NASS, 2011b).

Soybean yield trends in the U.S. indicate th at yield growth rate s have not reached a plateau. Average soybean yi eld 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 possi ble. 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 st resses effectively controlled (Evans and Fischer, 1999; Specht et al., 1999). Conve ntional soybean yield potential has beenSeedYield(bu/A)

Production yearMonsanto 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. averag e 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 aver age annual yield performance and yield potential. Actual yield performance is a comp lex outcome that is dependent on a number of genetic and environmental factors that in fluence a crop’s opportunity to realize its full yield potential. Growers are accustomed to ex periencing field-to-field and year-to-year yield variation based on environmental conditi ons 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 impr ovements such as genetic and agronomic innovations that provide producers with means for reducing “on-farm” yield c onstraints (Specht et al., 1999). Agricultural production depends on con tinuing infusions of genetic resources for yield stability and growth (Day-R ubenstein and Heisey, 2006).

Increased soybean production in the U.S. ha s been accomplished by both increasing the

area under cultivation and thr ough 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 chal lenge 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 pr oduction 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 18 th 

### century (Hoeft et al ., 2000b). During the 1930s, soybean started to be processed industria lly 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 pro ducts 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 larg est 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 gr owing soybean are very similar to corn. The soils and climate in the Midwestern, Easter n, 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 requireme nt for a high-yielding soybean crop is approximately 20 inches of wa ter during the growing season (Hoeft et al., 2000b). SoilMonsanto Company 11-SY-217U 145 of 467 

### texture and structure are key components dete rmining water availability in soils, where medium-textured soils hold more water, allo wing soybean roots to penetrate deeper in medium-textured soils than in clay soils. Ir rigation is used on appr oximately 9% of the soybean acreage to supplement the water s upply during dry periods in the Western and Southern soybean growing re gions (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 sy stem following winter wheat south of 35o North latitude (Wilcox, 2004). Howe ver, this percentage can va ry significantly from year to year. The decision to plant double-crop soybean is influenced by both agronomic and economic factors. Agronomic factors incl ude harvest date of the wheat crop, which determines the double-crop soybean planting date , and available soil mo isture. Economic factors include expected soybean price and anticipated economic return (Heatherly and Elmore, 2004).

The U.S. soybean acreage in the past 10 year s has varied from a pproximately 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 vari ed 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 V III-1). Soybean acreage and production in 2008 was up from 2007, mainly due to a decrease in corn ac reage. 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, re spectively (USDA-NASS, 2011a).

For purposes of this agronomic practices di scussion, soybean production is divided into three major soybean growing regions Midwes t/Great Plains region (IL, IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, SD, and WI), Sout heast region (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern Coastal regi on (DE, MD, NJ, NY, PA, and VA) (Table VIII-2). The vast majority of soybean wa s 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 averag e 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 fertiliz er to apply, and what herbicide program to use. The total operating cost for produc ing soybean in the U.S. in 2010 was $132.29 per acre, according to statistics compiled by th e USDA-Economic Research Service (USDAERS, 2011). The value of the production less operating cost was reported to be $317.03Monsanto 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’ decisi ons 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 partic ular 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 opera tion, plus all the operating costs associated with that crop.

Figure VIII-2 shows the average per acre ne t 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 a nd 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 operati ng cost is seed (USDA-ERS, 2011). As the data show, farming is often not profitable wh en 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), ma ny factors influence both operating costs and expected prices. Go vernment price supports can have a large effect on costs, and supply and demand governs prices.Monsanto Company 11-SY-217U 147 of 467 

### 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. Bu reau of Labor Statistics 2011



### Table VIII-1. Soybean Production in the U.S, 2001 – 2010 1



### 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 2005NetValue(dollarperacre)

Year Soybean

Corn

WheatMonsanto 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)Monsanto Company 11-SY-217U 149 of 467 

### Table VIII-3. U.S. Soybean Production Costs and Returns in 2010 1



### 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 sim ilar characteristics are cl assified as varieties. Historically breeders have deve loped desirable varieties by re taining for further breeding those plants that possess the desirable traits , as determined by visual inspection or by testing. In recent years breeders have used th e more direct methods of molecular breeding techniques, such as marker assisted breedi ng, to accelerate the pr ocess of identifying breeding lines containing a desired set of pos itive traits. These techniques rely on inventories of genomic re gions or genetic markers that have been positively associated with the desirable traits. Once the genetic markers a ssociated with the desired traits have been identified, molecular breeders can quickly se lect the offspring inheriting the genes for further development (Voosen, 2009).

Hundreds of soybean varieties ar e tested each year in performa nce trials (variety trials) conducted by universities and pr ivate companies in all the ma jor soybean growing states. The following information can typically be obt ained from the results of variety trials: maturity group, disease resistance, insecticide seed treatment, yield, maturity date, percent lodging (plants fallen over on th e 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 tolera nce or protein or oil content {Pedersen, 2008 #56136}.

Soybean is a self pollinating crop and the va st majority of commercial soybean currently under production was produced thro ugh 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 segregat ing 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. agricultu re. By the early 20t h 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 toMonsanto 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 we re 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 pres ence 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 agenci es to ensure that purchasers who received

certified seed could be assured that the se ed met established seed quality standards (Bradford, 2006). The federal government passed the U.S. Fe deral Seed Act of 1939 to recognize seed certification a nd the establishment of offi cial certifying agencies. Regulations first adopted in 1969 under the Fede ral Seed Act recognize land history, field isolation, and varietal purity standards for f oundation, registered, and ce rtified 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 Offici al Seed Certifying Agencies (AOSCA) represents state and private seed certification organizations in the U.S.,

and includes international memb er countries in North and South America, Australia, and

New Zealand.

Soybean seed is separated into four seed clas ses: 1) breeder, 2) foundation, 3) registered, and 4) certified (AOSCA, 2009). Breeder seed is seed directly controll ed by the originating or sponsoring plant breeding organization or fi rm. 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 f oundation seed that is handled to maintain satisfactory varietal purity and identity. Certified seed is the progeny of breeder, foundation or register ed seed, and is typically two generations removed from foundation seed. While not all soybean seed so ld to growers is o fficially 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 impr oved soybean varieties quickly deteriorated in the absence of monitoring for quali ty 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 see d. There are special land requirements, seed stock eligibility requirements, field inspect ions and seed labeling standards for seed

certification. Seed certific ation services are available through various state agencies affiliated with AOSCA. Large seed producers implement their own seed quality assurance programs. However, large seed producers ofte n will utilize the services of state certifying agencies as a third party source to perfor m certain field inspections and audits.

U.S soybean production for all purposes has vari ed from approximately 64.7 to 77.5 million acres in the past ten years (U SDA-NASS, 2011c). To plant th is 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 exce ss, seed returns, replants, and potential increases in soybean acreage. Assuming an average soybean yield of 45 bushels, or 54 unitsMonsanto Company 11-SY-217U 152 of 467 

### (50 lbs/unit) per acre, 1.9 to 2.3 million acres w ould be required to produce this volume of commercial certified soybean seed each year.

Certified soybean seed is pr oduced throughout most of the U. S. soybean-growing regions. Soybean varieties are developed and adapted to certain geographical zo nes 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 comp anies that produce and sell seed, such as Monsanto Company, Pioneer Hi-Bred Internatio nal, Syngenta Seeds, Kruger Seed Co., and Becks Hybrids. In addition, certified seed is produced by toll seed pr oducers, or tollers, which are companies that produce but do not dir ectly 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 soyb ean seed. Seed produ ction 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 soyb ean seed as well as monitor and inspect all the processes at the plant. Production plan ts typically produce between 100,000 units to 2,000,000 units of soybean seed. Production plants will produce the various soybean

varieties in different clim ates 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 Standard ization (ISO) certifica tion standards and; therefore, include inte rnal and external audi ts (ISO, 2009). ISO standards ensure desirable characteristics of seeds and services, such as quality, safety, reliabilit y, and efficiency. The ISO standards represent an international c onsensus on good management practices with the aim of ensuring that the organization can consis tently 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 obj ectives (ISO, 2009).

The field operations and mana gement 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 germinatio n rates, and high genetic purity (Helsel and Minor, 1993). General guidelines specific fo r 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 poten tial volunteer soybean plants (even though the risk of soybean volunteer pl ants 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 Mi nor, 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 fungici des should be considered when disease infestations areMonsanto Company 11-SY-217U 153 of 467 

### predicted in the area. Harves t should occur as soon as the mature soybean seed reaches 13% moisture content. Harvesting soybean seed with le ss than 13% moisture can cause damage to the seed coat and result in split soybean se ed that can affect germination and viability. Harvesting equipment must be adjusted to mi nimize or avoid seed damage. Harvesting equipment must be cleaned before entering the s eed fields to assure genetic purity. Certain seed handling equipment, such as auger elev ators, should be avoided because they can increase seed damage.

Field inspections are vital to ensure the soyb ean seed meets seed certification requirements, ISO certification standards, regulatory standard s, and trait licensing agreement standards. Field inspections are conducted on seed produc tion fields throughout the soybean growing season to visually evaluate variety purity, en sure 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 federa l isolation requirement of five feet as a physical barrier (AOSCA, 2009). So me states and seed producer s 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 baggi ng process. Specific methods are used to assure the genetic purity and the identity of th e seed is maintained throughout the handling and storage operation. Bin insp ections and sample collecti ons are conducted at storage locations at the seed producti on plant to examine the physical characteristics of the soybean seed and to ensure proper bin cleanout. S eed is inspected for appearance, disease, discoloration, seed coat, mechanical damage, in ert 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 tetrazoliu m staining tests to asse ss 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 (max imum), 0.05% weed seed (maximum, not to exceed 10 per lb.), 0.60% total of other cr op seeds (maximum), 0.5% other varieties (maximum, includes off-colo red beans and off-type se eds), 0.10% other crop seeds (maximum, not to exceed thre e per lb.), and 80% germinati on and hard seed (minimum) (AOSCA, 2009). State seed certif ication standards vary slightly from stat e to state and can

be more restrictive than the seed standards of AOSCA.

When deregulated, MON 87712 seed will be pr oduced in the same manner as commercially certified soybean seed, such that it will meet al l state and federal seed standards and labeling requirements.

VIII.D. Production Management Considerations

VIII.D.1. Pre-Season

Well in advance of planting a s oybean crop, decisions are made regarding the planned crop rotation, the tillage sy stem and row spacing that will be implemented, the planting equipment that will be used, the seed or vari ety that will be planted, and soil fertilityMonsanto Company 11-SY-217U 154 of 467 

### management requirements. Many of the decisi ons in this area are made prior to or immediately after harvest of the previous cr op. There are many bene fits to crop rotation, with the majority of the soybean acreage plante d in a two-year corn-s oybean 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 pa rticular economic or market opportunity. The decision to plant soybean in a conservation ti llage or no-till system may require special equipment and will be made long before plan ting. 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 lo ng-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 cost s, 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 emergen ce 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 cold er in no-till than conventional tillage systems, which can slow seedling emergence, but have little effect on soybean yield (Pedersen, 2008a). Improved planters for establishmen t of good soybean popula tions and planting Roundup Ready soybean allowing the use of glyphos ate to effectively control weeds in notill fields have made no-till a viable produc tion 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 soybe an, respond well to fertilizer when planted in soils with low fertility levels. Soybean requires 16 essen tial elements for growth and development. Deficiencies in any of these elements can re duce yields (Hoeft et al., 2000b). The primary or major essential nutrients are nitrogen, phosph orus 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 relations hip with the nitrogen- fixing Bradyrhizobia bacteria ( Bradyrhizobium japonicum ) that live in the nodules on its roots. Bradyrhizobia are unicellar, microscopic bacteria th at invade the soybean plant th rough its root hairs (Hoeft et al., 2000b). The plant responds to this invasi on by forming nodules wh ich contain colonies of bacteria. Once established on the soybean root, bacteria in the nodule take gaseous nitrogen from the atmosphere and fix it in fo rms easily used by the so ybean 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 decr ease 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 determin e 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 so ybean (Scott and Aldrich, 1970). In corn-soybean rotations in the Midwest, phosphorus and pot assium fertilizers are appliedMonsanto Company 11-SY-217U 155 of 467 

### prior to a corn crop in accord ance with soil test recommenda tions, 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 fertili zer application prior to planting soybean.

Although not common, deficiencies in soil ca n occur in secondary nutrients (calcium, magnesium, and sulfur) or micronutrients (boron, chloride, c opper, iron, manganese, molybdenum, and zinc). The availability of soil nutrients is dependent on soil acidity or pH level. Because soybean is adversely affect ed when the pH is below approximately 5.8 (Hoeft et al., 2000b), soil pH should be main tained at about 6.0 to 6.5 through the addition

of limestone.

Because soybean growth is so dependent on day/night length, diffe rent varieties are developed for different latitudes. In the U.S. ten geographically -designated “maturity groups” originally defined by Scott and Aldric h (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 th e major maturity groups. These are designated by a decimal value. For example, a variet y with maturity gro up 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 no rth and ending with Group VIII in the far

south. 4 

### Groups 00 and 0 are the earliest maturity groups and are adapte d 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 pl anted in the Midwest and Eastern Coastal regions. Groups II, III and IV, whic h extend from approxim ately the northern border of Iowa to the southern tip of Illinois, account for approximately 76% (24%, 36%, and 16%, respectively) of th e soybean planted in the U. S. (T. Schlueter, personal communication, August 2008). Because day lengt h delays maturity, a soybean cultivar suited to a southern maturity group would ma ture too late if planted too far north. Conversely, a northern cultivar would mature too early if pl anted in the south (Heatherly and Elmore, 2004).

Soybean variety selection is crucial for high yield and qualit y, and is the foundation of an effective management plan (Pedersen, 2008b). Ch aracteristics to consider in selecting a variety include maturity, yield potential, di sease and pest resistance, iron deficiency tolerance (chlorosis), lodging sc ore, height, and specific soyb ean quality traits, such as protein and oil content. If a field has a history of a partic ular disease or pest, planting soybean varieties that have resistance or tole rance to these pests a nd 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 herbicid e and insecticide applic ations. 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, inse ct pests, and diseases on soybean yield is dependent on growth stage. The system of soybean growth stages divides plant development into vegetative (V) and repr oductive (R) stages (P edersen, 2004). The vegetative stages begin with VE, which design ates emergence. V stages continue and are numbered according to how many fully deve loped 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 va rious growth stages in soybean are highly dependent on photoperiod (hours of daylight a nd darkness) and temperature (Major et al., 1975), and therefore, for the same soybean plant grown at different lat itudes, 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 lo nger (and days grow shor ter) than a critical length (Holshouser, 2010). Once flowering begins, temperatur e 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 th at 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 sp ring, soil temperature s hould not be the only criteria for optimum planting time. Planti ng into a good seedbed is the most important consideration. Planting into soil that is t oo wet will reduce emergence and plant population, and can lead to reduced yield.

Planting date has the greatest impact on yiel d, according to research conducted in the Northern states (Hoeft et al., 2000b). Highest yields are genera lly obtained wh en planting in early to mid May. Yields begin to decline qui te 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., pl anting 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 ea rly 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 yiel d. Row spacing is important to maximize soybean yield. Research in the Midwest over th e 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 bene ficial. In 2000, approxima tely 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 ov er a wide range of plant populations. Most soybean varieties have the ability to branch a nd adjust the number of pods on branches to compensate for large differences in seedi ng rate. Maximum yiel ds generally require planting rates that result in a bout 2.5 to 5 plants per square foot (Hoeft et al., 2000b). Therefore, a full stand of soybean is approximately eight to te n plants per foot of row at harvest for 40-inch rows, six to eight plants pe r 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 harv est. 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 loss es in germination, emergence, and seedling diseases. The accuracy of the planting equipm ent also can impact the decision on seeding rate. Soybean seed traditionally has been sold by weight. Th erefore, the grower must know the number of seeds per pound for the particular soybean variet ies being planted for acc urate 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 greates t 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 b ecause 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 an d 85oF (Hoeft et al., 2000b). Warmer temperatures result in larger plants and earlier fl owering. 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 va rieties adapted to the north. The extent of vegetative grow th occurring after the initiatio n of flowering depends not only on environmental factors but also the growth habit. Soybean varieties are described as

either indeterminate or determinate in thei r 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 af ter 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 f ourth or fifth node andMonsanto Company 11-SY-217U 158 of 467 

### progress upward. Flowering on determinate plan ts begins at the eight or tenth node and progresses both downward and upward.

The first appearance of flowers signals the be ginning of the reproductive stage, namely the R1 stage (Hoeft et al., 2000b). The reproductiv e 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 flow ers. 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 Aldric h, 1970). This charac teristic makes soyb ean less susceptible than corn to short periods of adverse weat her 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 hi gh yields (Hoeft et al., 2000b). Another critical requirement duri ng the seed-filling stages is a high rate of photosynthesis to maximize yield. High humidity and temperat ures during seed deve lopment and maturity can result in poor seed quality because th ese 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 mois ture 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 artifici al 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 stor age and transportation system for the crop. Because of the very high variability in crop production, storage fa cilities are not always adequate. Soybeans and other grain must sometimes be stored in temporary structures or in other existing buildings if storage facilities ar e overloaded, and this may result in additional costs for constructing or renting temporary f acilities and/or potential losses from exposure (Dorn, 2011; Hellevang, 1998). The same conditi ons can result when prices are low and growers want to hold on to their crops in th e 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, soybean s rarely are (Hurburgh, 2005). University extensions provide practical gu idelines for temporary storage of soybeans and other crops (Dorn, 2011; Harner et al., 1998; Helleva ng, 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 elev ator 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 soybean s or oil and meal are shippe d by rail, barge or truck. Approximately 24% of soybeans are transpor ted by rail, although hi gher percentages of meal and soybean oil are trans ported 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 sp ecifics were provided (Ash et al., 2006). Large crops can result in greater shipping competition and higher shipping costs, which translates to lower prices offere d 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 du ring the growth and development of soybean is important for protecting the yield of soybean (Aref and Pike, 1998b). Unders tanding the impact of insects on soybean growth is essential for proper ma nagement (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 re ceives an insecticide treatment (USDA-NASS, 2007).

Characterizing soybean responses to insect in jury 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-feedi ng, stem-feeding, leaf-f eeding, 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 de foliation 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 wi thout significant yield loss. By contrast, defoliation during the flowering and pod filling stag es poses a greater threat to yield because the soybean plant has less time to compensate fo r 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 leve l before yield is impacted.

VIII.F. Management of Diseases and Other Pests

More than 100 pathogens are known to affect so ybean, 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 pa rts of the soybean plant,Monsanto Company 11-SY-217U 160 of 467 

### resulting in reduced quality and yield. The ex tent of losses depends upon the pathogen, the state of plant development and health when in fection occurs, the severity of the disease on individual plants, and the number of plants affected (Bowers and Russin, 1999).

One or more diseases can gene rally be found in fields wher ever 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 ex tent and severity of soybean diseases depend on the degree of compatibility between the hos t and the pathogen and the influence of the

environment.

According to field surveys conducted in fift een 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 importa nce. As expected, yield losses varied by region. Sclerotinia stem rot caused yield losses in seve ral 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 t ool growers have for disease control (Bowers and Russin, 1999). Resistant varieties ma y have morphological or physiological characteristics that provide immunity, resi stance, tolerance or avoidance to certain pathogens. Cultural practices can also play an important role in di sease 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 r outinely recommended as a disease-management strategy. Rotating crops interrupts the diseas e 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 pe st impractical. Row spacing, pl ant 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. (Pederse n, 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 estimat ed loss of 50 million bushels of soybean in Iowa (Pedersen, 2008d). Soybean cyst nemat odes feed on the roots, causing severely stunted and yellow plants. Th e simplest, least expensive me thod 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. A lternating varieties with different sources of

resistance also is beneficial.

High-quality seed is essentia l for controlling seedling dis eases. The most important seedling diseases in soybean are Phytophthora sp

., Pythium sp

., Rhizoctonia sp ., and Fusarium sp . (Pedersen, 2008e). Many soybean varie ties demonstrate resi stance to specificMonsanto 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 bl ight) 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 dise ase 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, folia r application of fungici des 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). Howe ver, the economic return fr om a fungicide application may be limited to select soybean production systems; for example, high-yield environments or when producing soybean seed. Accordi ng 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). Soyb ean insects and diseases are rated less problematic but may reach economic thresholds requiring treatment. Weed control in soybean is essential to optimizing yields. W eeds 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 (Peder sen, 2007). The primary factors affecting soybean yield loss from weed compe tition are the weed species, weed density, and the duration of the competition. When weeds are left to compet e 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 w ith the soybean crop influences the level of yi eld loss. In general, early season weed competition will ha ve 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 th e 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 signifi cant impact on the adaptation and occurrence of weeds in soybea n. 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 th ese weeds (Nice and Johnson, 2005). In a recent survey of growers utilizing glyphosate -tolerant crops, pigw eed, morningglory ( Ipomoea

spp .), Johnsongrass ( Sorghum halepense ), ragweed spp. ( Ambrosia spp .), foxtail, and velvetleaf were mentioned as the most pr oblematic weeds, depending on the state andMonsanto Company 11-SY-217U 162 of 467 

### cropping system (Kruger et al., 2009). With the exception of morningglory and pigweed, the weed species identified as problematic we re present and problematic before glyphosatetolerant crops were introduced, but then we re to a reduced degr ee after implementing glyphosate-tolerant cropping systems (Kr uger et al., 2009). Common waterhemp ( Amaranthus rudis ) and ragweed were the most frequen tly mentioned problematic weeds in glyphosate-tolerant crops in Il linois, 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 c ontrol 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 c ontrol with selective herbicides in soybean production. Approximate ly 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 gl yphosate 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 acr eage (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 rota tion 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 br eak disease, insect, and weed cycles, reduce soil erosion, increase soil organic matter, impr ove 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 Economi c 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 rota tions 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 redu ce the risk of damage from diseases andMonsanto Company 11-SY-217U 163 of 467 

### 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 ro tated 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 ro tation than when either crop is grown continuously (Hoeft et al., 2000b).

A combination of conservation tillage practices and crop rotation has b een shown to be very effective in improving soil physical properties. Long-term studi es in the Midwest indicate that the corn-soybean rotation improves yield potential of no-till systems compared to continuous corn production (Al-Ka isi, 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 draine d soils under no-till practices.

Unique to the southern portion of the Midwes t and the Southeast regi ons, soybean is grown in a double-cropping system. Doub le-cropping refers to the pr actice 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 ca nopy most of the year (Hoeft et al., 2000b). In the Midwest,

winter wheat is harvested in late June or Ju ly, and then soybean is planted into the wheat residue in a no-till syst em 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 ha rvest. Soybean seeds also can remain in a field when pod placement on the plants is too close to the gr ound for the combine head to collect all the pods or when the combine is improperly adju sted for efficient ha rvesting. 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). Prep lant tillage is the first management tool for control of emerging vol unteer soybean in the spring. If volunteer soybean should emerge after planting, shallow cu ltivation 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 s oybean for several herbicides used in theMonsanto Company 11-SY-217U 164 of 467 

### 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 Co ntrol of Volunteer Soybean in Labeled Rotational Crops 1



### Product Rate (Product/Acre) Soybean

V2 – V3 Soybean

V4- V6

Corn 2


### 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 Sorghum 2,4


### 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 & Oats 2


### Buctril (bromoxynil) 1 pt E E Widematch (clopyralid/fluroxypyr) 0.25 pt E G

Cotton 3



### Envoke ® 

### (trifloxysulfuron) 0.1 oz E E

Rice 4



### 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).Monsanto Company 11-SY-217U 165 of 467 

### VIII.J. Stewardship of MON 87712

Monsanto Company develops effective produc ts and technologies and is committed to assuring that its products and technologies are safe and e nvironmentally responsible. Monsanto demonstrates this commitment by implementing product stewardship processes throughout the lifecycle of a product and by pa rticipation in the Excellence Through Stewardship SM 

### (ETS) Program 5 

### . 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 Guides 6 

### 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 Launch 7 

### . Monsanto continues to monitor other countries that are key importers of soybean from the U.S., for the development of formal biotechnology approval proce sses. If new functioning regulatory processes are developed, Monsanto will make appropria te and timely regulatory submissions.

Monsanto also commits to best industry practices on seed quality assurance and control to ensure the purity and integrity of M ON 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 ha ve minimal impact on current cultivation and management practices for soybean. MO N 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 agri cultural practices farmers use to produce a soybean crop. MON 87712 did not require an y 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 thei r farm’s productivity and profitability, and are accustom ed to the incremental yield improvements for varieties obtained through traditional breed ing. Growers are also accustom ed to field-to-field or year-to-year yield variation based on enviro nmental conditions and the varieties they

5 Excellence Through Stewardship Program can be found at: http://www.excellencethr oughstewardship.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/fact s/documents/Guide%20for%20Product%20Launch%20S tewardship.pdf

.Monsanto Company 11-SY-217U 166 of 467 

### select for planting. Therefore, farmers are cap able 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.Monsanto Company 11-SY-217U 167 of 467 

### 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 ag ronomic practices. USDA-APHIS has responsibility, under the Plant Protection Ac t (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 arti cle does not present a pl ant pest risk and no longer should be regulated. If APHIS dete rmines that the regulated article does not present a plant pest risk, the petition ma y be granted, thereby allowing unrestricted

introduction of the article.

The definition of “plant pest” in the Plan t PPA includes living organisms that could directly or indirectly injure, damage, or cau se disease in any plant or plant product (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for bi otechnology-derived crop products is not zero risk, but rather a determination that de regulation 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 appr oach based primarily on eight lines of evidence: 1) insertion of a single functional copy of th e BBX32 expression cassette, 2) characterization of BBX32 prot ein 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, agro nomic, and environmental interaction characteristics demonstrating no increased plan t pest potential compared to conventional soybean, 6) negligible risk to Non Targ et 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 analys is 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 CF R Part 340 that included provisions to utilize its noxious weed authority in regulat ing genetically engin eered 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 we ed” as defined by the Plant

Protection Act.Monsanto Company 11-SY-217U 168 of 467 

### IX.B. Plant Pest Assessment of MO N 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 safe ty evaluation of MON 87712.

IX.B.1. Characteristics of the Gene tic 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 vect or 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-1 α promoter, EF-1 α leader, EF-1 α intron, CTP2 targeting sequence, and the E9

3 ′ untranslated region. During transformati on, 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 br eeding methods and segr egation, along with a combination of analytical techniques, were us ed to isolate those pl ants 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 ar e 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 integrat ed 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 de monstrated 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 conclu sion that MON 87712 is unlik ely to be a plant pest.

IX.B.1.2. Protein Safety

MON 87712 is a biotechnology-derived soybean th at 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 speci es (e.g., germins, lipid transfer protein, profilins, and small molecular weight calcium binding proteins), no Arabidopsis proteins have been reported in a peer-revie wed database of known allergens.Monsanto Company 11-SY-217U 169 of 467 

### BBX32 is a member of a family of B-box-c ontaining proteins. B-box zinc finger family is found in many plant species including s oybean. Additionally, bioinformatic searches using the BBX32 amino acid sequence as a query yield homologous sequences from several different plant species , including the food cr ops citrus, grape, apple, soybean, rice, lettuce, and corn. Overall protein seque nce 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 rele vant to the risk assessment were determined by western bl ot 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 i ndicated 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 th at the expression levels in these tissues are very low.

Taken together, the low level of BBX32 pr otein expressed in M ON 87712 tissues along with the presence of homologous sequences of BBX32 in several pl ant species including soybean, support the conclusion that food and f eed products containing or derived from

MON 87712 are as safe for human and animal consumption as soybean currently on the market. Therefore, unintended environmenta l effects are not anticipated from dietary exposure to BBX32 in MON 87712, and suppor t the overall conclusion that MON 87712 is unlikely to be a plant pest

IX.B.2. Compositional Characteristics

Detailed compositional analys es 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 cont rol A3525 and several c onventional commercial reference varieties. Seed and forage were ha rvested from eight indi vidual sites in which MON 87712, the conventional control A3525, a nd a range of conventional commercial reference varieties were concurrently grown in the same field trial. The conventional

commercial reference varieties were used to es tablish a range of natural variability for the key nutrients and anti-n utrients in conventiona l soybean varieties that have a history of safe consumption.

The combined site analysis was conducted to determine statis tically significant differences (5% level of significance) betw een MON 87712 and the conventional control

A3525. The results from the combined-site da ta were reviewed using considerations relevant to food and feed safety and nutriti onal quality including the relative magnitudes

of the difference in the mean values of nutrient and anti-nut rient components of

MON 87712 and the conventional contro l A3525, whether the MON 87712 component mean value was within the range of natural va riability of that component as represented by the 99% tolerance interval of the conven tional commercial refe rence varieties grown concurrently in the same field trial, and analyses of the reproducibility of the statistically significant combined-site component di fferences at individual sites.Monsanto Company 11-SY-217U 170 of 467 

### Assessment of the analytical results confir med that the differences observed in the combined-site analysis were not meaningful to food and f eed safety or the nutritional quality of MON 87712 soybeans. In additi on, the levels of a ssessed components in MON 87712 were compositionally equivalent to the conventional control A3525 and within the range of variability of conven tional commercial soybeans that were grown concurrently in the same field trial. Th ese results support the overall conclusion that MON 87712 is unlikely to a lter plant pest potential.

IX.B 3. Phenotypic, Agronomic, and En vironmental Interaction Characteristics

Comparative plant characteriza tion 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 characteri stics; plant growth and development characteristics; observations for abiotic stress response, disease damage, arthropodrelated damage; pollen characteri stics; arthropod abundance; plant-symbiont interaction; volunteer potential and persistenc e outside of cultivation charact eristics. 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 characteris tics, nor increased susceptibility or tolerance to specific diseases, in sects, or abiotic stressors, or altered symbiont interactions compared to conventional soybean. Taken toge ther, the results of th e analysis support a

determination that MON 87712 is no more likel y 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 characteri zation demonstrated that MON 87712 seed had germination characteristics not different to those of th e conventional control (Section VII.C). In particular, the lack of hard seed, a well-accepted characteri stic often associated with plants that are weeds, supports a conclu sion of no increased w eediness or plant pest potential of MON 87712 compar ed 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 lodgi ng 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). Fi ve statistically significant differences were

detected between MON 87712 and the conventi onal control A3525 for early stand count, days to 50% senescence, days to physiologi cal maturity, final stand count, and yield. Although significantly different from the conve ntional control A3525, the mean values of MON 87712 for early stand coun t and final stand count we re within the range ofMonsanto Company 11-SY-217U 171 of 467 

### commercial reference varieties for each charac teristic and thus woul d not be adverse in terms of pest potential. Differences in days to 50% senescence, days to physiological maturity, and yield were consistent with th e mode of action. The increase in yield is agronomically desirable and would not contri bute to increased w eediness potential of MON 87712 without changes in a combination of other characteristic s associated with weediness (Baker, 1974). Thus, the observed differences are not considered to be biologically meaningful in te rms 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 conv entional control A3525 and responses to abiotic stressors, such as drought, mineral a nd nutrient toxicity, and temperature stress (Section VIII). The lack of significant biologically mean ingful differences in the MON 87712 response to abiotic stress support th e conclusion that the introduction of the BBX32 protein is unlikely to result in in creased weediness or plant pest potential compared to conventional soybean.

IX.B.3.4. Pollen Morphology and Viability

Evaluations of pollen morphology and viabili ty from field-grown plants provide information useful in a plant pe st assessment as it relates to the potential for gene flow and introgression of the biotec hnology-derived trait into othe r soybean varie ties and wild relatives. Pollen morphology and viability ev aluations demonstrated no statistically significant differences between MON 87712 a nd 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, observati onal data on environmental interactions

were collected for MON 87712 and the co nventional control A3525. In addition, multiple commercial reference varieties were in cluded in the analysis to establish a range of natural variability for each characteristic . The environmental interactions assessment (Section VIII) included data co llected on plant-arth ropod and plant-disease interactions.

The results of this assessment indicated that the presence of BBX32 did not meaningfully alter plant-arthropod interacti ons, including beneficial arth ropods and arthropod pests, nor did it alter disease suscep tibility of MON 87712 compared to conventional soybean. The lack of meaningful differences in di sease damage, arthropod-related damage, and pest and beneficial arthropod abundance dem onstrate that the intr oduction of the BBX32 in MON 87712 is unlikely to be biologically m eaningful in terms of increased plant pest potential.Monsanto Company 11-SY-217U 172 of 467 

### In the field, soybean forms a complex sy mbiotic relationship with members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae . This symbiosis results in the

formation of root nodules in which the b acteria 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 si gnificant differences were detected between

MON 87712 and the conventional control A3525 fo r the parameters m easured, indicating no impact on either the symbiotic relationshi p 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 char acterization, safety as sessments of BBX32,

results from the environmental assessment de scribed above, and the demonstration of compositional, agronomic and phenotypic equi valence to conventional soybean. Taken together, these data support the conclusi on 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 list ed threatened or endangered animal that occupies a habitat where it is likely to incl ude 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 Ea stern 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 loblol ly pine; buds and flowers of trees; fungi; insects; fruit; and an occasi onal 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 dive rsity of the Fox Squirrel diet, support a conclusion that no biologically significant chan ges to the habitat or diet of the Delmarva Peninsula Fox Squirrel are e xpected. Consequently, the planting of MON 87712 is not expected to affect the Delm arva Peninsula Fox Squirrel.

IX.B.3.4. Volunteer Potential and Pers istence Outside of Cultivation

Evaluations of volunteer potential and persistenc e outside of cultivation from field-grown plants provide information useful in assessi ng potential weedine ss characteristics of MON 87712 compared to the conventional co ntrol A3525 (Section VII.C). Volunteer potential evaluations demonstrated no stat istically significant differences between

MON 87712 and the conventional control A3525. The persistence outsi de of cultivation evaluations demonstrated a few statistically significant differences between MON 87712

and the conventional control A 3525, however these differences were not seen across the individual sites and were small in magn itude. 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 invadi ng established ecosystems. Soybean is not listed as a weed in the major weed references (Crockett, 19 77; Holm et al., 1979), nor is it present on the lists of noxious weed speci es 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, a nd become a dominant species in new or diverse landscapes or the ability to compete well with native vegetation. Due to the lack of dormancy, whic h 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 potenti al 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 woul d not compete well with the succeeding crop, and could be controlled readily by either m echanical or chemical means (OECD, 2000). In addition, since wi ld populations of Glycine species are not known to exist in the U.S., the potential does not exist for MON 87712 to ou tcross 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 inter action 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 conven tional control A3525 for characteristics potentially associated with weediness. Furthermore, comparative field observations

between MON 87712 and its conv entional control A3525 in th eir response to abiotic stressors, such as drought, mineral and nutrien t toxicity, and temperature stress, indicated no biologically meaningful diffe rences and, therefore, no incr eased 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 suppor t 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 pollina tion event, environmental factors, sexual compatibility of pollen donor and recipient plants, pollination biology, flowering phenology, hybrid stability and fert ility, 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 (Sut herland 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 recipien t plants will result in increased plant pest potential. The potential for gene introgression fr om 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 M ON 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-pollinate d species, low levels of natural crosspollination can occur (Caviness, 1966; OECD, 2000; Ray et al., 2003; Yoshimura et al., 2006). In studies with cultiv ated soybean, where conditions have been optimized to ensure close proximity and flowering synchr ony, natural cross-pollin ation generally has been found to be very low. Most outcros sing occurred with su rrounding plants, and cross-pollination frequencie s 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; Eric kson, 1975; Jaycox, 1970a; 1970b; 1970c) .

Numerous studies on soybean cross-pollina tion have been conducted, and the published results, with and without supplemental pollina tors, are summarized in Table IX-1. Under natural conditions, cross-pollination among adja cent 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-pol lination 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-pollinat ion 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 th e U.S., which permit any distance between different soybean cultivars in the field as l ong 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; th erefore the presence of BBX32 confers no increased plant pest po tential 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). Hybr idization 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. maxMonsanto 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 F 1 

### hybrids of G. max and G. soja carry similar genomes and are fertile (Si ngh 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 report ed.” This is also supported by work from Kuroda et al. (2008) in which molecular ma rkers were used and no gene flow from G.

max to G. soja was detected. Many barriers to natural hybridizatio n exist between soybean and wild relatives, including the hi ghly selfing nature of both plants, required proximity of wild soybean to cultivated s oybean, synchrony of flower ing, and presence of pollinators. As such, it is highly unlikely that naturally occurring, pollen-mediated gene flow and transgene introgression into wild s oybean 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 fert ile 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 Hy mowitz, 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 un likely 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 Austra lia; West, Central and South Pacific Islands; China; Papua New Guin ea; Philippines; and Taiwan (Hymowitz et al., 1992; Hymowitz and Singh, 1992). Therefore, the onl y opportunities for intersubgeneric hybridization would occur in areas where thos e species are endemic. Nonetheless, the likelihood of in terspecific 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 b een 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. Furt hermore, species dist antly relate d usually produce nonviable F1 seeds that either have premature death of the germinating seedlings or suffer from seedling and vegetative lethal ity (Kollipara et al., 1993). In North and South America, it is not possi ble for gene transfer to o ccur 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 regardi ng the unaided transfer of genetic material from soybean species to other sexually-inc ompatible plant species. The likelihood for horizontal gene flow to occur is exceedingly small. Therefore, potential ecological riskMonsanto 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 agronom ic 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 li kely to be used in common rotations on land previously used for agri cultural purposes. Certified seed production

will continue to use well-established indus try practices to deliver high quality seed containing MON 87712 to growers. Cultivatio n of MON 87712 is not expected to differ from typical soybean cultivation.

MON 87712 is comparable to conventional soybean in its agr onomic, phenotypic, ecological, and compositional characteristics and has levels of resistance to insects and diseases comparable to conventional soyb ean. Therefore, no significant impacts on current cultivation and management practi ces 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 th e Plant Protection Act as cert ain living organisms that can directly or indirectly injure , cause damage to, or cause di sease to any plant or plant product (7 U.S.C. § 7702[14]). Characterizati on 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 increase d 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 comp ared to conventional soybean, followed by

a risk assessment on detected differences. Th e risk assessment considered various factors including: 1) insertion of a single functional copy of the BBX32 expression cassette, 2) characterization of BBX32 pr otein 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, agro nomic, and environmental interaction characteristics demonstrating no increased plan t pest potential compared to conventional soybean, 6) negligible risk to Non Targ et Organisms (NTO) and threatened or endangered species, 7) familiarity with soybean as a cultivated crop and the inherentlyMonsanto Company 11-SY-217U 177 of 467 

### low plant pest potential of soybean, and 8) no unexpected impact to agronomic practices, including land use, cultivati on 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 dere gulated biotechnology-derived soybean, MON 87712 is highly unlikely to be a plant pest. Therefore, Monsanto Company requests a determination from APHIS th at MON 87712 and any progeny derived from

crosses between MON 87712 and other commer cial 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 si ngle-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 resu lts or observations associ ated with MON 87712 or the BBX32 protein indicating that there would be an adverse environmental consequence

from the introduction of MON 87712. MON 87712 c ontains BBX32 protein that interacts with one or more endogenous tr anscription factors to regu late the plant’s day/night processes and results in increas ed availability of assimilate s in the plant resulting in increased yield of MON 87712 when compared to a comparator without the introduced gene. As demonstrated by field results a nd laboratory tests, the difference between MON 87712 and conventional soybean is th e increased yield opportunity in MON 87712.

The data and information presented in th is 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 fr om a detailed characterizatio n of the product compared to conventional soybean, followed by risk as sessment on detected differences. The

characterization evaluations included molecula r 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 expres sion 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 di fferent plant species , including the food crops citrus, grape, apple, s oybean, rice, lettuce, and corn, wh ere a history of safe use is established. Compositional analysis of key nutrients and antinutrients from seed and forage demonstrate that MON 87712 is comp ositionally equivalent to conventional soybean. Finally, extensive characterization of the plant phenotype and environmental

interactions indicate that MON 87712 is comparable to conv entional 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 cultivatio n 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 he lp 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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Wilcox, J.R. 2004. World distribution and trad e of soybean. Pages 1-13 in Soybeans: Improvement, Production and Uses. Third Edition. H.R. Boerma and J. E. Specht (eds.). American Society of Agronomy, Inc., Crop Sc ience Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

Woodworth, C.M. 1922. The extent of natural cross-pollination in soybeans. Journal of the American Society of Agronomy 14:278-283.

Wrather, J.A., W.C. Stienstra and S.R. Koenning. 2000. Soybean disease loss estimates

for the United States from 1996 to 1998. Can. J. Plant Pathol. 23:122-131.Monsanto Company 11-SY-217U 195 of 467 

### Wrather, J.A., W.C. Stienstra and S.R. Koenning. 2001. Soybean disease loss estimates

for the United States from 1996 to 1998. Ca nadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 23:122-131.

Yates, B., A. De Soyza, R. Harkawat a nd C. Stenton. 2008. Occupational asthma caused by Arabidopsis thaliana : A case of laboratory plant al lergy. European Respiratory

Journal 32:1111-1112.

York, A.C., J.B. Beam and A.S. Culpe pper. 2005. Control of volunteer glyphosateresistant soybean in cotton. Journal of Cotton Science 9:102-109.

Yoshimura, Y., K. Matsuo and K. Yasuda . 2006. Gene flow from GM glyphosatetolerant to conventional soybeans under field conditions in Japan. Environmental Biosafety Research 5:169-173.

Zambryski, P., A. Depicker, K. Kruger and H.M. Goodman. 1982. Tumor induction by Agrobacterium tumefaciens : Analysis of the boundaries of T-DNA. Journal of Molecular and Applied Genetics 1:361-370.

Zollinger, R., M. McMullen, J. Knodel, J. Gr ay, D. Jantzi, G. Kimmet, K. Hagemeister and C. Schmitt. 2009. Statewide pesticide usage for North Dakota. Page 23 in Pesticide Use and Pest Management Practices in North Dakota 2008. North Dakota State University Extension Service, North Dakot a Agricultural Experiment Station, Fargo,

North Dakota.Monsanto Company 11-SY-217U 196 of 467 

### APPENDICESMonsanto Company 11-SY-217U 197 of 467 

### Appendix A: USDA Notifications

Field trials of MON 87712 have been conducted in the U.S. since 2006. The protocols

for these trials include fiel d 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, observati onal 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 prep aration. A list of trials conducted under USDA notifications and the status of the final reports for th ese trials are provide d in Table A-1.Monsanto Company 11-SY-217U 198 of 467 

### Table A-1. USDA Notifications and Perm itsApproved 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 ProgressMonsanto Company 11-SY-217U 199 of 467 

### Table A-1. USDA Notifications and Perm itsApproved for MON 87712 and Status of

Trials Conducted under These No tifications (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 ProgressMonsanto Company 11-SY-217U 200 of 467 

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

Crop yield results from a sequ ential growth and developmen t 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 geneti c and environmental factors, that influence a crop’s opportuni ty to realize its full yield po tential. Improvements in crop yield have been a primary focus of conve ntional breeding. The genetic changes that resulted in crop domestication and yield improve ment in conventional varieties have been shown by modern molecular biology analysis to have been typical ly 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 gene tic elements that use or modify existing pathways in the plant. The yield in crease 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 re gulate the plant’s day/night processes and

results in increased availabil ity 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 improveme nt (Kumudini, 2002; Sinclair et al., 2004). Increased assimilate availability in M ON 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 dur ing the reproductive phase of the soybean plant, as well as by the significantly hi gher yield of MON 87712 when compared to control, as observed in multisite field studies in the U.S.

To understand the mode-of-acti on of BBX32 protein in MON 87712, it is important to understand the biological proc esses associated with yiel d and the impact of diurnal biology on those processes in soybean. An alysis of yield improvement and yield

determination in conventional soybean has shown that the same basic principles and mechanisms apply for MON 87712. Thr ough highlighting yield limitation in conventional soybean as a function of carbon and nitrogen availability that are controlled by diurnal processes, we demonstrate how th e 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 fo r Soybean Breeders

Soybean crop yields have risen consistently in North America since the 1920s. In the U.S., soybean yield rose at an average a nnual rate of approxima tely 0.35 bu/A (0.8%) between 1924 – 2010 (Figure B-1) , and similar yield increase s have been reported in Canada. A survey of soybean yield in Canada between 1934 – 1992 revealed an average increase in yield of 0.5%, w ith 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 attributab le 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).Monsanto Company 11-SY-217U 201 of 467 

### 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 con ducted 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 th at yield growth rate s have not reached a plateau. Average soybean yi eld 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 possi ble. The concept of yield potential of soybean is defined as the yield of a cultivar when grown in environments where it is adapted, with nutri ents 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 perf ormance and yield potential.SeedYield(bu/A)

Production yearMonsanto Company 11-SY-217U 202 of 467 

### B.2. Soybean Yield is Fre quently Limited by the Ava ilability 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; Sincla ir et al., 2004). Soybe an yield is the net result of metabolic assimilate availability wi thin the plant (the s ource) and translocation of these assimilates to the developing seed (t he 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 in to dry weight (Egli, 1999). This division recognizes the two major de velopmental processes involved in the accumulation of yield, the production of assi milate in the leaves (the source) and

utilization of this assimilate by the developing seed (the sink).

Soybean plant development can be separa ted into two major generally overlapping developmental phases: vegetative and repr oductive. The duration of these phases is controlled primarily by genetics, temperatur e, and day length (Pedersen, 2007). Soybean producers influence the duration of these ph ases through variety selection, geographic location, and planting date. Yi eld production begins with vegetative growth when the formation of organs for nutrient absorption and photosynthesis provide s the machinery to produce yield. The reproductive phase is ty pically the most important for yield determination and is divided into eight repr oductive (R) stages (Fehr and Caviness, 1981) (Table B-1). Fehr and Caviness (1981) cl assify 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 wh en 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.Monsanto Company 11-SY-217U 203 of 467 

### 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 s eeds produced (Figure B-2). The period from R1 to R6 stages is critical fo r yield, because this is when both pod and seed number are set. The period between the R5 stag e 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 gr owth 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 translocat e 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.Monsanto Company 11-SY-217U 204 of 467 

### Figure B-2. Determination of yiel d 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 wei ght are determined. The developing seed (the sink) receives assimilates fr om the plant (the source).

Soybean plants are able to compensate for canopy gaps by increasing branch production per plant, improving leaf area index, therefor e even large increases in plant stand may result in little to no yield increase (Boa rd 2000; Carpenter and Board 1997). A wide range of seeding rates (100,000-200,000 pl ants/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 fo r a final plant stand increase of 80% (De Bruin and Pedersen 2008). Thus, minor differe nces in plant stand w ould not be expected to substantially affect yield when plant sta nds are near agronomically acceptable levels.

Under a broad range of environmental cond itions, soybean yield is more frequently limited by the amount of translocated assim ilates 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 (R 3 to R4) decreases seed nu mber while reducing source capacity during later growth st ages (R5 to R6.5) decreases individual seed size (BoardMonsanto 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 A ssimilate 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 (H armer et al, 2000; Schaffer et al, 2001). The day/night cycling of plant proc esses is called a diurnal rhyt hm and is achieved primarily by two mechanisms: first, by light, and second, by an internal circadian clock (Schaffer et al, 2001). Coordination of diurnal processe s such as nitrogen and carbon metabolism which affect assimilate availability and plant growth have been show n to be necessary for maintaining plant productivity (Smith and S titt, 2007; Gutierrez et al, 2008; Graf et al, 2010).

The first mechanism to consider in diurnal processes is light signaling. Li ght is one of the most important environmental factors for pl ants as it provides the source of energy for plant life. The ability of plan ts to respond to light is ac hieved through photoreceptors (Jiao et al, 2007). Plants detect a range of light intensities and wavelengths via photoreceptors, and subsequently convert th e light signal into physiological responses through transcriptional regulation (Jiao et al, 2007). Light-responsive transcription factors, such as LONG HYPOCOTYL5 (HY5 ), mediate light si gnaling through the coordinated activation and repression of speci fic plant genes (Lee et al, 2007). The ability to sense a light signal allows the plant to re spond to recurring diur nal cycles and thus control growth and development throughout vege tative and reproductive stages (Jiao et al, 2007).

The second mechanism in plant diurnal processes is the circ adian 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 environmen t, 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 signali ng network between environmental response pathways and internal programs (Millar, 200 4). 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 rela ted transcription factor proteins CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE EL ONGATED HYPOCOTYL (LHY), and a response regulator protein TIMING OF CAB EXPRESSION 1 (TOC1) (Salome and McClung, 2004). Diurnal regulatio n 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 expressi on at different times of the day/night and

are associated with circad ian biology, nitrogen and carbon metabolism (Harmer et al, 2000).Monsanto Company 11-SY-217U 206 of 467 

### Figure B-3. Diurnal cycling of processes su ch 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 th e circadian clock. The circadian cl ock is continually modified by light input, which helps the pl ant 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 di urnal 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 diur nal processes such as carbon metabolism. During the day, plants absorb light and stomates open to facilitate CO 2 

### 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 suppl y of assimilated carbon is not used immediately for growth.Monsanto Company 11-SY-217U 207 of 467 

### Newly assimilated carbon provides for bot h 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 assimila tion 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 propor tion of assimila te partitioned as starch for use at night is greater when the day length is shortened (Chatterton and Silvius 1979). This concept is demonstr ated when soybean plants are grown under different light/dark cycles. Conventional soybe an 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 Sl afer, 2005). Under field conditions, Kantolic and Slafer (2005) showed that artificially ex tended 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 Sl afer, 2005). Alteration of diurnal processes through genetic means therefore offers the potential for future yield improvements through optimization of post-flower ing 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 ge ne expression operate through regulation of endogenous plant pathways and processes. Normally occurring va riations in these pathways are likely to occur in response to genetic and environmental factors (Kier and Petrick, 2008). In addition, molecular bi ology 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 tran scription 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 c ontext 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 s oybean 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. M ode-of-action studies

were conducted to understand BBX32 functi on in MON 87712 using two controls: the parental conventional cont rol 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 trai t 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 controll ing variability when measuring impacts on yield, our primary focus for the determinati on 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 le af area, light interception, and leaf photosynthetic rate demonstrates that M ON 87712 has higher canopy- level assimilate availability during th e seed-filling period.

To examine the effect of BBX32 protein e xpression on diurnal pr ocesses in MON 87712, targeted analysis was conducted on a group of soybean genes and metabolites associated with diurnal biology and primary carbon a nd nitrogen metabolism. BBX32 protein expression is shown to modulate the respons e 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 meta bolism, metabolites indicative of source capacity, and the activity of an enzyme involved in carbon metabolism were observed when MON 87712 was compared to A3525 a nd MON 87712(-) controls. Increased plant assimilate availability is a ssociated 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 tr aditional breeding methods.

B.5.1. MON 87712 Delivered Significantly In creased Yield when Compared to
Conventional Soybeans of th e Same Genetic Background

Soybean seed yield is expressed as a func tion of the primary yield components: final stand count per area, seed num ber per plant, and individual seed weight. According to Mullen (1996) yield is expressed as:

൬ Yield ݐ݄݁݅݃ݓ

݁ܽݎܽ ൬ ݐ݊ݑ݋ܿ ܽ݊݀ݐܵ ൌ ൰ ݏݐ݈ܽ݊݌

ܽݎ݁ܽ ൰ ݔ Seed number per plant ൬ ݏ݁݁݀ݏ

ݐ݈ܽ݊݌ ൬ weight seed Individual ݔ ൰ ݐ݄݁݅݃ݓ

݁݁݀ݏ ൰



### Yield and yield component characteristic s of MON 87712 were compared to two controls: the parental conventional c ontrol population, A3525, and a near isogenicMonsanto 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 co mparator in the safety assessment of MON 87712, including the plant pest risk evaluation. The MON 87712(-) control line was identified at the R1 generation dur ing 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 productio n 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 statis tically significant increase in yield was observed for MON 87712 compared to M ON 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 s eed weight per plant. Similar results were observed when comparing MON 87712 and th e A3525 parental conventional control (Table B-3).

Results of this study across a range of site s 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 num erical 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 a nd Yield Components of MON 87712 Compared to the Negative Segregant Control [MON 87712 (-)] fr om 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 a nd 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 significan t 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 pl ant 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 s oybean yield (Board 2000; Butler et al. 2010; De Bruin and Pedersen 2009). The means of the final st and counts of MON 87712 and the control plots ranged from 123,379 plants/acre (283.2 plan ts/plot) to 127,234 pl ants/acre (292.1 plants/plot). These values are within the wide range of seeding rates and stand counts that have relatively little influence on s oybean yield (De Bruin and Pedersen, 2009). Based on the compensatory as pect of soybean growth, the increase in yield for

MON 87712 observed in the U.S. field study in 2009 was greate r 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 fo r seed number and seed weight are consistent with the overall increase in yield.

As MON 87712 exhibited statistical ly 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 conventi onal control A3525 would indicate that MON 87712 may be more li kely to become a weed than conventional soybean. The phenotypic, agronomic and enviro nmental interactions assessment (Section VII) showed few statistically signifi cant 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 st and (296.8 vs. 286.4 plants per plot) were

observed. These differences were of sma ll relative magnitude when compared to

commercial conventional referenc e varieties grown concurrentl y, therefore, there were no changes indicative of increased weediness or plant pest potential (Baker, 1974). Not unexpectedly from the trait, there were statis tically significant increases in yield (52.6 vs. 49.0 bu/ac). Although the yield difference is biologically meaningf ul from a grower perspective, the increase in yield is not i ndicative of increased we ediness 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 (assim ilate availability) during the reproductive peri od (Kumudini, 2002). The sour ce capacity of a crop canopy is linearly related to the ability of a canopy to intercept solar radia tion, and the ability to convert intercepted light into biomass (Montei th, 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 meas uring the photosynthetic photon flux density above and below the canopy and calculating th e proportion of light interception. Light interception is closely related to leaf area index, defined as the amount of leaf area perMonsanto Company 11-SY-217U 213 of 467 

### unit land area measured in m 2 

### leaf area per m 2 

### land area. Conversion efficiency of intercepted light into biomass is typi cally estimated based on light-saturated photosynthetic rate at a single st age of crop development, howe ver it is better to estimate conversion efficiency based on integrat ed 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 inte rception efficiency, and photosynthetic rate were collected during the seed-fill period (R 4 

### , R 5 

### and R 6 

### 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 so ybean (Figure B-4 and Figur e B-5) (Appendix C). MON 87712 had significantly higher leaf area index than the MON 87712(-) at the R 4 

### stage, and significantly higher leaf area inde x than both controls at the R 5 

### and R 6 

### stages (p<0.05 in all cases) (Figure B-4). MON 87712 had significantly higher light interception than the MON 87712(-) at the R 5 

### stage, and had higher light interception than both controls at the

R 6 

### stage. At the R 4 

### growth stage, the increased leaf area index of MON 87712 is counteracted by a decrease in midday leaf photo synthetic rate (Figure B-5). At midday of

the R 5 

### growth stage, MON 87712 had significantly higher photosynthetic rate. At the R 6 

### growth stage, MON 87712 had significantly high er photosynthetic rate than both controls at all of the measurement timepoints at the R 6 

### stage (Figure B-5).

Taken together, the increased leaf area i ndex and light interception combined with increased leaf photosynthetic rate late in crop development indicat e that MON 87712 had higher canopy-level assimilate availabi lity during the seed -filling period (R 5 

### -R 6 

### /R 7 

### ) that is critical to yield determination in s oybean. Increased source capacity has been associated with improved yield in convent ional 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 increas ed yield, increased ca nopy level assimilate availability in MON 87712 is related to two st atistically significan t differences observed between MON 87712 and the parental convent ional 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 physio logical maturity (280.8 vs. 277.5 days) were observed. Although these differences could be expected and are biologically meaningful with regard to the in creased source capacity of MON 87712 , they were of small relative magnitude when compared to natural variab ility and are therefore not indicative of increased weediness or plant pest potential fr om an environmental risk perspective, as

discussed in Section VII.Monsanto Company 11-SY-217U 214 of 467Figure B-4. Leaf Area Index and Light InterceptionLeaf Area Index and Light Interception Efficiency data for MON 87712 (open circles), A3525 (dark circles), and MON 87712 (-)(dark triangles) at the R4, R5 and R6-R7 growth stages within the thinned treatment. Subsamples of 2 measurements per plot werecollected on each of the measurement dates. Values represent arithmetic means± 1 SE. Significant differences between MON 87712and MON 87712 (-) (α=0.05) at a given measurement date are denoted by†. Significant differences between MON 87712 and A3525(α=0.05) at a given measurement date are denoted by *.Growth StageR4 R5 R6-R7

Leaf Area Index (m 2 /m 2 )02468Leaf Area IndexR4 R5 R6-R7 Light Interception0.00.20.40.60.81.0A3525MON 87712MON 87712 (-)Light Interception***†††††Monsanto Company 11-SY-217U 215 of 467Figure B-5. Leaf Photosynthetic Rate DataLeaf photosynthetic rate of MON 87712 (opencircles), parental conventional controlA3525 (dark circles) and MON 87712(-) (darktriangles) measured at morning, midday andafternoon timepoints at the R4, R5 and R6-R7 stages. Values represent arithmetic means± 1 SE. Significant differences betweenMON 87712 and the MON 87712 negative segregant(p<0.05) at a given measurement dateare denoted by †. Significant differencesbetween MON 87712 and the conventional control A3525 (p<0.05) at a given measurementdate are denoted by *.R4morning midday afternoon

Photosynthetic Rate (  mol CO 2 /m 2 /s)010203040A3525MON 87712MON 87712 (-)*R5Approximate Time of Daymorning midday afternoon*R6-R7morning midday afternoon***†††††Monsanto Company 11-SY-217U 216 of 467 

### B.5.3. Plant BBX32 Protein Modulates Diur nally 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 finge r proteins that contain one or more B-box domains with specialized tertia ry structures that are stabili zed by binding zinc ions. The B-box domain is predicted to be involved in pr otein-protein interacti ons (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 fa mily 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 agrono mically-important species, suggesting that the function of BBX32 in its source plant, Arabidopsis thaliana , is conserved in other plant species.

Transcriptional regulation of e ukaryotic genes is an orchestr ated process that requires the concerted functions of multiple proteins . In addition to the ubiquitous general transcription factors, such as RNA polymer ase II, there are targ et-specific DNA-binding transcription regulators which m odulate 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 re gulation of genes involved in plant diurnal processes. BBX32 protein is a represso r 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 functi on of another protein through protein-protein interactions, and, while not directly involved in contacting DNA, aid transcriptional re gulation (Martin, 1991).

Mode of action analyses in Arabidopsis thaliana show that BBX32 ex pression represses plant responses to the transition from dark to light via interaction with the HY5 transcriptional complex (H oltan et al, 2011). The rela tionship between BBX32 and repression of the HY5 func tion is evidenced by the phenotypic similarity between Arabidopsis thaliana seedlings overexpressing BBX32 and a hy5 loss of function mutant. Both BBX32 overexp ression lines and hy5 mutants exhibit el ongated 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 (H oltan et al. 2011).

To investigate the function of BBX32 in M ON 87712, targeted analys is of a group of soybean genes was conducted to determine the impact of expression of BBX32 on the

mRNA level of certain diur nally regulated genes in MON 87712. The genes analyzed

were selected based on their potential involvement in th e mode of action for BBX32 leading to increased yield, and included ge nes associated with circadian biology, carbon and nitrogen metabolism, and phytohormone function. Expression analysis was conducted on tissue samples taken from growth chamber and field gr own plants, and the expression level of each gene in MON 87712 wa s 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 fiel d and the growth chamber studies wereMonsanto 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 compone nt of the circadian clock, and the BBX13 gene, a B-box gene with sim ilarity 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 BBX 32 activity can result in ei ther up-regulation or downregulation of endogenous genes. These obs ervations support a role for BBX32 in modulating the response of MON 87712 to the plant’s transition from night to day by repressing light signal ing. At other time points, expr ession patterns of the soybean LHY

and BBX13 genes in MON 87712 were not significan tly different from the expression pattern observed in control soybean, thus demonstrating that the introduced BBX32 protein functions within an existing fr amework of gene regulation in soybean.Monsanto Company 11-SY-217U 218 of 467 

### Figure B-6. Gene Expression Patterns fo r 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 “pr e” and “post” are relative to dawn.

B.5.4. BBX32 Impacts Existing Metabo lic Processes in MON 87712, Improving
Assimilate Availability

The impact of BBX32 expression in M ON 87712 on selected metabolite and enzyme activities was investigated. L eaf samples from the 2009 growi ng season were collected at two developmental stages (R1 and R6) at th ree different time poi nts 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 de termined 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 act ivity analyses included allantoate amidohydrolase, glutamine synthetase, mala te dehydrogenase, Nicotinamide Adenine Dinucleotide Phosphate (NADP) Mali c Enzyme, Phosphoenolpyruvate (PEP) Carboxylase, and Sucrose Phosphate Synthase (SPS). Metabolites and enzymes selected for evaluation are generally associated w ith diurnal biology and primary carbon and nitrogen metabolism resulting in increa sed capacity for growth and reproductive development or correlated with yield or senesc ence (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 gene rated, and metabolites where statistically significant differences (p<0.05) were obs erved for both comparisons (MON 87712 vs. MON 87712(-) and MON 87712 vs. A3525) are repor ted (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 tiss ue at the R1 developmental stage Data is shown as percent change from control, and represents metabo lites 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) a cross 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 A 3525) leaf tissue collected at the R6 developmental
stage Data is shown as percent change from contro l, and represents enzyme/metabolites that
exhibited significant differences that were cons istent 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 55.56 a 

### 236.91 a 

### 51.59 a 

### 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) a cross comparisons to both controls.

a 

### Significantly different (p < 0.05 compa red 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 availableMonsanto Company 11-SY-217U 222 of 467 

### Evaluation of the overall dataset indicates te mporal-specific differences in levels of metabolites involved in carbon and nitrogen me tabolism, metabolites in dicative 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, sh ikimic 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 al lantoic 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 (NH 3 

### ) assimilation (Re ynolds 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 n itrogen 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 metabo lism (Bourguignon et al., 1999) Fructose and shikimic acid are downstr eam 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 nitr ogen metabolism, possibly as storage and/or transport of nitrogen by assimilating carbons from glutamate to generate C:N fluxes that enter the tricarboxylic acid cy cle, 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 we re observed for values of th ree amino acids (isoleucine, leucine, and valine), one organic acid (mal ate), 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 time point, 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 differe nces indicate changes in car bon and nitrogen metabolism, and support the hypothesis of in creased transport of the am ino 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 in creased 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 in creased yield.

These temporal-specific differe nces in levels of metabol ites involved in carbon and nitrogen metabolism, and indicative of source capacity, and the increased activity of SPSMonsanto Company 11-SY-217U 223 of 467 

### enzyme observed in MON 87712 are consiste nt with the proposed mode of action hypothesis for the increased yield in M ON 87712 when compared to conventional soybean with the same genetic background. Specifically, the expr ession of BBX32 in MON 87712 increases carbon and nitrogen assim ilate availability and translocation to reproductive parts during development stages wh en 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 thre e sampling time points, supporting theproposal that these differences in carbon and nitrogen metabolites were temporalspecific. These temporal differences in me tabolism observed at se lect timepoints during the plant’s reproductive phase are consistent with the ultimate increased yield phenotype

of MON 87712 and the associated numerical increases in s eed number and seed weight observed when compared to the conventional c ontrol (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 cy stine (3.07% relative decrease), and small decreases in two fatty acids (palmitic acid an d stearic acid, 1.42 and 3.04% relative decreases, respectively) were observed. Thes e few significant differences were of small relative magnitude when compared to natural variability, and thus indicated that, although the small changes in metabolism obser ved in the green tissues increased seed number and seed weight, the ch anges did not affect composition of the seed from a food and feed safety or nutritional perspective.

B.5.5. BBX32 Protein F unction: 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 associ ated with increased photosynthetic rate at canopy level and a result of al tered diurnal plant metabolism. Plant BBX32-mediated changes in soybean gene expression impact ex isting 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.Monsanto Company 11-SY-217U 224 of 467 

### Figure B-7: BBX32 expression in MON 87712 leads to increased yieldMonsanto Company 11-SY-217U 225 of 467 

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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 l eaf tissue of the R3 generation of MON 87712 and the convent ional 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 referen ce 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 a dditional positive hybridization controls. As additional reference standards, the 1 Kb DNA Extension Ladder and λ DNA/ Hin d III Fragments from Invitrogen (Carlsbad, CA) we re used for size estimations on Southern blots and agarose gels. The 1 Kb DNA Ladde r 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 South ern Blot and PCR Analyses

MON 87712 and conventional control genom ic DNA samples were isolated from soybean leaf tissue. Prior to extraction, l eaf tissue was processe d to a fine powder by mortar and pestle using liquid nitroge n. 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 chlorofo rm 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 preci pitated 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). Fo r further purification of the DNA samples, polyethylene glycol (PEG) precipitation was performed. An equal volume of 20% (w/v) PEG precipitation buffer was a dded 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 supernat ant was removed, the pellets were

washed at least twice with 80% ethanol and th en air dried. The pellets were resuspendedMonsanto Company 11-SY-217U 231 of 467 

### 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 Qu ant 200 Fluorometer (Hoefer, Inc., Holliston, MA). Molecular Size Marker IX (R oche, Indianapolis, IN) was used as the

calibration standard.

C.5. Restriction Enzyme Digestion of DNA

Approximately 10 micrograms (μg) of ge nomic DNA extracted from MON 87712 and conventional control was digest ed with restriction enzyme Nco I (Fermentas, Glen Burnie, MD or New England Biolabs, Beverl y, 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.). A ll 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 hybrid ization controls, ~10 μg of genomic DNA extracted from the conventional control was dige sted 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) agaros e gels. For all Southern blot analyses except insert stability, i ndividual 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 allowe d 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 geno mic 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 fo r Southern Blot Analyses

Probe templates were prepared by PCR amplification using PV-GMAP5779 as the template. The PCR products were separate d on an agarose gel by electrophoresis and purified from the gel using QIAquick Ge l Extraction Kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. The probe temp lates were designed based on the nucleotide composition (% GC) of the sequen ce, 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 te mplate were radiolabeled with [ α

32 

### P] deoxycytidine triphosphate (dCTP) (6000 C i/mmol) using the random priming methodMonsanto Company 11-SY-217U 232 of 467 

### (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 subs tances was evaluated using Southern blot analyses (Southern, 1975). In Southern blots hybrid ized 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 geno me 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 ag arose 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 generate d using Kodak Biomax MS film (Eastman Kodak, Rochester, NY) in conjunction with one Kodak Biomax MS intensifying screen

in a -80°C freezer.Monsanto Company 11-SY-217U 233 of 467 

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

Probe DNA Probe Probe labeled with dNTP ( 32 P) 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 60Monsanto Company 11-SY-217U 234 of 467 

### C.9. DNA Sequence Analyses of the Insert

Overlapping PCR products, denoted as Pr oduct 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 we re sequenced to determine the nucleotide sequence of the insert in MON 87712, as we ll 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 c onducted using 100 ng of genomic DNA template in a 50 μl reaction volume containing a final concentration of 2 mM MgSO 4 

### , 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 Produc t 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 performe d 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) ag arose gels and visualized by ethidium staining to veri fy that the products were th e expected size. Prior to sequencing, each verified PCR product was purified using the QIAquick PCR Purification Kit (Qiagen) according to manuf acturer’s instructi on. The purified PCR products were sequenced using multiple pr imers, including primers used for PCR amplification. All sequencing was perfor med by the Monsanto Genomics Sequencing Center using BigDye terminator chemistr y (Applied Biosystems, Foster City, CA).

A consensus sequence was generated by compiling multiple sequencing reactions performed on the overlapping PCR products. Th is consensus sequence was aligned to the PV-GMAP5779 sequence to determ ine 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 pa ired 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 fina l concentration of 2 mM MgSO 4 

### , 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 amplifica tion 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.Monsanto Company 11-SY-217U 235 of 467 

### Aliquots of each PCR product were separated on 1.0% (w/v) ag arose 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 se quenced using multiple primers, including primers used for PCR amplification. All sequencing was performed by the Monsanto Genomics Sequencing Center usi ng BigDye terminator chemistry.

A consensus sequence was generated by compiling multiple sequencing reactions performed on the verified PCR product. Th is consensus sequence was aligned to the 5' and 3' sequences flanking the MON 87712 in sert to determine the integrity and organization of the insertion site.Monsanto Company 11-SY-217U 236 of 467 

### References for Appendix C

Southern, E.M. 1975. Detection of specifi c sequences among DNA fragments separated by gel electrophoresis. Jour nal of Molecular Biology 98:503-517.Monsanto Company 11-SY-217U 237 of 467 

### 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 M ON 87712 as a component of a cation exchange (CEX)- fractionated leaf extract (l ot 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 112958 30) 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 prepar ed 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. Th e records describing the preparation ofthe 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 prev iously characterized. Records pertaining to the purificati on and the characterization of this E. coli -produced reference protein are archived under Or ion lot 11267091 in the Monsanto Regulatory

Archives.

D.1.2. Description of Assay Control

Protein molecular weight standards (Preci sion Plus Dual Color Standards, Bio-Rad, Hercules, CA) were used to verify protein tr ansfer to nitrocellulose membranes, and to approximate the position of BBX32 on the west ern blots. BSA (Pierce, Rockford, IL) was used to generate a standard cu rve for total protei n 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 a nd filtration, di afiltration, and cation exchange chromatography. A brief description of the is olation process is

below.Monsanto Company 11-SY-217U 238 of 467 

### The MON 87712 and the A3525 leaf tissues were harvested and imme diately transferred to containers and frozen on dry ice. L eaf 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 mate rial for the isolation process. Approximately 135 g of processed ground M ON 87712 leaf tissue was resuspended with 1 liter of chilled (4 °C) extraction buffe r [50 mM Tris-HCl, pH 8.0, 150mM NaCl, 0.50% Igepal-630 (v/v), 4mM TCEP, 5m M sodium fluoride, 5mM sodium orthovanadate, 1 mM magnesium chloride, 2 mM Benzamidine, 10 μM Bestatin, 20μM E64, 500 μM AEBSF]. The leaf tissue susp ension 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 homoge nization 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 stirri ng. After 2 h the extract was centrifuged at 18,600 ×g for 45 minutes at 4 °C. The clarif ied extract supernatant was carefully decanted from pellet and the resultant supe rnatant poured through Miracloth (EMD Chemicals, Gibbstown, NJ) into a beaker chil led on ice to remove any loose pellet. The supernatant was then filtered through a Whatman 0.8/0.2μm 820cm 2 

### EFA Polycap filter (Whatman Inc., Piscataway, NJ). The filtered extract supernatant was kept chilled on ice.

The filtered extract supernatant of appr oximately 950 ml was concentrated to approximately 200 ml using a Millipore Pellicon-2 unit equipped with 2 x 0.1 m 2


### 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 we re chilled on ice during the operation. Following diafiltration and ri nsing 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 Healthca re, 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 CE X pool. The MON 87712 CEX pool was then aliquoted and frozen on dry ice. The fro zen 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 A 3525 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 an alysis were performed to c onfirm the identity, and to estimate the molecular weight and purity of MON 87712 BBX32.

Aliquots of the MON 87712 CEX pool (l ot 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 gi ve 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 curv e (10, 40, 80, 120 pg BBX32). All samples, including the MON 87712 CEX pool, the A3525 CEX pool, and th e reference protein- spiked samples, were loaded at 40 μg of total protein per la ne. 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 fo r four minutes, and were loaded onto a precast 10% polyacrylamide Bis-Tris, 10-we ll 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 (In vitrogen) 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 M ON 87712 CEX pool. For immunodetection, the

membrane was incubated for 14 h at 4 °C w ith 10% (w/v) non-fat dr ied 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 pur ified mouse monoclona l 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. Th e membrane was then incubated with horseradish peroxidase (HRP)-conjugated ho rse 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 si x 15 min washes with TBST. All antibody incubations and TBST washes were perf ormed at RT. Immunoreactive bands wereMonsanto Company 11-SY-217U 240 of 467 

### visualized using the ECL (Enhanced Ch emiluminescence) detection system (GE Healthcare) and exposed to Amersham Hype rfilm (GE Healthcare ). The film was developed using a Konica SRX-101A automa ted film processor (Tokyo, Japan).

Analysis of the blot was performed usi ng 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 so ftware. The mass of BBX32 present in the duplicate lanes of the MON 87712 CEX pool was qua ntified 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 M ON 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 Wester n 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 vi sual analysis (Figure D-1) . Purity of BBX32 in the MON 87712 CEX pool relative to total protein loaded wa s 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 (Fi gure 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 mo lecular weight of BBX32, ~25 kDa. The second band, which migrat ed at ~16 kDa, coul d be due to some degradation of the BBX32 protein. No a dditional bands unique to the MON 87712 CEX pool were observed. This western blot analysis confirmed the identity of the MON 87712 BBX32 protein.Monsanto Company 11-SY-217U 241 of 467 

### 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 comp arison 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. Approxima te MWs (in kDa) are shown on the left and right sides and correspond to the markers load ed 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/AMonsanto Company 11-SY-217U 242 of 467 

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

The purity of MON 87712-produced BBX32 was ca lculated using dens itometric analysis of western blot shown in Figure D-1, and the total protein loaded.

Total Protein Loaded per lane Mass of BBX32 (pg) BBX32 Purity 1
(pg BBX32/ μg total protein) Lane 2 Lane 3

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.Monsanto Company 11-SY-217U 243 of 467 

### D.1.5. BBX32 Coding Sequence Comparison

Sequence alignment showed that translati on of the BBX32 coding sequence from the

vector used to transform conventional soybean to produce M ON 87712, PV-GMAP5779,

and from the E. coli BBX32 expression vector, pM ON102114, resulted in the same amino acid sequence (Figure D-2). As repor ted in Section IV.D, the DNA sequence of the T-DNA I region in MON 87712 is identi cal 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 c oding 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 ARAMTQQLRRWRVDSEEGWAENDNVMonsanto Company 11-SY-217U 244 of 467 

### 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 conventio nal control A3525 were harvested from eight field sites in the U.S. during the 2009 growing seas on from starting seed lots 11223539 and 11223542, respectively. An E. coli -produced BBX32 protein (lot 11267091) was used as the an alytical reference standard.

E.2. Characterization of the Materials

The identities of MON 87712 and the convent ional control samples were confirmed by event specific polymerase chain reaction (P CR) analyses that were conducted on the

harvested seed from each site. The PCR an alyses and the resulting Verification of

Identities were archiv ed in the Monsanto Regulatory Ar chives under the st arting seed lot

numbers.

E.3. Field Design and Tissue Collection

Field trials were initia ted during the 2009 planting s eason 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; Cl inton County, Illinois; Madison County, Illinois; Stark Count y, Illinois; York County, Nebras ka; Boone County, Indiana; and Pawnee County, Kansas. These field sites were representative of soybean producing regions suitable for commercial production. At each site, four replicat ed plots of plants containing MON 87712 and the conventiona l control A3525 were planted using a randomized complete block field design. OS L-1 - OSL-4, OSR-3, forage-1, and seed samples were collected from each replicated pl ot 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 tissu e 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, Sw itzerland) with the appropriate amount of BBX32 extraction buffer [50 mM Tris-HCl bu ffer, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) IGEPAL CA-630, 5 mM NaF, and 5 mM NaVO 4 

### , supplemented with 4 mM TCEP, neutralized with 0.65 M Na 2 

### HPO 4 

### 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, andMonsanto Company 11-SY-217U 245 of 467 

### was rotated at 4  C for 120 minutes. Each extract wa s clarified by cent rifugation 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 an tibody 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 Prot ein G affinity chroma tography and was used as the primary antibody in the BBX32 western blot. The antibody was then aliquoted and assigned lot number, G 863954. The concentrat ion of the purified monoclonal antibody was determined to be 1.60 mg/ml by spectr ophotometric 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 West ern Blot Method

Extracts were analyzed by Sodium Dodecy l Sulphate Polyacrylam ide Gel Electrophoresis (SDS PAGE) using a Bis-Tris 10% polyacrylamide Midi Ge l (Invitrogen, Carlsbad, CA, Cat # WG1202BOX). Prior to loading, the sa mples including the conventional controls were diluted in 2 × Laemmli Buffer (Bio Ra d, 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 stan dards were prepared in tissue-specific

conventional control extracts and d iluted in 1 × Laemmli buffer.

Preliminary analysis indicated that the BBX 32 protein was not detectable in seed and forage tissues. Therefore, two different a pproaches for running samples and reference standards were taken depending on the expression level.. Fo r tissues where the protein was detectable (leaf and root ), the reference standards we re diluted in a conventional

tissue extract and loaded at concentrations ra nging 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. Fo r seed and forage, only a single reference standard, diluted in conventional tissue extrac t, 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 sele cted tissue specific sample. Additionally,

the Precision Plus Protein Dual Color Sta ndards (Bio Rad, Cat # 161 0374) was loaded on the gel to demonstrate the transfer of prot ein to the membrane and for the approximate molecular weight determination. Elec trophoresis was conduc ted at 100 V for approximately 120 minutes in MES running buffer (Invitrogen, Cat# NP0002), pH 7.0, with 4 mM TCEP (Thermo Sc ientific, Cat # 20491).

Proteins separated by SDS PAGE were electrophoretically tr ansferred to 0.45 μ m Invitrolon Polyvinylidene fl uoride (PVDF) membrane (Invitrogen, Cat # LC2007) using 1 × NuPage transfer buffer (I nvitrogen, Cat # NP0006 1) cont aining 20% methanol at 300Monsanto Company 11-SY-217U 246 of 467 

### mA for approximately one hour. After transf er, 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 × Phospha te Buffered Saline with 0.05% (v/v) Tween 20 (1 × PBST). The membrane was probed fo r the presence of th e 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 rem oved by washing four times for 10 minuteseach in 1 × PBST. Bound antibody was probed with a 1:5000 dilution of horse antimouse IgG antibody conjugated to horseradi sh peroxidase (Cell Signaling Technology, Cat # 7076, lot 22) in 1 × PB ST 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 Enha ncer solution and the SuperSignal Western

Dura Stable Peroxide Buffer were added to th e membrane at a 1:2 ra tio according to the

manufacturer’s instructions. The membra ne was exposed to Amersham Hyperfilm enhanced chemiluninescence (ECL) (GE H ealthcare, Cat # 28906839) and the film was developed using a Konica SRX 101A auto mated 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 Moistu re 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 averag e percent moisture for each TSSP was calculated from triplicate an alyses. 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 fres h 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 repor ted on a dry weight basis.Monsanto Company 11-SY-217U 247 of 467 

### E.8. Data Analyses

The standards present on the deve loped film were used to esta blish a standard curve. At least three standard points were utilized to generate the standard curve for sample quantification. Anti BBX32 antibodies de tected 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 duplicat e bands were visible for a sample they

were used if each value was within 33% of th e average. For leaf and root tissues where BBX32 was not detected, the levels were re ported as below the limit of detection (<LOD). LOD is the protein amount correspond ing 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 blot s 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 th e standard curve of the west ern 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 conve rted 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 samp le means, standard deviations (SDs), and ranges were also calculated by Microsoft Ex cel 2007. All protein expression levels were rounded to two signi ficant figures.

For forage and seed samples each extract was loaded in a single well and a single

reference standard was used. Western blot analysis confir med that the BBX32 protein was not detected in forage and seed. Separate ly, the LOD of the BBX32 protein in forage and seed tissues was determined by seria lly 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 we re analyzed using the aforementioned

materials and methods with th e exception of the standard curve preparation which was

done as follows: Prior to lo ading, the samples were dilute d in 2 × Laemmli Buffer (BioRad, Hercules, CA, Cat # 161-0737). The seed tissue extract was furt her diluted 2-fold

with 1× Laemmli Buffer to reduce matrix eff ects. The BBX32 protein reference standard (lot 11267091) was serially dilu ted into the diluted extract s to generate six protein concentrations to be loaded on the ge l 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, coole d, and then 10 μl of each sample was loaded on to the gel in tr iplicate. The LOD of BBX32 on the western blots was defined by the lowest amount of BB X32 protein that resulted in a visible band

on the western blot.Monsanto Company 11-SY-217U 248 of 467 

### Appendix F: Materials, Methods, and I ndividual-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 a nd 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 assess ment was conducted on seed and forage samples harvested from a single growing s eason conducted in the U.S. during 2009 under typical agronomic practices.

The materials and methods for compositional an alysis, 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 co mpositionally assessed. The conventional

commercial reference varieties are listed in Table F-1.

Table F-1. Commercial Ref erence 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 NEYOMonsanto Company 11-SY-217U 249 of 467 

### 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 th e study by confirming the chain-of-custody documentation supplied with the seed and fora ge harvested from the field plots. The harvested seed of MON 87712, the conventiona l 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), C linton County, Illinoi s (ILCY), Madison County, Illinois (ILHI), Stark County, Illinoi s (ILWY), Boone County, Indiana (INSH), Pawnee County, Kansas (KSLA), and York C ounty, Nebraska (NEYO). The starting seeds were planted in a randomized complete block design with four blocks per site. MON 87712, the conventional control, and th e commercial referen ce 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 obtai ned from each tissue sample collected. These subsamples were ground and stor ed in a freezer set to maintain

 20°C until their shipment on dry ice to Covance Laboratori es, Inc. (Madison, W I) 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 a nd 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 incl uded raffinose, stachyose, lectin, phytic acid, trypsin inhibitors, and is oflavones (daidzein, genist ein, and glycitein).

All compositional analyses were performed at Covance Laboratories, Inc. (Madison, WI). Methods for analysis were based on internat ionally-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, carbohydrat e, and ash. An acetone wash removed the fats and pigments. The lignocellulose fracti on was collected on the frit and determined gravimetrically (Goering and Van Soest, 1970) . The limit of quantitation (LOQ) for this study was 0.100%.Monsanto Company 11-SY-217U 250 of 467 

### 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. Th e sulfur-containing ami no 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-Internat ional, 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-Tryptopha n, 100%, Lot Number 097K0119

 Sigma-Aldrich, L-Cysteic Acid Mo nohydrate, 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 gravimetrical ly 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 ac id 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 s odium sulfate column. The hexane extract was then evaporated again on a steambat h under nitrogen, dried, and weighed (AOACInternational, current edition). The lim it of quantitation for this study was 0.100%.

F.4.6. Fat by Soxhlet Extraction

The sample was weighed into a cellulose thim ble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the sample to re move the fat. The extract was then evaporated, dried, and wei ghed (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 in ternal standard. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation (AOAC-International, 2005c; AOC S, 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 Linole nate, >99%, Lot Number U-63M-08-T

 Nu Chek Prep, Methyl Tridecanoate , >99%, Lot Number N-13M-MA25-TMonsanto 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 syst em with ultraviolet detection and was compared to an external standard curve of known standards for quantitation (Pettersson and Kiessling, 1984; Seo and Morr, 1984). Th e limit of quantitation for each component was 10.0 ppm ( μ g/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 c ontaining PBS. A 10% hematocrit of lyophilized ra bbit blood in PBS was added to each dilution. After 2.5 hours, the absorbance of each d ilution of the sample and lec tin control was measured on a spectrophotometer at 620 nm, using PBS to ze ro the instrument. One hemagglutinating unit (H.U.) was defined as the level that caus ed 50% of the standard cell suspension to sediment in 2.5 hours (Klurf eld 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 qua ntitation 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, carbohydr ate, enzyme, and ash. An acetone wash removed the fats and pigments. Hemicellu lose, cellulose, and li gnin 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-performan ce liquid chromatography column PRP-1,

5  m (150 x 4.1mm) with a refractive index de tector (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 sulfur ic acid containing a catalyst mixture. The acid digest was

made alkaline. The ammonia was distille d and then titrated with a previously standardized acid. The per cent nitrogen was calculated a nd converted to equivalent protein using the factor 6.25 (AOAC-International, 2005d; AOC S, 1997a). The limit of quantitation for this study was 0.100%.

F.5.14. Raffinose and Stachyose

Sugars in the sample are ex tracted with a 50:50 water:meth anol solution. Aliquots are taken, dried under inert gas, and then rec onstituted with a hydroxylamine hydrochloride solution in pyridine containing phenyl- β -D-glucopyranoside as the internal standard. The resulting oximes were converted to si lyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization detector (Brobst, 1972; Mas on and Slover, 1971). The quantitation limit for this study was 0.0500%.

Reference Standards:

 Sigma-Aldrich, D-(+)-Raffinose Pent ahydrate, 99% Lot Number 037K1059

 Sigma-Aldrich, Stachyose Hydrate, 98%, Lot Number 049K3800

F.5.15. Trypsin Inhibitor

The sample was ground and defatted with petr oleum ether. A sample of matrix was extracted with 0.01N sodium hydroxide. Varyi ng aliquots of the sample suspension were exposed to a known amount of trypsin a nd 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 a ddition of acetic acid. The solution was centrifuged, then the absorban ce was determined at 410 nm. Trypsin inhibitor activity was determined by photometrically measuring th e inhibition of trypsin’s reaction with benzoyl-DL-arginine~p~nitroani lide hydrochloride (Kakade et al, 1997). The limit of quantitation for this study was 1.00 Tr ypsin 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 hi gh-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 revi ewed, formatted, and sent to Certus International, Inc. (Chesterfield, MO) for statistical analysis.

The following formulas were used for re-e xpression of soybean composition data for statistical analysis (Table F-2):

Table F-2. Re-expression Formulas for St atistical Analysis of Composition Data

Component From (X) To Formula 1
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)X j /  X, for

each FA j where

 X is over all

the FA

1 ‘X’ is the individual sample value; ‘d’ is th e fraction of the sample that is dry matter.



### In order to complete a statisti cal 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 observati ons below the assay LOQ were excluded from summaries and analysis. The following 14 anal ytes 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 pentad ecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecen oic acid, 18:3 gamma-linolenic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, a nd 20:4 arachidonic acid.Monsanto Company 11-SY-217U 255 of 467 

### If less than 50% of the observations for a component we re below the LOQ, individual analyses that were below the LOQ were assign ed a value equal to one-half the LOQ. The following analyte was assigne d 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 Assigned N (%) Seed Fatty Acid

20:1 Eicosenoic % fwt 25 13.2 190 0.020 0.010

The data were assessed for potential outli ers 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 va lues are expected to be between  3. Extreme data points that are also outside of the  6 studentized PRESS resi dual 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 st atistically 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) Y ij 

### = U + T i 

### + B j 

### + e ij 

### ,

where Y ij 

### = unique individual observa tion, U = overall mean, T i 

### = substance effect,

B j 

### = random block effect, and e ij 

### = residual error.

Combined-site analyses used model (2)

.

(2) Y ijk 

### = U + T i 

### + L j 

### + B(L) jk



### + LT ij 

### + e ijk,



### where Y ijk 

### = unique individua l observation, U = overall mean, T i 

### = substance effect,

L

j 

### = random site effect, B(L) jk



### = random block within site effect,

LT ij 

### = random site by substance interaction effect, and e ijk 

### = residual error.

A range of observed values from the refe rence varieties was determined for each analytical component. Additionally, the re ference varieties were used to develop population tolerance intervals. A tolerance interval is an interval that one can claim, with a specified degree of co nfidence, contains at least a specified propor tion, p, of an entire sampled population for the parameter measured.Monsanto Company 11-SY-217U 256 of 467 

### For each compositional component, 99% tolera nce intervals were calculated that are expected to contain, with 95% confidence, 99% of the quantitie s expressed in the population of reference varieties. Because negative quantities are not possible, negative

calculated lower to lerance bounds were set to zero.

SAS  

### software was used to generate all summa ry 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 257 of 467Table F-4. Summary of Site ARNE Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.90 (0.13)5.20 (0.13)-0.30 (0.18)-0.75, 0.150.1524.43, 5.89(4.71 - 5.24)(4.86 - 5.66)(-0.78 - -0.060)(4.43 - 6.14)Carbohydrates36.05 (0.51)35.66 (0.51)0.40 (0.65)-1.21, 2.000.56632.36, 41.63(34.74 - 37.51)(34.72 - 36.61)(-0.087 - 0.91)(33.43 - 40.39)Moisture (% fwt)7.57 (0.23)7.94 (0.23)-0.37 (0.31)-1.13, 0.390.2825.41, 10.36(7.21 - 7.89)(7.54 - 8.21)(-0.68 - -0.11)(5.43 - 9.86)Protein41.06 (0.25)40.87 (0.25)0.19 (0.32)-0.59, 0.970.57135.06, 43.58(40.44 - 41.58)(40.39 - 41.75)(-0.42 - 1.09)(35.11 - 42.16)Total Fat17.96 (0.32)18.28 (0.32)-0.31 (0.41)-1.32, 0.690.47113.15, 23.90(17.35 - 18.78)(17.54 - 19.05)(-0.45 - -0.19)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber17.99 (0.87)18.71 (0.87)-0.72 (1.19)-3.63, 2.190.5669.99, 22.21(17.24 - 19.11)(17.80 - 19.68)(-2.40 - 0.71)(11.74 - 22.13)Monsanto Company 11-SY-217U 258 of 467Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber19.42 (0.54)19.39 (0.54)0.033 (0.76)-1.82, 1.890.96711.03, 23.27(18.32 - 20.74)(17.80 - 20.37)(-1.83 - 1.47)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.77 (0.015)1.76 (0.015)0.0092 (0.021)-0.042, 0.0600.6721.54, 1.88(1.76 - 1.79)(1.73 - 1.81)(-0.024 - 0.039)(1.58 - 1.84)Arginine3.08 (0.025)3.03 (0.025)0.053 (0.036)-0.035, 0.140.1892.51, 3.33(3.05 - 3.12)(2.96 - 3.11)(0.0071 - 0.16)(2.57 - 3.24)Aspartic Acid4.81 (0.037)4.72 (0.037)0.090 (0.053)-0.039, 0.220.1394.04, 5.07(4.73 - 4.91)(4.63 - 4.79)(-0.0027 - 0.24)(4.06 - 4.89)Cystine0.60 (0.013)0.62 (0.013)-0.027 (0.018)-0.072, 0.0180.1990.51, 0.67(0.56 - 0.63)(0.61 - 0.63)(-0.052 - -0.0029)(0.54 - 0.69)Glutamic Acid7.68 (0.062)7.54 (0.062)0.14 (0.088)-0.071, 0.360.1526.28, 8.18(7.53 - 7.81)(7.44 - 7.67)(0.056 - 0.36)(6.40 - 7.94)Monsanto Company 11-SY-217U 259 of 467Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.80 (0.012)1.78 (0.012)0.015 (0.017)-0.027, 0.0560.4221.52, 1.90(1.77 - 1.81)(1.76 - 1.81)(-0.013 - 0.050)(1.54 - 1.85)Histidine1.07 (0.0077)1.07 (0.0077)0.0053 (0.011)-0.021, 0.0320.6450.91, 1.17(1.06 - 1.08)(1.05 - 1.08)(-0.0058 - 0.030)(0.93 - 1.16)Isoleucine1.93 (0.025)1.90 (0.025)0.028 (0.035)-0.058, 0.110.4591.62, 2.03(1.91 - 1.97)(1.82 - 1.97)(-0.058 - 0.089)(1.60 - 2.00)Leucine3.16 (0.022)3.13 (0.022)0.031 (0.031)-0.045, 0.110.3552.71, 3.38(3.12 - 3.20)(3.08 - 3.19)(-0.023 - 0.11)(2.77 - 3.29)Lysine2.67 (0.016)2.67 (0.016)0.0057 (0.022)-0.049, 0.0600.8062.33, 2.81(2.65 - 2.70)(2.63 - 2.70)(-0.031 - 0.069)(2.36 - 2.74)Methionine0.56 (0.012)0.57 (0.012)-0.0071 (0.017)-0.048, 0.0340.6820.51, 0.59(0.52 - 0.60)(0.56 - 0.58)(-0.031 - 0.017)(0.51 - 0.60)Monsanto Company 11-SY-217U 260 of 467Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.17 (0.018)2.14 (0.018)0.029 (0.026)-0.034, 0.0930.3011.81, 2.33(2.15 - 2.19)(2.10 - 2.17)(-0.016 - 0.082)(1.81 - 2.25)Proline2.01 (0.020)1.99 (0.020)0.025 (0.029)-0.046, 0.0950.4231.70, 2.13(1.99 - 2.06)(1.95 - 2.04)(-0.046 - 0.088)(1.69 - 2.09)Serine2.16 (0.016)2.16 (0.016)0.0022 (0.023)-0.053, 0.0570.9241.86, 2.33(2.11 - 2.20)(2.13 - 2.20)(-0.065 - 0.071)(1.90 - 2.30)Threonine1.60 (0.016)1.56 (0.016)0.034 (0.023)-0.021, 0.0900.1771.40, 1.69(1.57 - 1.63)(1.50 - 1.59)(-0.0077 - 0.13)(1.36 - 1.68)Tryptophan0.44 (0.0089)0.45 (0.0089)-0.012 (0.013)-0.043, 0.0190.3670.36, 0.50(0.41 - 0.46)(0.44 - 0.46)(-0.043 - 0.021)(0.38 - 0.48)Tyrosine1.48 (0.016)1.49 (0.016)-0.014 (0.022)-0.069, 0.0400.5501.28, 1.57(1.45 - 1.52)(1.47 - 1.51)(-0.045 - 0.021)(1.28 - 1.55)Monsanto Company 11-SY-217U 261 of 467Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine1.99 (0.029)1.96 (0.029)0.030 (0.041)-0.069, 0.130.4841.71, 2.13(1.95 - 2.02)(1.85 - 2.02)(-0.069 - 0.13)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.69 (0.043)12.01 (0.043)-0.32 (0.057)-0.46, -0.180.0017.76, 13.14(11.54 - 11.80)(11.96 - 12.07)(-0.44 - -0.21)(9.00 - 12.03)18:0 Stearic3.85 (0.033)4.16 (0.033)-0.31 (0.031)-0.38, -0.23<0.0013.06, 5.10(3.78 - 3.89)(4.07 - 4.23)(-0.34 - -0.22)(3.49 - 4.97)18:1 Oleic24.18 (0.40)21.03 (0.40)3.15 (0.53)1.85, 4.450.00117.37, 26.86(23.08 - 25.71)(20.80 - 21.18)(2.07 - 4.66)(18.93 - 25.33)18:2 Linoleic51.50 (0.33)53.62 (0.33)-2.12 (0.46)-3.25, -0.990.00350.14, 57.81(50.26 - 52.27)(53.44 - 53.90)(-3.63 - -1.28)(51.57 - 56.25)18:3 Linolenic8.06 (0.089)8.39 (0.089)-0.32 (0.12)-0.61, -0.0400.0315.60, 11.61(7.87 - 8.29)(8.17 - 8.59)(-0.68 - -0.064)(5.89 - 10.16)Monsanto Company 11-SY-217U 262 of 467Table F-4. Summary of Site ARNE Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.29 (0.0084)0.32 (0.0084)-0.031 (0.012)-0.060, -0.00180.0400.22, 0.39(0.26 - 0.30)(0.30 - 0.34)(-0.077 - -0.0061)(0.23 - 0.38)20:1 Eicosenoic0.19 (0.0040)0.18 (0.0040)0.0010 (0.0057)-0.013, 0.0150.8630.094, 0.23(0.18 - 0.19)(0.17 - 0.19)(-0.011 - 0.019)(0.072 - 0.21)22:0 Behenic0.25 (0.019)0.29 (0.019)-0.049 (0.026)-0.11, 0.0140.1050.18, 0.43(0.22 - 0.31)(0.24 - 0.32)(-0.092 - -0.00071)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E1.78 (0.091)2.16 (0.091)-0.39 (0.13)-0.70, -0.0730.0230.10, 2.85(1.58 - 1.88)(2.00 - 2.23)(-0.64 - -0.12)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 263 of 467Table F-5. Summary of Site ARNE Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)2.53 (0.24)2.99 (0.24)-0.45 (0.32)-1.23, 0.320.2040, 6.11(2.23 - 3.17)(2.53 - 3.66)(-1.43 - 0.031)(0.60 - 6.99)Phytic Acid (% dwt)1.14 (0.087)1.22 (0.087)-0.075 (0.12)-0.37, 0.230.5650.50, 1.92(0.86 - 1.45)(0.97 - 1.32)(-0.46 - 0.15)(0.66 - 1.74)Raffinose (% dwt)0.82 (0.016)0.73 (0.016)0.098 (0.0097)0.074, 0.12<0.0010.39, 1.01(0.77 - 0.86)(0.69 - 0.75)(0.082 - 0.14)(0.45 - 0.93)Stachyose (% dwt)4.13 (0.078)3.80 (0.078)0.33 (0.11)0.068, 0.600.0212.45, 5.34(3.94 - 4.33)(3.52 - 3.99)(0.077 - 0.53)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)28.47 (2.28)36.40 (2.28)-7.93 (2.78)-14.74, -1.110.02920.97, 50.01(24.54 - 34.92)(31.47 - 42.82)(-15.39 - -1.56)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein603.87 (30.50)646.83 (30.50)-42.96 (38.60)-137.42, 51.490.3080, 1756.99(549.34 - 687.57)(537.10 - 728.12)(-178.78 - 63.51)(138.15 - 1548.98)Monsanto Company 11-SY-217U 264 of 467Table F-5. Summary of Site ARNE Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein532.00 (35.50)610.70 (35.50)-78.70 (43.31)-184.67, 27.270.11987.22, 1792.07(503.75 - 559.33)(508.77 - 778.42)(-219.09 - 39.80)(335.67 - 1409.07)Glycitein116.05 (10.94)116.47 (10.94)-0.42 (14.75)-36.51, 35.670.9788.13, 299.67(106.50 - 131.79)(86.28 - 162.85)(-46.46 - 23.21)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 265 of 467Table F-6. Summary of Site ARNE Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.42 (0.38)5.94 (0.38)0.48 (0.43)-0.57, 1.530.3054.29, 8.65(5.35 - 7.91)(5.32 - 6.54)(-1.19 - 1.62)(4.82 - 8.98)Carbohydrates66.80 (0.56)67.35 (0.56)-0.55 (0.79)-2.49, 1.390.51655.73, 77.45(65.75 - 67.72)(65.78 - 69.37)(-2.29 - 1.94)(54.40 - 72.96)Moisture (% fwt)75.53 (0.54)72.63 (0.54)2.90 (0.65)1.30, 4.500.00465.61, 80.67(74.60 - 77.50)(71.60 - 73.70)(0.90 - 3.90)(64.50 - 79.80)Protein22.25 (0.31)21.38 (0.31)0.87 (0.40)-0.10, 1.850.07113.77, 26.51(21.60 - 22.99)(20.35 - 22.32)(0.12 - 1.62)(16.56 - 27.76)Total Fat4.41 (0.31)5.39 (0.31)-0.97 (0.44)-2.05, 0.100.0680.54, 13.11(3.64 - 4.96)(5.04 - 6.10)(-2.46 - -0.076)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber32.48 (1.30)31.88 (1.30)0.60 (1.36)-2.73, 3.930.67323.12, 38.15(30.71 - 34.07)(26.14 - 35.21)(-2.21 - 6.09)(22.60 - 41.29)Monsanto Company 11-SY-217U 266 of 467Table F-6. Summary of Site ARNE Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber37.01 (1.72)36.71 (1.72)0.30 (1.99)-4.56, 5.160.88424.96, 43.33(33.46 - 41.73)(33.69 - 39.79)(-4.41 - 6.39)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 267 of 467Table F-7. Summary of Site ILCY Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.86 (0.14)5.05 (0.14)-0.19 (0.19)-0.66, 0.280.3574.43, 5.89(4.48 - 5.27)(4.64 - 5.24)(-0.73 - 0.62)(4.43 - 6.14)Carbohydrates37.26 (0.54)37.66 (0.54)-0.40 (0.76)-2.26, 1.450.61432.36, 41.63(35.09 - 38.66)(37.17 - 38.16)(-3.07 - 1.01)(33.43 - 40.39)Moisture (% fwt)8.34 (0.27)8.06 (0.27)0.29 (0.38)-0.63, 1.200.4765.41, 10.36(7.78 - 9.13)(7.44 - 8.40)(-0.18 - 0.73)(5.43 - 9.86)Protein41.34 (0.37)40.95 (0.37)0.39 (0.51)-0.85, 1.640.46535.06, 43.58(39.62 - 42.09)(40.59 - 41.27)(-1.65 - 1.22)(35.11 - 42.16)Total Fat16.56 (0.41)16.34 (0.41)0.22 (0.58)-1.19, 1.630.71713.15, 23.90(14.77 - 17.60)(15.83 - 16.75)(-1.88 - 1.47)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber15.30 (0.51)13.46 (0.51)1.84 (0.42)0.82, 2.860.0049.99, 22.21(13.99 - 16.40)(12.87 - 14.08)(1.13 - 2.47)(11.74 - 22.13)Monsanto Company 11-SY-217U 268 of 467Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber16.42 (0.46)14.58 (0.46)1.84 (0.54)0.52, 3.170.01411.03, 23.27(15.64 - 17.27)(13.40 - 15.37)(1.28 - 2.76)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.81 (0.016)1.76 (0.016)0.049 (0.020)-0.00087, 0.0990.0521.54, 1.88(1.78 - 1.85)(1.74 - 1.78)(0.038 - 0.069)(1.58 - 1.84)Arginine3.28 (0.034)3.11 (0.034)0.18 (0.045)0.070, 0.290.0072.51, 3.33(3.23 - 3.38)(3.03 - 3.20)(0.11 - 0.23)(2.57 - 3.24)Aspartic Acid4.84 (0.035)4.75 (0.035)0.097 (0.049)-0.023, 0.220.0964.04, 5.07(4.77 - 4.91)(4.69 - 4.83)(0.078 - 0.13)(4.06 - 4.89)Cystine0.61 (0.0072)0.63 (0.0072)-0.024 (0.010)-0.049, 0.00110.0580.51, 0.67(0.59 - 0.62)(0.61 - 0.66)(-0.051 - 0.00050)(0.54 - 0.69)Glutamic Acid7.78 (0.065)7.57 (0.065)0.21 (0.081)0.010, 0.410.0426.28, 8.18(7.63 - 7.87)(7.46 - 7.66)(0.17 - 0.29)(6.40 - 7.94)Monsanto Company 11-SY-217U 269 of 467Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.84 (0.011)1.79 (0.011)0.044 (0.016)0.0041, 0.0830.0351.52, 1.90(1.80 - 1.85)(1.77 - 1.81)(0.029 - 0.061)(1.54 - 1.85)Histidine1.10 (0.0083)1.08 (0.0083)0.024 (0.012)-0.0043, 0.0530.0820.91, 1.17(1.08 - 1.12)(1.07 - 1.09)(0.011 - 0.033)(0.93 - 1.16)Isoleucine1.94 (0.018)1.87 (0.018)0.071 (0.025)0.010, 0.130.0291.62, 2.03(1.87 - 1.97)(1.84 - 1.91)(0.029 - 0.13)(1.60 - 2.00)Leucine3.23 (0.021)3.15 (0.021)0.073 (0.028)0.0038, 0.140.0412.71, 3.38(3.18 - 3.27)(3.12 - 3.20)(0.059 - 0.087)(2.77 - 3.29)Lysine2.71 (0.018)2.68 (0.018)0.036 (0.026)-0.027, 0.0980.2112.33, 2.81(2.66 - 2.74)(2.66 - 2.70)(-0.0052 - 0.053)(2.36 - 2.74)Methionine0.58 (0.0049)0.59 (0.0049)-0.0082 (0.0067)-0.025, 0.00820.2650.51, 0.59(0.57 - 0.59)(0.58 - 0.60)(-0.025 - 0.012)(0.51 - 0.60)Monsanto Company 11-SY-217U 270 of 467Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.22 (0.016)2.15 (0.016)0.070 (0.023)0.014, 0.130.0221.81, 2.33(2.17 - 2.26)(2.13 - 2.17)(0.039 - 0.083)(1.81 - 2.25)Proline2.04 (0.029)2.00 (0.029)0.036 (0.040)-0.063, 0.130.4091.70, 2.13(1.91 - 2.09)(1.95 - 2.06)(-0.15 - 0.14)(1.69 - 2.09)Serine2.22 (0.028)2.20 (0.028)0.018 (0.035)-0.069, 0.100.6321.86, 2.33(2.21 - 2.22)(2.18 - 2.24)(-0.024 - 0.039)(1.90 - 2.30)Threonine1.63 (0.013)1.62 (0.013)0.011 (0.019)-0.035, 0.0560.5911.40, 1.69(1.58 - 1.66)(1.59 - 1.65)(-0.0065 - 0.040)(1.36 - 1.68)Tryptophan0.43 (0.015)0.46 (0.015)-0.036 (0.022)-0.089, 0.0170.1460.36, 0.50(0.36 - 0.47)(0.44 - 0.49)(-0.094 - 0.010)(0.38 - 0.48)Tyrosine1.50 (0.031)1.45 (0.031)0.057 (0.044)-0.050, 0.160.2421.28, 1.57(1.42 - 1.57)(1.40 - 1.51)(-0.090 - 0.18)(1.28 - 1.55)Monsanto Company 11-SY-217U 271 of 467Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine2.05 (0.022)1.96 (0.022)0.083 (0.032)0.0052, 0.160.0401.71, 2.13(1.95 - 2.08)(1.92 - 2.01)(0.018 - 0.15)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.79 (0.044)11.85 (0.044)-0.065 (0.038)-0.16, 0.0290.1417.76, 13.14(11.73 - 11.86)(11.76 - 11.97)(-0.17 - 0.013)(9.00 - 12.03)18:0 Stearic3.77 (0.049)4.00 (0.049)-0.22 (0.039)-0.32, -0.130.0013.06, 5.10(3.71 - 3.91)(3.94 - 4.14)(-0.25 - -0.18)(3.49 - 4.97)18:1 Oleic19.99 (0.29)20.31 (0.29)-0.32 (0.34)-1.15, 0.510.38017.37, 26.86(19.88 - 20.04)(19.60 - 21.45)(-1.57 - 0.44)(18.93 - 25.33)18:2 Linoleic54.93 (0.21)54.34 (0.21)0.59 (0.23)0.017, 1.160.04550.14, 57.81(54.84 - 55.03)(53.49 - 54.78)(0.059 - 1.50)(51.57 - 56.25)18:3 Linolenic8.89 (0.15)8.76 (0.15)0.13 (0.17)-0.29, 0.550.4695.60, 11.61(8.69 - 9.13)(8.17 - 9.09)(-0.38 - 0.52)(5.89 - 10.16)Monsanto Company 11-SY-217U 272 of 467Table F-7. Summary of Site ILCY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.27 (0.0090)0.30 (0.0090)-0.027 (0.013)-0.058, 0.00450.0810.22, 0.39(0.25 - 0.29)(0.29 - 0.31)(-0.042 - -0.018)(0.23 - 0.38)20:1 Eicosenoic0.14 (0.012)0.16 (0.012)-0.027 (0.017)-0.068, 0.0140.1550.094, 0.23(0.077 - 0.16)(0.16 - 0.17)(-0.082 - -0.0059)(0.072 - 0.21)22:0 Behenic0.23 (0.021)0.29 (0.021)-0.062 (0.029)-0.13, 0.00940.0770.18, 0.43(0.18 - 0.28)(0.29 - 0.30)(-0.11 - -0.012)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E1.03 (0.056)1.18 (0.056)-0.15 (0.079)-0.34, 0.0430.1060.10, 2.85(0.89 - 1.14)(1.09 - 1.28)(-0.28 - -0.0056)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 273 of 467Table F-8. Summary of Site ILCY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)2.24 (0.44)2.60 (0.44)-0.36 (0.39)-1.32, 0.600.3940, 6.11(1.71 - 2.56)(1.42 - 3.69)(-1.35 - 0.94)(0.60 - 6.99)Phytic Acid (% dwt)1.22 (0.038)1.25 (0.038)-0.029 (0.054)-0.16, 0.100.6100.50, 1.92(1.10 - 1.28)(1.22 - 1.30)(-0.20 - 0.048)(0.66 - 1.74)Raffinose (% dwt)0.78 (0.016)0.70 (0.016)0.076 (0.019)0.030, 0.120.0060.39, 1.01(0.72 - 0.83)(0.68 - 0.72)(0.036 - 0.12)(0.45 - 0.93)Stachyose (% dwt)4.02 (0.091)4.24 (0.091)-0.22 (0.12)-0.52, 0.0700.1112.45, 5.34(3.77 - 4.43)(4.18 - 4.31)(-0.41 - 0.12)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)32.64 (2.52)33.68 (2.52)-1.04 (3.56)-9.76, 7.680.78020.97, 50.01(30.15 - 36.92)(29.00 - 37.06)(-6.91 - 4.12)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein1056.89 (32.01)1049.27 (32.01)7.63 (45.27)-103.14, 118.400.8710, 1756.99(980.67 - 1126.05)(943.09 - 1120.78)(-140.11 - 182.96)(138.15 - 1548.98)Monsanto Company 11-SY-217U 274 of 467Table F-8. Summary of Site ILCY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein915.27 (22.00)863.79 (22.00)51.48 (30.69)-23.62, 126.580.14487.22, 1792.07(877.25 - 957.41)(802.44 - 912.95)(-31.11 - 142.13)(335.67 - 1409.07)Glycitein92.70 (6.02)97.78 (6.02)-5.08 (8.52)-25.93, 15.770.5738.13, 299.67(82.10 - 107.24)(89.62 - 121.18)(-23.24 - 16.92)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 275 of 467Table F-9. Summary of Site ILCY Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash7.68 (0.20)7.22 (0.20)0.47 (0.23)-0.11, 1.040.0934.29, 8.65(7.49 - 7.88)(7.10 - 7.45)(0.28 - 0.70)(4.82 - 8.98)Carbohydrates65.33 (0.77)65.29 (0.77)0.039 (0.91)-2.18, 2.260.96755.73, 77.45(63.14 - 66.31)(64.37 - 66.28)(-1.35 - 1.94)(54.40 - 72.96)Moisture (% fwt)72.85 (0.43)74.70 (0.43)-1.85 (0.46)-2.97, -0.730.00665.61, 80.67(71.80 - 74.50)(73.80 - 75.50)(-3.50 - -1.00)(64.50 - 79.80)Protein22.12 (0.59)22.05 (0.59)0.069 (0.74)-1.74, 1.880.92813.77, 26.51(21.06 - 24.04)(21.72 - 22.49)(-1.04 - 1.55)(16.56 - 27.76)Total Fat4.83 (0.30)5.39 (0.30)-0.56 (0.42)-1.58, 0.450.2240.54, 13.11(4.69 - 4.93)(4.57 - 6.15)(-1.33 - 0.12)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber29.44 (0.60)28.68 (0.60)0.76 (0.85)-1.31, 2.830.40323.12, 38.15(28.24 - 30.21)(28.09 - 30.32)(-0.11 - 1.68)(22.60 - 41.29)Monsanto Company 11-SY-217U 276 of 467Table F-9. Summary of Site ILCY Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber33.99 (1.04)31.13 (1.04)2.86 (1.48)-0.76, 6.470.10124.96, 43.33(33.35 - 34.71)(28.38 - 32.98)(1.20 - 5.66)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A35255With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 277 of 467Table F-10. Summary of Site ILHI Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.67 (0.12)4.80 (0.12)-0.13 (0.17)-0.54, 0.290.4804.43, 5.89(4.56 - 4.91)(4.46 - 5.34)(-0.73 - 0.22)(4.43 - 6.14)Carbohydrates35.62 (0.34)35.84 (0.34)-0.22 (0.48)-1.39, 0.940.65732.36, 41.63(34.64 - 36.63)(35.50 - 36.10)(-1.27 - 1.13)(33.43 - 40.39)Moisture (% fwt)8.34 (0.16)8.20 (0.16)0.14 (0.21)-0.37, 0.640.5365.41, 10.36(8.00 - 8.49)(7.89 - 8.49)(-0.020 - 0.38)(5.43 - 9.86)Protein42.14 (0.36)41.39 (0.36)0.75 (0.50)-0.49, 1.980.18835.06, 43.58(40.98 - 42.79)(41.09 - 42.12)(-1.15 - 1.56)(35.11 - 42.16)Total Fat17.56 (0.23)17.97 (0.23)-0.41 (0.22)-0.94, 0.120.10813.15, 23.90(16.83 - 18.14)(17.70 - 18.28)(-0.88 - 0.13)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber14.48 (0.58)16.40 (0.58)-1.91 (0.82)-3.92, 0.100.0599.99, 22.21(13.21 - 16.72)(15.89 - 16.91)(-3.70 - 0.83)(11.74 - 22.13)Monsanto Company 11-SY-217U 278 of 467Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber15.77 (0.35)16.45 (0.35)-0.68 (0.50)-1.90, 0.530.21811.03, 23.27(14.35 - 16.83)(15.96 - 17.05)(-1.61 - 0.83)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.81 (0.018)1.79 (0.018)0.019 (0.025)-0.043, 0.0810.4821.54, 1.88(1.76 - 1.82)(1.75 - 1.80)(-0.041 - 0.073)(1.58 - 1.84)Arginine3.21 (0.060)3.16 (0.060)0.046 (0.084)-0.16, 0.250.6052.51, 3.33(3.05 - 3.31)(3.11 - 3.26)(-0.20 - 0.19)(2.57 - 3.24)Aspartic Acid4.84 (0.060)4.73 (0.060)0.11 (0.085)-0.10, 0.310.2604.04, 5.07(4.68 - 4.92)(4.65 - 4.83)(-0.15 - 0.24)(4.06 - 4.89)Cystine0.58 (0.0096)0.59 (0.0096)-0.011 (0.011)-0.038, 0.0170.3750.51, 0.67(0.57 - 0.60)(0.57 - 0.60)(-0.031 - 0.0059)(0.54 - 0.69)Glutamic Acid7.79 (0.11)7.62 (0.11)0.17 (0.15)-0.19, 0.540.2916.28, 8.18(7.49 - 7.96)(7.49 - 7.74)(-0.25 - 0.38)(6.40 - 7.94)Monsanto Company 11-SY-217U 279 of 467Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.82 (0.018)1.80 (0.018)0.025 (0.025)-0.037, 0.0860.3691.52, 1.90(1.77 - 1.84)(1.77 - 1.81)(-0.041 - 0.062)(1.54 - 1.85)Histidine1.10 (0.015)1.07 (0.015)0.021 (0.021)-0.030, 0.0730.3510.91, 1.17(1.06 - 1.11)(1.05 - 1.09)(-0.024 - 0.051)(0.93 - 1.16)Isoleucine1.94 (0.025)1.92 (0.025)0.022 (0.035)-0.064, 0.110.5531.62, 2.03(1.88 - 1.98)(1.89 - 1.96)(-0.063 - 0.073)(1.60 - 2.00)Leucine3.23 (0.038)3.18 (0.038)0.046 (0.053)-0.085, 0.180.4242.71, 3.38(3.12 - 3.30)(3.12 - 3.24)(-0.12 - 0.12)(2.77 - 3.29)Lysine2.68 (0.025)2.66 (0.025)0.029 (0.036)-0.059, 0.120.4542.33, 2.81(2.62 - 2.72)(2.62 - 2.69)(-0.073 - 0.068)(2.36 - 2.74)Methionine0.57 (0.0074)0.57 (0.0074)-0.00079 (0.0086)-0.022, 0.0200.9290.51, 0.59(0.56 - 0.59)(0.56 - 0.58)(-0.011 - 0.012)(0.51 - 0.60)Monsanto Company 11-SY-217U 280 of 467Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.22 (0.033)2.17 (0.033)0.044 (0.047)-0.070, 0.160.3801.81, 2.33(2.13 - 2.26)(2.14 - 2.20)(-0.073 - 0.11)(1.81 - 2.25)Proline2.03 (0.038)2.05 (0.038)-0.013 (0.039)-0.11, 0.0820.7451.70, 2.13(1.96 - 2.14)(1.99 - 2.11)(-0.095 - 0.056)(1.69 - 2.09)Serine2.21 (0.024)2.16 (0.024)0.044 (0.035)-0.040, 0.130.2481.86, 2.33(2.16 - 2.25)(2.11 - 2.23)(-0.019 - 0.14)(1.90 - 2.30)Threonine1.63 (0.019)1.59 (0.019)0.035 (0.027)-0.031, 0.100.2391.40, 1.69(1.59 - 1.65)(1.55 - 1.66)(-0.074 - 0.083)(1.36 - 1.68)Tryptophan0.46 (0.011)0.46 (0.011)0.00096 (0.016)-0.038, 0.0400.9540.36, 0.50(0.44 - 0.49)(0.44 - 0.48)(-0.037 - 0.028)(0.38 - 0.48)Tyrosine1.49 (0.027)1.48 (0.027)0.010 (0.038)-0.082, 0.100.7901.28, 1.57(1.43 - 1.56)(1.43 - 1.50)(-0.063 - 0.072)(1.28 - 1.55)Monsanto Company 11-SY-217U 281 of 467Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine2.03 (0.027)2.00 (0.027)0.028 (0.039)-0.067, 0.120.5031.71, 2.13(1.96 - 2.07)(1.97 - 2.05)(-0.063 - 0.087)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.37 (0.055)11.55 (0.055)-0.18 (0.036)-0.27, -0.0920.0027.76, 13.14(11.30 - 11.49)(11.46 - 11.72)(-0.23 - -0.070)(9.00 - 12.03)18:0 Stearic4.37 (0.073)4.50 (0.073)-0.12 (0.074)-0.30, 0.0580.1473.06, 5.10(4.16 - 4.58)(4.32 - 4.64)(-0.36 - 0.0014)(3.49 - 4.97)18:1 Oleic21.58 (0.25)20.84 (0.25)0.74 (0.34)-0.083, 1.570.07017.37, 26.86(21.05 - 21.95)(20.62 - 20.98)(0.26 - 1.32)(18.93 - 25.33)18:2 Linoleic54.24 (0.23)54.41 (0.23)-0.16 (0.32)-0.94, 0.620.62950.14, 57.81(53.94 - 54.60)(54.19 - 54.61)(-0.44 - 0.052)(51.57 - 56.25)18:3 Linolenic7.65 (0.10)7.92 (0.10)-0.27 (0.13)-0.59, 0.0440.0795.60, 11.61(7.46 - 8.01)(7.77 - 8.17)(-0.53 - 0.23)(5.89 - 10.16)Monsanto Company 11-SY-217U 282 of 467Table F-10. Summary of Site ILHI Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.32 (0.011)0.32 (0.011)-0.0063 (0.014)-0.041, 0.0280.6740.22, 0.39(0.28 - 0.34)(0.30 - 0.34)(-0.043 - 0.047)(0.23 - 0.38)20:1 Eicosenoic0.17 (0.0035)0.17 (0.0035)0.0014 (0.0047)-0.010, 0.0130.7800.094, 0.23(0.16 - 0.18)(0.16 - 0.18)(-0.0097 - 0.019)(0.072 - 0.21)22:0 Behenic0.30 (0.017)0.29 (0.017)0.0039 (0.024)-0.055, 0.0630.8760.18, 0.43(0.23 - 0.33)(0.23 - 0.32)(-0.073 - 0.096)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E1.65 (0.16)1.85 (0.16)-0.20 (0.21)-0.73, 0.320.3760.10, 2.85(1.30 - 2.05)(1.61 - 2.06)(-0.76 - 0.44)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 283 of 467Table F-11. Summary of Site ILHI Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)1.84 (0.34)2.66 (0.34)-0.81 (0.37)-1.71, 0.0800.0670, 6.11(1.49 - 2.46)(1.88 - 4.15)(-1.69 - 0.035)(0.60 - 6.99)Phytic Acid (% dwt)0.89 (0.072)1.14 (0.072)-0.25 (0.10)-0.50, 0.00380.0520.50, 1.92(0.65 - 1.11)(1.02 - 1.23)(-0.58 - 0.091)(0.66 - 1.74)Raffinose (% dwt)0.78 (0.017)0.90 (0.017)-0.12 (0.024)-0.18, -0.0610.0020.39, 1.01(0.76 - 0.80)(0.84 - 0.93)(-0.17 - -0.047)(0.45 - 0.93)Stachyose (% dwt)4.50 (0.11)4.21 (0.11)0.29 (0.16)-0.097, 0.680.1152.45, 5.34(4.44 - 4.58)(3.69 - 4.48)(0.051 - 0.90)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)35.70 (2.98)32.16 (2.98)3.54 (4.22)-6.79, 13.860.43320.97, 50.01(32.45 - 40.72)(26.40 - 35.84)(-3.39 - 14.32)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein911.92 (40.49)946.01 (40.49)-34.09 (57.27)-174.22, 106.040.5730, 1756.99(811.93 - 1017.39)(827.27 - 1050.16)(-150.09 - 190.12)(138.15 - 1548.98)Monsanto Company 11-SY-217U 284 of 467Table F-11. Summary of Site ILHI Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein750.00 (30.31)754.26 (30.31)-4.26 (37.35)-95.65, 87.130.91287.22, 1792.07(679.71 - 829.24)(661.17 - 849.09)(-76.63 - 122.53)(335.67 - 1409.07)Glycitein86.98 (8.44)96.44 (8.44)-9.47 (11.94)-38.68, 19.750.4588.13, 299.67(78.13 - 99.75)(77.73 - 115.62)(-32.22 - 17.24)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 285 of 467Table F-12. Summary of Site ILHI Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.99 (0.20)5.55 (0.20)0.44 (0.21)-0.081, 0.960.0844.29, 8.65(5.66 - 6.28)(4.79 - 6.11)(-0.11 - 0.87)(4.82 - 8.98)Carbohydrates63.53 (0.73)62.90 (0.73)0.63 (0.48)-0.54, 1.800.23555.73, 77.45(61.74 - 64.50)(61.46 - 64.97)(-0.66 - 1.52)(54.40 - 72.96)Moisture (% fwt)69.00 (0.54)68.35 (0.54)0.65 (0.58)-0.76, 2.060.30265.61, 80.67(67.60 - 70.20)(66.60 - 69.90)(-1.30 - 2.30)(64.50 - 79.80)Protein22.29 (0.55)22.15 (0.55)0.14 (0.28)-0.53, 0.820.62613.77, 26.51(21.30 - 23.56)(20.96 - 23.15)(-0.45 - 0.83)(16.56 - 27.76)Total Fat8.15 (0.27)9.36 (0.27)-1.21 (0.32)-1.98, -0.440.0080.54, 13.11(7.44 - 8.62)(9.06 - 9.80)(-1.89 - -0.63)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber26.24 (0.68)25.53 (0.68)0.71 (0.97)-1.66, 3.070.49223.12, 38.15(24.36 - 27.87)(24.69 - 25.95)(-1.58 - 2.45)(22.60 - 41.29)Monsanto Company 11-SY-217U 286 of 467Table F-12. Summary of Site ILHI Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber29.16 (0.84)26.91 (0.84)2.25 (0.93)-0.023, 4.520.05124.96, 43.33(28.30 - 30.60)(24.92 - 29.59)(-0.96 - 4.19)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 287 of 467Table F-13. Summary of Site ILWY Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.41 (0.22)5.33 (0.22)0.084 (0.31)-0.67, 0.840.7944.43, 5.89(4.98 - 6.36)(4.94 - 5.60)(-0.45 - 1.42)(4.43 - 6.14)Carbohydrates40.46 (0.46)39.22 (0.46)1.24 (0.40)0.27, 2.210.02032.36, 41.63(39.98 - 41.50)(37.73 - 40.20)(-0.024 - 2.47)(33.43 - 40.39)Moisture (% fwt)8.38 (0.15)8.16 (0.15)0.22 (0.19)-0.25, 0.690.2915.41, 10.36(7.95 - 8.70)(7.72 - 8.55)(-0.32 - 0.94)(5.43 - 9.86)Protein39.78 (0.26)40.09 (0.26)-0.31 (0.35)-1.17, 0.540.40735.06, 43.58(38.87 - 40.53)(39.57 - 40.42)(-1.55 - 0.39)(35.11 - 42.16)Total Fat14.35 (0.35)15.35 (0.35)-1.00 (0.38)-1.94, -0.0660.03913.15, 23.90(13.91 - 14.60)(14.41 - 16.73)(-2.38 - 0.15)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber16.59 (1.06)16.54 (1.06)0.047 (1.43)-3.44, 3.540.9749.99, 22.21(15.47 - 17.49)(14.65 - 20.81)(-4.82 - 2.76)(11.74 - 22.13)Monsanto Company 11-SY-217U 288 of 467Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber16.76 (0.89)17.50 (0.89)-0.75 (1.26)-3.83, 2.340.57611.03, 23.27(15.22 - 19.93)(16.84 - 18.31)(-1.62 - 1.61)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.73 (0.016)1.73 (0.016)-0.0013 (0.015)-0.039, 0.0360.9331.54, 1.88(1.72 - 1.75)(1.68 - 1.76)(-0.028 - 0.039)(1.58 - 1.84)Arginine3.03 (0.044)3.04 (0.044)-0.015 (0.036)-0.10, 0.0720.6932.51, 3.33(2.93 - 3.08)(2.96 - 3.10)(-0.065 - 0.058)(2.57 - 3.24)Aspartic Acid4.55 (0.051)4.59 (0.051)-0.038 (0.049)-0.16, 0.0820.4634.04, 5.07(4.44 - 4.64)(4.45 - 4.70)(-0.16 - 0.093)(4.06 - 4.89)Cystine0.56 (0.0064)0.57 (0.0064)-0.015 (0.0082)-0.035, 0.00530.1220.51, 0.67(0.56 - 0.56)(0.57 - 0.58)(-0.021 - -0.0036)(0.54 - 0.69)Glutamic Acid7.34 (0.091)7.44 (0.091)-0.10 (0.083)-0.31, 0.100.2626.28, 8.18(7.18 - 7.46)(7.18 - 7.70)(-0.37 - 0.097)(6.40 - 7.94)Monsanto Company 11-SY-217U 289 of 467Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.74 (0.013)1.76 (0.013)-0.015 (0.013)-0.046, 0.0160.2791.52, 1.90(1.72 - 1.76)(1.72 - 1.78)(-0.041 - 0.013)(1.54 - 1.85)Histidine1.04 (0.012)1.06 (0.012)-0.017 (0.0098)-0.041, 0.00650.1250.91, 1.17(1.03 - 1.05)(1.03 - 1.09)(-0.045 - -0.00034)(0.93 - 1.16)Isoleucine1.82 (0.021)1.84 (0.021)-0.020 (0.025)-0.082, 0.0410.4521.62, 2.03(1.79 - 1.85)(1.80 - 1.88)(-0.072 - 0.024)(1.60 - 2.00)Leucine3.05 (0.028)3.08 (0.028)-0.026 (0.027)-0.091, 0.0400.3782.71, 3.38(3.00 - 3.09)(2.99 - 3.13)(-0.082 - 0.026)(2.77 - 3.29)Lysine2.59 (0.026)2.62 (0.026)-0.038 (0.022)-0.092, 0.0170.1452.33, 2.81(2.54 - 2.63)(2.55 - 2.69)(-0.11 - 0.0034)(2.36 - 2.74)Methionine0.55 (0.0054)0.56 (0.0054)-0.0028 (0.0077)-0.022, 0.0160.7310.51, 0.59(0.54 - 0.56)(0.55 - 0.57)(-0.026 - 0.016)(0.51 - 0.60)Monsanto Company 11-SY-217U 290 of 467Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.09 (0.027)2.10 (0.027)-0.014 (0.022)-0.069, 0.0400.5471.81, 2.33(2.05 - 2.11)(2.04 - 2.15)(-0.062 - 0.025)(1.81 - 2.25)Proline1.98 (0.035)1.94 (0.035)0.037 (0.037)-0.053, 0.130.3501.70, 2.13(1.83 - 2.04)(1.85 - 1.98)(-0.025 - 0.079)(1.69 - 2.09)Serine2.08 (0.031)2.12 (0.031)-0.042 (0.024)-0.10, 0.0170.1351.86, 2.33(2.01 - 2.13)(2.05 - 2.19)(-0.13 - 0.0028)(1.90 - 2.30)Threonine1.57 (0.022)1.55 (0.022)0.017 (0.026)-0.047, 0.0820.5341.40, 1.69(1.51 - 1.61)(1.52 - 1.61)(-0.0063 - 0.046)(1.36 - 1.68)Tryptophan0.42 (0.010)0.43 (0.010)-0.011 (0.015)-0.047, 0.0240.4680.36, 0.50(0.39 - 0.44)(0.40 - 0.46)(-0.047 - 0.042)(0.38 - 0.48)Tyrosine1.45 (0.027)1.44 (0.027)0.0089 (0.037)-0.081, 0.0980.8151.28, 1.57(1.40 - 1.47)(1.37 - 1.49)(-0.027 - 0.080)(1.28 - 1.55)Monsanto Company 11-SY-217U 291 of 467Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine1.93 (0.023)1.95 (0.023)-0.017 (0.027)-0.083, 0.0490.5481.71, 2.13(1.89 - 1.96)(1.91 - 2.00)(-0.072 - 0.035)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.23 (0.031)11.28 (0.031)-0.049 (0.044)-0.16, 0.0590.3067.76, 13.14(11.21 - 11.26)(11.22 - 11.33)(-0.12 - -0.0020)(9.00 - 12.03)18:0 Stearic4.09 (0.062)4.27 (0.062)-0.18 (0.075)-0.36, 0.00230.0523.06, 5.10(3.99 - 4.21)(4.11 - 4.44)(-0.29 - -0.075)(3.49 - 4.97)18:1 Oleic20.03 (0.21)19.84 (0.21)0.19 (0.30)-0.54, 0.920.54917.37, 26.86(19.88 - 20.16)(19.41 - 20.17)(-0.0059 - 0.54)(18.93 - 25.33)18:2 Linoleic54.42 (0.19)54.42 (0.19)0.0039 (0.26)-0.64, 0.650.98850.14, 57.81(54.31 - 54.55)(53.98 - 54.81)(-0.26 - 0.32)(51.57 - 56.25)18:3 Linolenic9.61 (0.078)9.50 (0.078)0.11 (0.11)-0.15, 0.370.3275.60, 11.61(9.45 - 9.78)(9.34 - 9.71)(-0.087 - 0.44)(5.89 - 10.16)Monsanto Company 11-SY-217U 292 of 467Table F-13. Summary of Site ILWY Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.29 (0.0070)0.30 (0.0070)-0.014 (0.0099)-0.038, 0.0100.2030.22, 0.39(0.27 - 0.31)(0.28 - 0.32)(-0.042 - 0.013)(0.23 - 0.38)20:1 Eicosenoic0.081 (0.016)0.12 (0.016)-0.036 (0.020)-0.085, 0.0120.1180.094, 0.23(0.080 - 0.083)(0.080 - 0.16)(-0.077 - 0.0032)(0.072 - 0.21)22:0 Behenic0.25 (0.023)0.28 (0.023)-0.024 (0.032)-0.10, 0.0550.4850.18, 0.43(0.21 - 0.30)(0.22 - 0.30)(-0.082 - 0.060)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E0.89 (0.11)1.19 (0.11)-0.30 (0.15)-0.68, 0.0690.0930.10, 2.85(0.81 - 0.94)(1.02 - 1.37)(-0.44 - -0.13)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 293 of 467Table F-14. Summary of Site ILWY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)1.91 (0.23)1.99 (0.23)-0.082 (0.33)-0.89, 0.720.8120, 6.11(1.37 - 2.23)(1.02 - 2.43)(-1.05 - 1.07)(0.60 - 6.99)Phytic Acid (% dwt)1.37 (0.072)1.33 (0.072)0.038 (0.10)-0.21, 0.290.7190.50, 1.92(1.33 - 1.48)(1.14 - 1.44)(-0.12 - 0.34)(0.66 - 1.74)Raffinose (% dwt)0.68 (0.023)0.66 (0.023)0.013 (0.032)-0.065, 0.0910.6890.39, 1.01(0.63 - 0.69)(0.64 - 0.70)(-0.065 - 0.057)(0.45 - 0.93)Stachyose (% dwt)4.29 (0.055)4.21 (0.055)0.084 (0.078)-0.11, 0.270.3222.45, 5.34(4.14 - 4.37)(4.15 - 4.31)(-0.025 - 0.20)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)35.39 (4.06)37.57 (4.06)-2.18 (4.78)-13.87, 9.520.66420.97, 50.01(32.35 - 39.32)(27.58 - 53.09)(-20.74 - 8.70)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein1397.02 (59.70)1252.03 (59.70)144.98 (71.95)-31.08, 321.050.0900, 1756.99(1226.73 - 1609.37)(1126.30 - 1362.69)(-20.09 - 384.84)(138.15 - 1548.98)Monsanto Company 11-SY-217U 294 of 467Table F-14. Summary of Site ILWY Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein1039.09 (47.68)933.81 (47.68)105.29 (61.87)-46.10, 256.670.13987.22, 1792.07(929.19 - 1204.29)(810.28 - 1044.37)(-14.02 - 266.93)(335.67 - 1409.07)Glycitein117.88 (4.74)96.68 (4.74)21.19 (5.09)8.75, 33.640.0058.13, 299.67(112.20 - 122.62)(85.40 - 110.11)(9.72 - 33.87)(66.83 - 280.71)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-biotechnologyderived, conventional control, A35255With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 295 of 467Table F-15. Summary of Site ILWY Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.18 (0.18)6.24 (0.18)-0.059 (0.26)-0.69, 0.570.8254.29, 8.65(5.82 - 6.53)(5.90 - 6.51)(-0.38 - 0.35)(4.82 - 8.98)Carbohydrates65.05 (0.44)64.69 (0.44)0.36 (0.47)-0.79, 1.510.47355.73, 77.45(63.87 - 66.05)(64.08 - 66.17)(-0.37 - 1.97)(54.40 - 72.96)Moisture (% fwt)72.18 (0.81)70.08 (0.81)2.10 (1.07)-0.52, 4.720.09765.61, 80.67(71.30 - 72.90)(66.60 - 71.60)(1.00 - 4.70)(64.50 - 79.80)Protein21.74 (0.46)21.90 (0.46)-0.16 (0.49)-1.35, 1.030.75113.77, 26.51(20.70 - 22.67)(21.11 - 22.57)(-1.87 - 0.92)(16.56 - 27.76)Total Fat7.09 (0.30)7.19 (0.30)-0.091 (0.35)-0.94, 0.760.8020.54, 13.11(6.94 - 7.35)(6.69 - 8.16)(-1.12 - 0.44)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber29.54 (1.33)29.48 (1.33)0.052 (0.99)-2.38, 2.490.96023.12, 38.15(26.09 - 32.03)(27.01 - 31.44)(-2.34 - 1.96)(22.60 - 41.29)Monsanto Company 11-SY-217U 296 of 467Table F-15. Summary of Site ILWY Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber30.73 (0.99)32.78 (0.99)-2.05 (1.40)-5.49, 1.390.19424.96, 43.33(29.85 - 31.55)(29.47 - 35.33)(-4.21 - 2.08)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 297 of 467Table F-16. Summary of Site INRC Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.69 (0.19)4.92 (0.17)-0.23 (0.17)-0.68, 0.220.2464.43, 5.89(4.52 - 5.04)(4.58 - 5.46)(-0.68 - -0.028)(4.43 - 6.14)Carbohydrates38.77 (0.46)39.38 (0.40)-0.61 (0.58)-2.09, 0.870.33532.36, 41.63(38.33 - 39.39)(38.13 - 40.43)(-1.87 - 0.72)(33.43 - 40.39)Moisture (% fwt)8.04 (0.14)7.45 (0.12)0.59 (0.18)0.13, 1.040.0215.41, 10.36(7.91 - 8.11)(7.17 - 7.95)(-0.040 - 0.94)(5.43 - 9.86)Protein41.36 (0.31)40.71 (0.28)0.65 (0.33)-0.19, 1.500.10335.06, 43.58(40.37 - 42.01)(39.86 - 41.17)(0.42 - 1.29)(35.11 - 42.16)Total Fat15.11 (0.39)14.99 (0.34)0.12 (0.46)-1.07, 1.300.81113.15, 23.90(15.02 - 15.31)(13.91 - 16.42)(-1.19 - 0.58)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber14.25 (0.70)14.96 (0.61)-0.71 (0.84)-2.87, 1.450.4359.99, 22.21(13.93 - 14.69)(13.25 - 16.31)(-2.38 - 1.19)(11.74 - 22.13)Monsanto Company 11-SY-217U 298 of 467Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber15.95 (0.64)16.12 (0.55)-0.17 (0.84)-2.35, 2.000.84411.03, 23.27(15.53 - 16.43)(14.23 - 17.57)(-1.40 - 1.30)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.76 (0.012)1.76 (0.011)0.00036 (0.016)-0.042, 0.0420.9831.54, 1.88(1.75 - 1.77)(1.74 - 1.80)(-0.055 - 0.034)(1.58 - 1.84)Arginine3.09 (0.037)3.10 (0.033)-0.011 (0.042)-0.12, 0.0960.8082.51, 3.33(3.02 - 3.18)(3.02 - 3.14)(-0.12 - 0.086)(2.57 - 3.24)Aspartic Acid4.70 (0.031)4.71 (0.027)-0.00071 (0.041)-0.11, 0.110.9864.04, 5.07(4.65 - 4.73)(4.66 - 4.75)(-0.031 - 0.069)(4.06 - 4.89)Cystine0.59 (0.011)0.60 (0.0095)-0.010 (0.013)-0.045, 0.0250.4910.51, 0.67(0.59 - 0.60)(0.56 - 0.63)(-0.034 - 0.030)(0.54 - 0.69)Glutamic Acid7.55 (0.063)7.54 (0.054)0.0074 (0.083)-0.21, 0.220.9326.28, 8.18(7.49 - 7.60)(7.43 - 7.62)(-0.058 - 0.16)(6.40 - 7.94)Monsanto Company 11-SY-217U 299 of 467Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.78 (0.010)1.77 (0.0089)0.0050 (0.014)-0.030, 0.0400.7281.52, 1.90(1.77 - 1.78)(1.76 - 1.80)(-0.023 - 0.029)(1.54 - 1.85)Histidine1.06 (0.0089)1.07 (0.0077)-0.014 (0.012)-0.044, 0.0160.2870.91, 1.17(1.04 - 1.08)(1.05 - 1.09)(-0.046 - 0.022)(0.93 - 1.16)Isoleucine1.87 (0.016)1.87 (0.014)-0.0016 (0.022)-0.057, 0.0540.9431.62, 2.03(1.83 - 1.90)(1.83 - 1.90)(-0.073 - 0.051)(1.60 - 2.00)Leucine3.13 (0.018)3.14 (0.016)-0.0098 (0.024)-0.072, 0.0520.7002.71, 3.38(3.10 - 3.15)(3.10 - 3.16)(-0.053 - 0.043)(2.77 - 3.29)Lysine2.68 (0.017)2.69 (0.015)-0.015 (0.023)-0.075, 0.0440.5332.33, 2.81(2.67 - 2.69)(2.65 - 2.73)(-0.055 - 0.038)(2.36 - 2.74)Methionine0.57 (0.011)0.56 (0.0095)0.00094 (0.015)-0.036, 0.0380.9500.51, 0.59(0.56 - 0.57)(0.53 - 0.59)(-0.033 - 0.036)(0.51 - 0.60)Monsanto Company 11-SY-217U 300 of 467Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.13 (0.019)2.15 (0.016)-0.020 (0.025)-0.083, 0.0440.4601.81, 2.33(2.08 - 2.17)(2.11 - 2.16)(-0.077 - 0.054)(1.81 - 2.25)Proline2.00 (0.025)2.03 (0.022)-0.036 (0.032)-0.12, 0.0460.3091.70, 2.13(1.94 - 2.06)(1.99 - 2.08)(-0.077 - 0.075)(1.69 - 2.09)Serine2.17 (0.027)2.20 (0.024)-0.021 (0.036)-0.11, 0.0710.5791.86, 2.33(2.13 - 2.21)(2.16 - 2.25)(-0.12 - 0.033)(1.90 - 2.30)Threonine1.59 (0.020)1.58 (0.017)0.0074 (0.026)-0.060, 0.0750.7881.40, 1.69(1.55 - 1.63)(1.56 - 1.61)(-0.060 - 0.064)(1.36 - 1.68)Tryptophan0.44 (0.0096)0.46 (0.0084)-0.023 (0.013)-0.055, 0.0100.1370.36, 0.50(0.43 - 0.45)(0.45 - 0.47)(-0.040 - -0.0074)(0.38 - 0.48)Tyrosine1.48 (0.018)1.50 (0.016)-0.020 (0.024)-0.082, 0.0410.4351.28, 1.57(1.47 - 1.49)(1.49 - 1.52)(-0.054 - -0.0066)(1.28 - 1.55)Monsanto Company 11-SY-217U 301 of 467Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine1.97 (0.019)1.95 (0.016)0.015 (0.025)-0.049, 0.0790.5691.71, 2.13(1.93 - 1.99)(1.91 - 1.97)(-0.040 - 0.085)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.23 (0.067)11.24 (0.063)-0.0071 (0.049)-0.13, 0.120.8897.76, 13.14(11.10 - 11.35)(11.13 - 11.44)(-0.093 - 0.091)(9.00 - 12.03)18:0 Stearic3.67 (0.11)3.96 (0.11)-0.29 (0.048)-0.41, -0.160.0013.06, 5.10(3.54 - 3.84)(3.78 - 4.21)(-0.37 - -0.24)(3.49 - 4.97)18:1 Oleic19.32 (0.43)19.60 (0.41)-0.27 (0.27)-0.97, 0.430.36117.37, 26.86(18.72 - 20.01)(18.72 - 20.50)(-0.49 - -0.0016)(18.93 - 25.33)18:2 Linoleic55.27 (0.36)54.55 (0.33)0.72 (0.34)-0.15, 1.580.08550.14, 57.81(54.66 - 55.60)(53.75 - 55.02)(0.46 - 0.91)(51.57 - 56.25)18:3 Linolenic9.85 (0.17)9.98 (0.16)-0.14 (0.072)-0.32, 0.0490.1165.60, 11.61(9.49 - 10.22)(9.66 - 10.31)(-0.31 - 0.0045)(5.89 - 10.16)Monsanto Company 11-SY-217U 302 of 467Table F-16. Summary of Site INRC Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.27 (0.0097)0.29 (0.0085)-0.020 (0.011)-0.049, 0.00850.1290.22, 0.39(0.25 - 0.29)(0.27 - 0.31)(-0.023 - -0.017)(0.23 - 0.38)20:1 Eicosenoic0.13 (0.019)0.12 (0.017)0.0068 (0.020)-0.045, 0.0590.7500.094, 0.23(0.079 - 0.16)(0.079 - 0.16)(-0.0043 -0.00068)(0.072 - 0.21)22:0 Behenic0.25 (0.026)0.26 (0.023)-0.0089 (0.035)-0.098, 0.0800.8060.18, 0.43(0.19 - 0.29)(0.18 - 0.29)(-0.014 - 0.0073)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E0.96 (0.050)0.86 (0.043)0.092 (0.066)-0.077, 0.260.2190.10, 2.85(0.90 - 1.03)(0.79 - 0.95)(0.077 - 0.13)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 303 of 467Table F-17. Summary of Site INRC Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)2.03 (0.45)2.35 (0.39)-0.32 (0.59)-1.85, 1.200.6080, 6.11(1.22 - 3.41)(1.43 - 3.05)(-1.84 - 1.21)(0.60 - 6.99)Phytic Acid (% dwt)1.08 (0.065)1.17 (0.057)-0.089 (0.075)-0.28, 0.100.2910.50, 1.92(0.96 - 1.17)(1.03 - 1.24)(-0.12 - -0.066)(0.66 - 1.74)Raffinose (% dwt)0.63 (0.046)0.59 (0.042)0.035 (0.049)-0.090, 0.160.5000.39, 1.01(0.54 - 0.70)(0.52 - 0.66)(0.015 - 0.062)(0.45 - 0.93)Stachyose (% dwt)4.59 (0.12)4.70 (0.11)-0.11 (0.13)-0.45, 0.230.4372.45, 5.34(4.46 - 4.76)(4.48 - 4.88)(-0.17 - -0.019)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)31.45 (2.42)32.94 (2.15)-1.49 (2.65)-8.31, 5.330.59820.97, 50.01(32.21 - 32.86)(24.37 - 38.89)(-6.64 - -1.61)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein1036.00 (48.75)878.45 (42.22)157.54 (64.50)-8.25, 323.330.0580, 1756.99(978.39 - 1120.78)(770.23 - 1050.52)(-72.13 - 350.56)(138.15 - 1548.98)Monsanto Company 11-SY-217U 304 of 467Table F-17. Summary of Site INRC Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein918.56 (57.59)759.54 (49.88)159.02 (76.19)-36.83, 354.870.09187.22, 1792.07(852.43 - 974.97)(583.34 - 924.50)(-72.07 - 391.63)(335.67 - 1409.07)Glycitein97.52 (5.54)89.93 (4.80)7.59 (7.17)-10.85, 26.020.3388.13, 299.67(90.56 - 104.90)(80.16 - 102.96)(-3.52 - 24.74)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 305 of 467Table F-18. Summary of Site INRC Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.65 (0.19)6.17 (0.17)0.47 (0.25)-0.16, 1.100.1134.29, 8.65(6.38 - 6.93)(5.63 - 6.57)(-0.089 - 0.97)(4.82 - 8.98)Carbohydrates60.79 (0.98)61.41 (0.93)-0.63 (0.70)-2.42, 1.160.40855.73, 77.45(59.41 - 62.73)(58.14 - 63.43)(-2.60 - 1.27)(54.40 - 72.96)Moisture (% fwt)73.13 (0.48)73.35 (0.41)-0.22 (0.63)-1.84, 1.410.74565.61, 80.67(72.90 - 73.60)(72.90 - 74.20)(-1.30 - 0.50)(64.50 - 79.80)Protein24.85 (0.76)23.91 (0.74)0.94 (0.45)-0.23, 2.100.09213.77, 26.51(23.06 - 26.20)(21.87 - 26.12)(0.075 - 1.35)(16.56 - 27.76)Total Fat7.87 (0.37)8.51 (0.32)-0.64 (0.45)-1.80, 0.510.2120.54, 13.11(7.60 - 8.18)(7.84 - 9.22)(-1.61 - 0.34)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber27.04 (1.79)29.14 (1.57)-2.10 (2.11)-7.54, 3.330.36523.12, 38.15(25.61 - 29.37)(24.46 - 31.30)(-5.70 - -0.94)(22.60 - 41.29)Monsanto Company 11-SY-217U 306 of 467Table F-18. Summary of Site INRC Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber31.87 (1.00)32.74 (0.86)-0.87 (1.32)-4.25, 2.520.53924.96, 43.33(29.59 - 34.10)(30.22 - 34.42)(-3.39 - 0.76)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A35255With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 307 of 467Table F-19. Summary of Site INSH Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.65 (0.081)4.77 (0.081)-0.12 (0.11)-0.40, 0.160.3484.43, 5.89(4.56 - 4.73)(4.55 - 4.94)(-0.21 - 0.0049)(4.43 - 6.14)Carbohydrates38.06 (0.64)38.18 (0.64)-0.12 (0.76)-1.97, 1.730.88132.36, 41.63(37.20 - 38.51)(37.34 - 39.61)(-1.29 - 1.16)(33.43 - 40.39)Moisture (% fwt)8.24 (0.43)8.60 (0.43)-0.37 (0.61)-1.85, 1.120.5705.41, 10.36(7.60 - 8.93)(7.09 - 10.30)(-1.69 - 1.84)(5.43 - 9.86)Protein41.55 (0.50)41.15 (0.50)0.40 (0.71)-1.34, 2.150.59435.06, 43.58(41.02 - 42.28)(39.72 - 42.14)(-0.56 - 2.56)(35.11 - 42.16)Total Fat15.75 (0.39)15.92 (0.39)-0.17 (0.47)-1.33, 0.990.72913.15, 23.90(14.71 - 16.63)(15.14 - 16.88)(-1.26 - 0.87)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber15.91 (0.71)14.90 (0.71)1.01 (0.72)-0.75, 2.780.2099.99, 22.21(14.97 - 16.96)(12.85 - 17.01)(-0.86 - 2.24)(11.74 - 22.13)Monsanto Company 11-SY-217U 308 of 467Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber16.38 (0.57)16.78 (0.57)-0.41 (0.81)-2.38, 1.560.63211.03, 23.27(15.58 - 17.62)(15.38 - 18.40)(-2.59 - 2.23)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.79 (0.020)1.78 (0.020)0.014 (0.019)-0.032, 0.0610.4761.54, 1.88(1.78 - 1.82)(1.73 - 1.83)(-0.047 - 0.057)(1.58 - 1.84)Arginine3.19 (0.054)3.21 (0.054)-0.021 (0.072)-0.20, 0.150.7762.51, 3.33(3.13 - 3.29)(3.11 - 3.31)(-0.16 - 0.18)(2.57 - 3.24)Aspartic Acid4.79 (0.056)4.79 (0.056)-0.0033 (0.063)-0.16, 0.150.9594.04, 5.07(4.75 - 4.84)(4.67 - 4.89)(-0.14 - 0.14)(4.06 - 4.89)Cystine0.56 (0.0084)0.58 (0.0084)-0.025 (0.012)-0.054, 0.00440.0830.51, 0.67(0.55 - 0.58)(0.57 - 0.62)(-0.072 - 0.010)(0.54 - 0.69)Glutamic Acid7.74 (0.12)7.76 (0.12)-0.022 (0.13)-0.34, 0.300.8746.28, 8.18(7.69 - 7.80)(7.54 - 7.97)(-0.27 - 0.25)(6.40 - 7.94)Monsanto Company 11-SY-217U 309 of 467Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.81 (0.019)1.80 (0.019)0.014 (0.020)-0.035, 0.0640.5061.52, 1.90(1.80 - 1.82)(1.74 - 1.84)(-0.036 - 0.079)(1.54 - 1.85)Histidine1.08 (0.013)1.09 (0.013)-0.0072 (0.013)-0.040, 0.0250.6030.91, 1.17(1.06 - 1.09)(1.06 - 1.12)(-0.038 - 0.038)(0.93 - 1.16)Isoleucine1.93 (0.025)1.90 (0.025)0.036 (0.028)-0.033, 0.100.2471.62, 2.03(1.92 - 1.95)(1.86 - 1.96)(-0.038 - 0.075)(1.60 - 2.00)Leucine3.19 (0.041)3.19 (0.041)0.0030 (0.045)-0.11, 0.110.9482.71, 3.38(3.17 - 3.21)(3.10 - 3.26)(-0.088 - 0.11)(2.77 - 3.29)Lysine2.69 (0.026)2.69 (0.026)-0.0058 (0.025)-0.066, 0.0550.8232.33, 2.81(2.67 - 2.70)(2.63 - 2.75)(-0.070 - 0.064)(2.36 - 2.74)Methionine0.55 (0.0069)0.57 (0.0069)-0.016 (0.0097)-0.039, 0.00810.1570.51, 0.59(0.54 - 0.57)(0.55 - 0.59)(-0.054 - 0.0071)(0.51 - 0.60)Monsanto Company 11-SY-217U 310 of 467Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.18 (0.031)2.18 (0.031)0.0020 (0.034)-0.081, 0.0850.9561.81, 2.33(2.13 - 2.22)(2.12 - 2.24)(-0.063 - 0.098)(1.81 - 2.25)Proline1.99 (0.038)2.02 (0.038)-0.038 (0.053)-0.17, 0.0920.4991.70, 2.13(1.87 - 2.11)(1.98 - 2.07)(-0.20 - 0.13)(1.69 - 2.09)Serine2.16 (0.037)2.22 (0.037)-0.061 (0.035)-0.15, 0.0240.1291.86, 2.33(2.13 - 2.18)(2.14 - 2.34)(-0.16 - 0.032)(1.90 - 2.30)Threonine1.62 (0.012)1.59 (0.012)0.032 (0.017)-0.0085, 0.0720.1011.40, 1.69(1.60 - 1.64)(1.56 - 1.63)(-0.00038 - 0.064)(1.36 - 1.68)Tryptophan0.46 (0.014)0.44 (0.014)0.015 (0.020)-0.034, 0.0640.4780.36, 0.50(0.44 - 0.48)(0.38 - 0.47)(-0.023 - 0.10)(0.38 - 0.48)Tyrosine1.50 (0.026)1.51 (0.026)-0.015 (0.037)-0.11, 0.0770.7071.28, 1.57(1.47 - 1.55)(1.42 - 1.56)(-0.095 - 0.13)(1.28 - 1.55)Monsanto Company 11-SY-217U 311 of 467Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine2.04 (0.025)1.99 (0.025)0.044 (0.028)-0.026, 0.110.1741.71, 2.13(2.02 - 2.06)(1.96 - 2.07)(-0.050 - 0.095)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic10.99 (0.075)11.19 (0.075)-0.19 (0.11)-0.45, 0.0670.1207.76, 13.14(10.78 - 11.21)(11.16 - 11.22)(-0.44 - 0.050)(9.00 - 12.03)18:0 Stearic4.62 (0.057)4.57 (0.057)0.045 (0.080)-0.15, 0.240.5933.06, 5.10(4.45 - 4.72)(4.45 - 4.65)(-0.20 - 0.25)(3.49 - 4.97)18:1 Oleic22.26 (0.13)21.25 (0.13)1.01 (0.18)0.57, 1.450.00117.37, 26.86(22.07 - 22.50)(21.07 - 21.54)(0.90 - 1.19)(18.93 - 25.33)18:2 Linoleic52.72 (0.14)53.25 (0.14)-0.52 (0.19)-0.99, -0.0540.03450.14, 57.81(52.33 - 53.25)(53.21 - 53.27)(-0.92 - 0.037)(51.57 - 56.25)18:3 Linolenic8.70 (0.098)8.98 (0.098)-0.28 (0.14)-0.62, 0.0600.0905.60, 11.61(8.54 - 8.87)(8.83 - 9.18)(-0.64 - -0.0039)(5.89 - 10.16)Monsanto Company 11-SY-217U 312 of 467Table F-19. Summary of Site INSH Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.31 (0.0096)0.33 (0.0096)-0.016 (0.013)-0.048, 0.0160.2690.22, 0.39(0.27 - 0.33)(0.31 - 0.34)(-0.066 - 0.0050)(0.23 - 0.38)20:1 Eicosenoic0.14 (0.016)0.15 (0.016)-0.018 (0.014)-0.051, 0.0160.2430.094, 0.23(0.081 - 0.16)(0.15 - 0.16)(-0.066 - 0.0011)(0.072 - 0.21)22:0 Behenic0.26 (0.019)0.29 (0.019)-0.029 (0.024)-0.088, 0.0300.2760.18, 0.43(0.22 - 0.31)(0.21 - 0.32)(-0.091 - 0.022)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E1.32 (0.053)1.35 (0.053)-0.024 (0.075)-0.21, 0.160.7650.10, 2.85(1.28 - 1.37)(1.20 - 1.47)(-0.19 - 0.15)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 313 of 467Table F-20. Summary of Site INSH Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)1.58 (0.22)2.17 (0.22)-0.60 (0.31)-1.35, 0.160.1030, 6.11(1.10 - 1.97)(1.63 - 3.05)(-1.40 - 0.30)(0.60 - 6.99)Phytic Acid (% dwt)0.99 (0.042)0.94 (0.042)0.048 (0.060)-0.098, 0.190.4530.50, 1.92(0.90 - 1.08)(0.87 - 1.03)(-0.13 - 0.21)(0.66 - 1.74)Raffinose (% dwt)0.66 (0.034)0.68 (0.034)-0.024 (0.048)-0.14, 0.0940.6380.39, 1.01(0.58 - 0.75)(0.62 - 0.74)(-0.12 - 0.13)(0.45 - 0.93)Stachyose (% dwt)4.12 (0.078)4.05 (0.078)0.069 (0.11)-0.20, 0.330.5462.45, 5.34(3.91 - 4.29)(3.78 - 4.15)(-0.21 - 0.28)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)30.60 (4.05)30.29 (4.05)0.31 (5.69)-13.62, 14.230.95820.97, 50.01(28.25 - 32.72)(20.34 - 41.47)(-8.75 - 9.85)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein981.19 (40.89)1055.58 (40.89)-74.39 (45.24)-185.08, 36.300.1510, 1756.99(926.76 - 1105.15)(910.56 - 1132.28)(-187.49 - 44.95)(138.15 - 1548.98)Monsanto Company 11-SY-217U 314 of 467Table F-20. Summary of Site INSH Soybean Seed Anti-nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein728.13 (30.47)773.66 (30.47)-45.53 (32.81)-125.82, 34.760.21487.22, 1792.07(674.24 - 809.72)(675.92 - 836.15)(-125.71 - 37.81)(335.67 - 1409.07)Glycitein106.91 (6.85)108.31 (6.85)-1.40 (9.69)-25.11, 22.310.8898.13, 299.67(83.78 - 131.31)(102.05 - 116.49)(-22.23 - 22.61)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 315 of 467Table F-21. Summary of Site INSH Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.95 (0.21)5.50 (0.21)-0.55 (0.27)-1.20, 0.110.0864.29, 8.65(4.55 - 5.28)(4.98 - 5.93)(-1.04 - 0.11)(4.82 - 8.98)Carbohydrates63.40 (0.91)63.49 (0.91)-0.086 (1.20)-3.02, 2.840.94455.73, 77.45(62.62 - 64.84)(62.18 - 64.82)(-1.80 - 2.66)(54.40 - 72.96)Moisture (% fwt)68.85 (0.66)68.38 (0.66)0.48 (0.92)-1.78, 2.730.62465.61, 80.67(68.00 - 69.50)(67.30 - 69.30)(-0.40 - 1.40)(64.50 - 79.80)Protein23.23 (0.65)22.59 (0.65)0.64 (0.83)-1.40, 2.670.47213.77, 26.51(22.52 - 23.57)(20.98 - 24.01)(-1.49 - 2.53)(16.56 - 27.76)Total Fat8.45 (0.52)8.41 (0.52)0.039 (0.63)-1.50, 1.580.9520.54, 13.11(8.16 - 8.59)(7.59 - 9.33)(-0.83 - 1.00)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber25.96 (1.24)27.36 (1.24)-1.40 (1.76)-5.70, 2.900.45523.12, 38.15(24.43 - 27.31)(24.68 - 29.02)(-4.54 - 0.90)(22.60 - 41.29)Monsanto Company 11-SY-217U 316 of 467Table F-21. Summary of Site INSH Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber28.21 (1.41)30.37 (1.41)-2.16 (1.78)-6.51, 2.190.27024.96, 43.33(26.56 - 30.13)(26.89 - 32.60)(-4.37 - 1.04)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 317 of 467Table F-22. Summary of Site KSLA Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.63 (0.092)5.55 (0.092)0.082 (0.13)-0.24, 0.400.5534.43, 5.89(5.25 - 5.85)(5.33 - 5.71)(-0.41 - 0.38)(4.43 - 6.14)Carbohydrates37.84 (0.65)38.65 (0.65)-0.81 (0.90)-3.01, 1.400.40632.36, 41.63(36.54 - 38.71)(37.51 - 40.40)(-2.05 - 0.88)(33.43 - 40.39)Moisture (% fwt)6.80 (0.35)6.86 (0.35)-0.055 (0.50)-1.28, 1.170.9165.41, 10.36(5.72 - 8.32)(6.44 - 7.33)(-0.72 - 1.62)(5.43 - 9.86)Protein39.25 (0.33)38.62 (0.33)0.62 (0.35)-0.23, 1.480.12435.06, 43.58(38.90 - 39.92)(37.73 - 39.23)(0.013 - 1.20)(35.11 - 42.16)Total Fat17.31 (0.41)17.18 (0.41)0.13 (0.58)-1.28, 1.530.83413.15, 23.90(16.57 - 17.89)(16.25 - 17.81)(-1.16 - 0.95)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber14.62 (0.84)14.30 (0.84)0.32 (1.19)-2.59, 3.230.7969.99, 22.21(12.94 - 15.27)(12.41 - 17.85)(-2.58 - 2.81)(11.74 - 22.13)Monsanto Company 11-SY-217U 318 of 467Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber15.22 (0.58)16.08 (0.58)-0.86 (0.68)-2.53, 0.820.25611.03, 23.27(13.68 - 17.02)(15.28 - 16.62)(-1.60 - 0.62)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.70 (0.010)1.67 (0.010)0.037 (0.013)0.0045, 0.0690.0311.54, 1.88(1.69 - 1.72)(1.65 - 1.68)(0.021 - 0.051)(1.58 - 1.84)Arginine3.05 (0.025)2.90 (0.025)0.15 (0.028)0.083, 0.220.0012.51, 3.33(3.03 - 3.08)(2.85 - 2.98)(0.074 - 0.23)(2.57 - 3.24)Aspartic Acid4.56 (0.031)4.45 (0.031)0.10 (0.039)0.0079, 0.200.0374.04, 5.07(4.51 - 4.62)(4.36 - 4.49)(0.030 - 0.18)(4.06 - 4.89)Cystine0.62 (0.0072)0.63 (0.0072)-0.016 (0.010)-0.041, 0.00900.1690.51, 0.67(0.60 - 0.63)(0.61 - 0.65)(-0.053 - 0.014)(0.54 - 0.69)Glutamic Acid7.24 (0.057)7.03 (0.057)0.21 (0.066)0.046, 0.370.0206.28, 8.18(7.19 - 7.36)(6.86 - 7.10)(0.10 - 0.34)(6.40 - 7.94)Monsanto Company 11-SY-217U 319 of 467Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.72 (0.010)1.69 (0.010)0.031 (0.011)0.0032, 0.0590.0341.52, 1.90(1.70 - 1.75)(1.66 - 1.70)(0.013 - 0.052)(1.54 - 1.85)Histidine1.04 (0.0082)1.01 (0.0082)0.031 (0.010)0.0064, 0.0550.0210.91, 1.17(1.03 - 1.06)(1.00 - 1.03)(0.013 - 0.044)(0.93 - 1.16)Isoleucine1.80 (0.019)1.77 (0.019)0.029 (0.026)-0.035, 0.0920.3161.62, 2.03(1.78 - 1.84)(1.70 - 1.81)(-0.023 - 0.10)(1.60 - 2.00)Leucine3.02 (0.020)2.96 (0.020)0.063 (0.025)0.0023, 0.120.0432.71, 3.38(3.00 - 3.05)(2.90 - 2.99)(0.017 - 0.12)(2.77 - 3.29)Lysine2.57 (0.016)2.53 (0.016)0.036 (0.016)-0.0029, 0.0750.0632.33, 2.81(2.55 - 2.61)(2.49 - 2.56)(0.015 - 0.055)(2.36 - 2.74)Methionine0.57 (0.0058)0.57 (0.0058)0.0048 (0.0081)-0.015, 0.0250.5730.51, 0.59(0.55 - 0.59)(0.56 - 0.57)(-0.012 - 0.031)(0.51 - 0.60)Monsanto Company 11-SY-217U 320 of 467Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.04 (0.018)1.98 (0.018)0.055 (0.023)0.00024, 0.110.0491.81, 2.33(1.98 - 2.07)(1.95 - 2.02)(0.027 - 0.097)(1.81 - 2.25)Proline1.91 (0.028)1.93 (0.028)-0.017 (0.038)-0.11, 0.0760.6661.70, 2.13(1.90 - 1.93)(1.89 - 1.97)(-0.074 - 0.039)(1.69 - 2.09)Serine2.10 (0.019)2.04 (0.019)0.053 (0.025)-0.0086, 0.110.0801.86, 2.33(2.06 - 2.13)(2.01 - 2.08)(-0.027 - 0.091)(1.90 - 2.30)Threonine1.56 (0.012)1.55 (0.012)0.015 (0.017)-0.027, 0.0570.4101.40, 1.69(1.55 - 1.57)(1.51 - 1.58)(-0.029 - 0.063)(1.36 - 1.68)Tryptophan0.44 (0.0095)0.42 (0.0095)0.019 (0.012)-0.0096, 0.0480.1540.36, 0.50(0.43 - 0.45)(0.40 - 0.44)(-0.0065 - 0.038)(0.38 - 0.48)Tyrosine1.45 (0.016)1.40 (0.016)0.048 (0.018)0.0044, 0.0910.0351.28, 1.57(1.41 - 1.48)(1.37 - 1.44)(0.023 - 0.11)(1.28 - 1.55)Monsanto Company 11-SY-217U 321 of 467Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine1.91 (0.020)1.87 (0.020)0.045 (0.025)-0.018, 0.110.1291.71, 2.13(1.88 - 1.96)(1.78 - 1.91)(-0.013 - 0.11)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.89 (0.033)12.12 (0.033)-0.23 (0.046)-0.34, -0.110.0027.76, 13.14(11.84 - 11.94)(12.04 - 12.23)(-0.39 - -0.13)(9.00 - 12.03)18:0 Stearic3.97 (0.023)3.92 (0.023)0.053 (0.028)-0.014, 0.120.1023.06, 5.10(3.92 - 4.03)(3.87 - 4.01)(0.022 - 0.091)(3.49 - 4.97)18:1 Oleic19.70 (0.11)19.71 (0.11)-0.0098 (0.10)-0.27, 0.250.92817.37, 26.86(19.60 - 19.81)(19.45 - 19.99)(-0.20 - 0.15)(18.93 - 25.33)18:2 Linoleic54.62 (0.14)54.37 (0.14)0.25 (0.16)-0.16, 0.650.18650.14, 57.81(54.38 - 54.90)(53.90 - 54.70)(-0.038 - 0.48)(51.57 - 56.25)18:3 Linolenic9.08 (0.058)9.23 (0.058)-0.15 (0.082)-0.35, 0.0490.1135.60, 11.61(8.96 - 9.19)(9.03 - 9.41)(-0.45 - 0.033)(5.89 - 10.16)Monsanto Company 11-SY-217U 322 of 467Table F-22. Summary of Site KSLA Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.30 (0.0084)0.26 (0.0084)0.032 (0.0078)0.013, 0.0510.0060.22, 0.39(0.29 - 0.30)(0.25 - 0.30)(0.0020 - 0.049)(0.23 - 0.38)20:1 Eicosenoic0.16 (0.0030)0.15 (0.0030)0.0054 (0.0042)-0.0047, 0.0160.2390.094, 0.23(0.15 - 0.16)(0.15 - 0.16)(-0.0062 - 0.011)(0.072 - 0.21)22:0 Behenic0.29 (0.018)0.23 (0.018)0.052 (0.016)0.013, 0.0920.0170.18, 0.43(0.28 - 0.30)(0.20 - 0.30)(-0.0048 - 0.079)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E1.33 (0.092)1.23 (0.092)0.094 (0.10)-0.16, 0.340.3920.10, 2.85(1.17 - 1.45)(1.01 - 1.37)(-0.084 - 0.23)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 323 of 467Table F-23. Summary of Site KSLA Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)2.17 (0.32)1.28 (0.32)0.88 (0.46)-0.23, 2.000.1010, 6.11(1.22 - 2.81)(0.58 - 2.39)(-1.17 - 1.63)(0.60 - 6.99)Phytic Acid (% dwt)1.72 (0.069)1.61 (0.069)0.11 (0.097)-0.12, 0.350.2800.50, 1.92(1.63 - 1.81)(1.39 - 1.77)(-0.034 - 0.42)(0.66 - 1.74)Raffinose (% dwt)0.87 (0.011)0.82 (0.011)0.043 (0.012)0.013, 0.0730.0110.39, 1.01(0.86 - 0.88)(0.80 - 0.85)(0.020 - 0.063)(0.45 - 0.93)Stachyose (% dwt)4.33 (0.065)4.21 (0.065)0.12 (0.092)-0.10, 0.350.2362.45, 5.34(4.27 - 4.42)(4.16 - 4.25)(0.064 - 0.26)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)34.65 (4.00)36.99 (4.00)-2.34 (5.66)-16.20, 11.520.69320.97, 50.01(31.85 - 39.35)(26.29 - 54.88)(-23.03 - 6.80)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein1311.42 (35.73)1341.83 (35.73)-30.41 (48.92)-150.10, 89.290.5570, 1756.99(1232.55 - 1393.21)(1268.27 - 1442.92)(-148.91 - 98.29)(138.15 - 1548.98)Monsanto Company 11-SY-217U 324 of 467Table F-23. Summary of Site KSLA Soybean Seed Anti-nutrients for MON 87712vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein1017.66 (44.06)1064.05 (44.06)-46.39 (59.96)-193.11, 100.330.46887.22, 1792.07(887.87 - 1093.13)(1002.79 - 1175.72)(-159.29 - 66.50)(335.67 - 1409.07)Glycitein98.83 (10.16)101.86 (10.16)-3.03 (12.66)-34.01, 27.950.8188.13, 299.67(75.71 - 125.39)(92.22 - 117.90)(-28.68 - 23.31)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 325 of 467Table F-24. Summary of Site ILCY Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash7.90 (0.30)7.32 (0.30)0.58 (0.42)-0.45, 1.620.2164.29, 8.65(6.93 - 8.75)(7.07 - 8.04)(-0.13 - 1.34)(4.82 - 8.98)Carbohydrates68.63 (1.06)67.21 (1.06)1.42 (1.49)-2.23, 5.080.37755.73, 77.45(66.67 - 70.88)(65.25 - 70.63)(-3.97 - 4.90)(54.40 - 72.96)Moisture (% fwt)75.45 (0.59)73.90 (0.59)1.55 (0.73)-0.24, 3.340.07865.61, 80.67(73.90 - 76.90)(73.10 - 75.00)(0.10 - 3.80)(64.50 - 79.80)Protein19.75 (0.61)19.41 (0.61)0.33 (0.86)-1.78, 2.440.71313.77, 26.51(18.45 - 21.17)(17.88 - 20.50)(-2.05 - 3.29)(16.56 - 27.76)Total Fat3.74 (0.42)6.14 (0.42)-2.40 (0.42)-3.42, -1.380.0010.54, 13.11(3.41 - 4.03)(4.57 - 7.18)(-3.15 - -0.86)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber33.37 (1.43)29.10 (1.43)4.26 (2.03)-0.70, 9.220.08023.12, 38.15(30.74 - 38.10)(26.28 - 32.84)(-0.081 - 11.81)(22.60 - 41.29)Monsanto Company 11-SY-217U 326 of 467Table F-24. Summary of Site ILCY Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber38.88 (1.58)34.35 (1.58)4.53 (2.18)-0.80, 9.870.08224.96, 43.33(33.84 - 41.65)(32.51 - 38.64)(1.33 - 7.93)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 327 of 467Table F-25. Summary of Site NEYO Soybean SeedNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash4.98 (0.16)4.94 (0.16)0.045 (0.19)-0.42, 0.510.8184.43, 5.89(4.54 - 5.30)(4.69 - 5.21)(-0.27 - 0.25)(4.43 - 6.14)Carbohydrates38.54 (0.60)39.13 (0.60)-0.60 (0.85)-2.68, 1.490.50932.36, 41.63(37.56 - 40.03)(37.69 - 40.86)(-1.37 - 1.05)(33.43 - 40.39)Moisture (% fwt)8.46 (0.20)7.88 (0.20)0.58 (0.25)-0.039, 1.200.0615.41, 10.36(7.98 - 8.94)(7.74 - 8.07)(-0.090 - 1.17)(5.43 - 9.86)Protein41.05 (0.14)40.14 (0.14)0.91 (0.15)0.54, 1.28<0.00135.06, 43.58(40.85 - 41.36)(40.03 - 40.21)(0.74 - 1.17)(35.11 - 42.16)Total Fat15.46 (0.55)15.80 (0.55)-0.34 (0.77)-2.22, 1.550.67713.15, 23.90(14.44 - 16.47)(14.09 - 16.95)(-2.32 - 0.53)(15.71 - 22.65)Fiber (% dwt)Acid Detergent Fiber14.94 (0.47)15.41 (0.47)-0.48 (0.67)-2.11, 1.160.5039.99, 22.21(14.11 - 15.87)(14.25 - 16.05)(-1.56 - 1.62)(11.74 - 22.13)Monsanto Company 11-SY-217U 328 of 467Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber16.28 (0.54)17.20 (0.54)-0.93 (0.76)-2.79, 0.940.26911.03, 23.27(14.95 - 17.17)(15.45 - 18.10)(-2.65 - 1.72)(12.18 - 22.88)Amino Acid (% dwt)Alanine1.78 (0.013)1.73 (0.013)0.052 (0.013)0.020, 0.0840.0071.54, 1.88(1.76 - 1.80)(1.70 - 1.74)(0.042 - 0.063)(1.58 - 1.84)Arginine3.19 (0.035)3.02 (0.035)0.17 (0.026)0.10, 0.23<0.0012.51, 3.33(3.16 - 3.24)(2.93 - 3.12)(0.095 - 0.23)(2.57 - 3.24)Aspartic Acid4.77 (0.025)4.64 (0.025)0.13 (0.031)0.053, 0.200.0054.04, 5.07(4.74 - 4.81)(4.57 - 4.67)(0.082 - 0.17)(4.06 - 4.89)Cystine0.60 (0.0069)0.62 (0.0069)-0.023 (0.0097)-0.046, 0.00130.0590.51, 0.67(0.58 - 0.62)(0.61 - 0.63)(-0.051 - 0.0037)(0.54 - 0.69)Glutamic Acid7.68 (0.056)7.41 (0.056)0.27 (0.068)0.11, 0.440.0076.28, 8.18(7.60 - 7.76)(7.28 - 7.48)(0.22 - 0.32)(6.40 - 7.94)Monsanto Company 11-SY-217U 329 of 467Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glycine1.79 (0.0080)1.74 (0.0080)0.052 (0.0096)0.028, 0.0760.0011.52, 1.90(1.78 - 1.80)(1.71 - 1.76)(0.033 - 0.081)(1.54 - 1.85)Histidine1.09 (0.0059)1.06 (0.0059)0.025 (0.0043)0.014, 0.0350.0010.91, 1.17(1.08 - 1.09)(1.04 - 1.07)(0.017 - 0.035)(0.93 - 1.16)Isoleucine1.92 (0.013)1.85 (0.013)0.066 (0.018)0.023, 0.110.0091.62, 2.03(1.90 - 1.93)(1.82 - 1.88)(0.020 - 0.096)(1.60 - 2.00)Leucine3.18 (0.016)3.09 (0.016)0.099 (0.020)0.050, 0.150.0022.71, 3.38(3.17 - 3.21)(3.05 - 3.11)(0.084 - 0.13)(2.77 - 3.29)Lysine2.68 (0.011)2.62 (0.011)0.058 (0.013)0.025, 0.0900.0042.33, 2.81(2.66 - 2.68)(2.59 - 2.65)(0.034 - 0.074)(2.36 - 2.74)Methionine0.57 (0.0048)0.57 (0.0048)0.0020 (0.0068)-0.015, 0.0190.7830.51, 0.59(0.55 - 0.58)(0.56 - 0.58)(-0.018 - 0.018)(0.51 - 0.60)Monsanto Company 11-SY-217U 330 of 467Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Phenylalanine2.17 (0.018)2.08 (0.018)0.095 (0.020)0.046, 0.140.0031.81, 2.33(2.15 - 2.19)(2.03 - 2.13)(0.064 - 0.15)(1.81 - 2.25)Proline2.02 (0.014)1.97 (0.014)0.051 (0.020)0.0011, 0.100.0461.70, 2.13(2.00 - 2.04)(1.95 - 1.99)(0.029 - 0.091)(1.69 - 2.09)Serine2.19 (0.027)2.12 (0.027)0.074 (0.029)0.0023, 0.140.0441.86, 2.33(2.17 - 2.24)(2.10 - 2.13)(0.052 - 0.11)(1.90 - 2.30)Threonine1.55 (0.016)1.58 (0.016)-0.028 (0.023)-0.084, 0.0280.2611.40, 1.69(1.52 - 1.59)(1.55 - 1.63)(-0.089 - 0.040)(1.36 - 1.68)Tryptophan0.44 (0.0084)0.45 (0.0084)-0.012 (0.012)-0.041, 0.0160.3240.36, 0.50(0.41 - 0.47)(0.44 - 0.47)(-0.030 - 0.0028)(0.38 - 0.48)Tyrosine1.47 (0.032)1.45 (0.032)0.028 (0.017)-0.013, 0.0700.1481.28, 1.57(1.41 - 1.54)(1.33 - 1.51)(-0.0033 - 0.078)(1.28 - 1.55)Monsanto Company 11-SY-217U 331 of 467Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Valine2.03 (0.017)1.95 (0.017)0.083 (0.024)0.025, 0.140.0131.71, 2.13(2.00 - 2.05)(1.91 - 1.99)(0.0091 - 0.13)(1.69 - 2.09)Fatty Acid (% Total FA)16:0 Palmitic11.63 (0.052)11.89 (0.052)-0.26 (0.073)-0.44, -0.0790.0127.76, 13.14(11.59 - 11.71)(11.83 - 12.00)(-0.41 - -0.11)(9.00 - 12.03)18:0 Stearic4.00 (0.033)4.02 (0.033)-0.021 (0.033)-0.10, 0.0600.5493.06, 5.10(3.92 - 4.10)(3.94 - 4.07)(-0.12 - 0.032)(3.49 - 4.97)18:1 Oleic19.45 (0.11)19.13 (0.11)0.32 (0.15)-0.056, 0.700.08217.37, 26.86(19.38 - 19.60)(18.73 - 19.38)(0.0019 - 0.68)(18.93 - 25.33)18:2 Linoleic54.44 (0.14)54.47 (0.14)-0.029 (0.20)-0.52, 0.460.89050.14, 57.81(54.10 - 54.61)(54.29 - 54.81)(-0.35 - 0.27)(51.57 - 56.25)18:3 Linolenic9.80 (0.091)9.82 (0.091)-0.017 (0.12)-0.30, 0.270.8895.60, 11.61(9.76 - 9.85)(9.59 - 9.99)(-0.14 - 0.20)(5.89 - 10.16)Monsanto Company 11-SY-217U 332 of 467Table F-25. Summary of Site NEYO Soybean Seed Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)20:0 Arachidic0.30 (0.0029)0.30 (0.0029)0.00053 (0.0039)-0.0089, 0.0100.8940.22, 0.39(0.29 - 0.31)(0.29 - 0.30)(-0.0097 - 0.0091)(0.23 - 0.38)20:1 Eicosenoic0.095 (0.016)0.095 (0.016)0.00010 (0.023)-0.057, 0.0570.9960.094, 0.23(0.075 - 0.14)(0.073 - 0.15)(-0.066 - 0.071)(0.072 - 0.21)22:0 Behenic0.29 (0.0028)0.29 (0.0028)0.0028 (0.0040)-0.0069, 0.0130.5000.18, 0.43(0.29 - 0.30)(0.28 - 0.30)(-0.00065 - 0.011)(0.16 - 0.37)Vitamin (mg/100g dwt)Vitamin E0.96 (0.045)1.13 (0.045)-0.17 (0.059)-0.31, -0.0270.0270.10, 2.85(0.94 - 0.99)(1.02 - 1.30)(-0.31 - -0.065)(0.86 - 2.73)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 333 of 467Table F-26. Summary of Site NEYO Soybean Seed Anti-nutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)AntinutrientLectin (H.U./mg dwt)2.18 (0.27)1.72 (0.27)0.45 (0.24)-0.12, 1.030.1030, 6.11(1.31 - 2.59)(1.25 - 2.27)(0.066 - 0.97)(0.60 - 6.99)Phytic Acid (% dwt)0.99 (0.064)0.97 (0.064)0.013 (0.073)-0.17, 0.190.8630.50, 1.92(0.88 - 1.12)(0.86 - 1.08)(-0.027 - 0.043)(0.66 - 1.74)Raffinose (% dwt)0.66 (0.011)0.71 (0.011)-0.046 (0.015)-0.084, -0.00870.0230.39, 1.01(0.65 - 0.66)(0.69 - 0.74)(-0.072 - -0.028)(0.45 - 0.93)Stachyose (% dwt)4.27 (0.030)4.11 (0.030)0.16 (0.042)0.057, 0.260.0092.45, 5.34(4.24 - 4.29)(4.05 - 4.16)(0.11 - 0.21)(2.57 - 4.68)Trypsin Inhibitor (TIU/mg dwt)33.52 (2.48)33.08 (2.48)0.44 (3.44)-7.98, 8.870.90120.97, 50.01(29.21 - 40.86)(29.37 - 36.43)(-7.22 - 11.49)(24.22 - 51.78)Isoflavone (μg/g dwt)Daidzein1425.70 (61.46)1262.35 (61.46)163.36 (86.92)-49.34, 376.050.1090, 1756.99(1378.10 - 1471.56)(1076.47 - 1533.78)(-121.04 - 363.95)(138.15 - 1548.98)Monsanto Company 11-SY-217U 334 of 467Table F-26. Summary of Site NEYO Soybean Seed Anti-nutrients for MON 87712vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein1050.47 (50.27)898.64 (50.27)151.83 (71.10)-22.14, 325.790.07687.22, 1792.07(1006.30 - 1085.77)(761.46 - 1087.78)(-81.48 - 324.31)(335.67 - 1409.07)Glycitein99.73 (9.66)99.40 (9.66)0.33 (13.66)-33.10, 33.750.9818.13, 299.67(89.15 - 107.95)(77.28 - 129.45)(-31.42 - 30.67)(66.83 - 280.71)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-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 335 of 467Table F-27. Summary of Site NEYO Soybean ForageNutrients for MON 87712 vs. Conventional ControlDifference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.76 (0.35)7.83 (0.35)-1.07 (0.50)-2.29, 0.150.0744.29, 8.65(6.14 - 7.33)(6.56 - 8.74)(-2.60 - 0.77)(4.82 - 8.98)Carbohydrates66.98 (0.83)65.75 (0.83)1.22 (1.17)-1.65, 4.100.33755.73, 77.45(64.83 - 68.67)(63.01 - 67.97)(-2.36 - 5.67)(54.40 - 72.96)Moisture (% fwt)75.38 (0.33)74.73 (0.33)0.65 (0.37)-0.26, 1.560.13265.61, 80.67(74.80 - 76.40)(74.40 - 75.40)(-0.30 - 1.70)(64.50 - 79.80)Protein21.01 (0.48)20.57 (0.48)0.44 (0.68)-1.23, 2.110.54213.77, 26.51(20.44 - 21.53)(18.71 - 22.24)(-1.79 - 2.40)(16.56 - 27.76)Total Fat5.37 (0.29)5.84 (0.29)-0.47 (0.41)-1.48, 0.540.2980.54, 13.11(4.84 - 6.53)(5.57 - 6.21)(-1.37 - 0.95)(2.73 - 12.11)Fiber (% dwt)Acid Detergent Fiber33.04 (1.31)31.24 (1.31)1.80 (1.85)-2.72, 6.320.36723.12, 38.15(30.84 - 35.04)(27.71 - 33.66)(-2.82 - 7.33)(22.60 - 41.29)Monsanto Company 11-SY-217U 336 of 467Table F-27. Summary of Site NEYO Soybean Forage Nutrients for MON 87712 vs. Conventional Control (continued)Difference (Test minus Control)Analytical Component (Units)1Test2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber36.33 (1.71)35.65 (1.71)0.67 (2.42)-5.26, 6.600.79024.96, 43.33(34.14 - 40.76)(32.73 - 41.41)(-6.68 - 8.04)(25.78 - 44.41)1dwt = dry weight; fwt = fresh weight.2Test refers to MON 87712.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnologyderived, conventional control, A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negative limitsset to zero.Monsanto Company 11-SY-217U 337 of 467 

### References for Appendix F

AACC. 1998. Approved methods of the Ameri can Association of Cereal Chemists.

American Association of Cereal Ch emists, Inc., St. Paul, Minnesota.

AOAC. 2005a. AOAC official method 982.30: Prot ein efficiency rati o. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005b. AOAC offi cial method 923.03: Ash of fl our. Official Methods of Analysis of AOAC International, Associati on 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 offici al method 955.04: Nitrogen (total) in fertilizers. Official Methods of Analysis of AOAC Internationa l, Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2007a. AOAC official method 960.39 a nd 948.22. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2007b. AOAC offi cial 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 Reco mmended 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 li quid 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). Agri culture Handbook No. 379. U.S. Department of Agriculture, Agricultural Res earch Service, Washington, D.C.Monsanto Company 11-SY-217U 338 of 467 

### Kakade, M.L., J.J. Rackis, J.E. McGhee a nd G. Puski. 1974. Determination of trypsin inhibitor activity of soy pr oducts: 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. Isol ation and quantitation of lectins from vegetable oils. Lipids 22:667-668.

Lehrfeld, J. 1989. High-performa nce liquid chromatography analysis of phytic acid on a pH-stable macroporous polymer column. Cereal Chemistry 66:510-515.

Lehrfeld, J. 1994. HPLC separation and quant itation of phytic acid and some inositol phosphates in foods: Problems and solutions. Jo urnal of Agricultural and Food Chemistry

42:2726-2731.

Liener, I.E. 1955. The photometric determina tion of the hemagglu tinating 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 chro matographic 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. Ener gy value of foods... Ba sis and derivation. Agricultural Handbook No. 74. US Department of Agriculture, Agri cultural Research Service, Washington, D.C.

OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean: Key food and feed nutrients and anti-nutrients. ENV/JM/MONO(2001)15. Series on the Safety of Novel Foods a nd Feeds No.2. Organisation for Economic Cooperation and Development, Paris, France.

Pettersson, H. and K.-H. Kiessling. 1984. Liqui d chromatographic determination of the plant estrogens coumestrol and isoflavones in animal feed. Journa l 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-performa nce liquid chromatography with fluorometric

detection. Journal of Food Science 50:121-124.Monsanto Company 11-SY-217U 339 of 467 

### Appendix G: Materials, Methods, an d Individual-Site Results for Seed Dormancy and Germination Assessment of MON 87712

G.1. Materials

Seed dormancy and germination characterist ics were assessed on seed from MON 87712, a conventional control A3525, and commercial re ference varieties produced at the Stark County, Illinois; Macon Count y, Missouri; and Butler Count y, Missouri sites in 2009 field trials (Appendix G). The seed fr om MON 87712, the conventional control A3525,

and the commercial reference varieties were harv ested from four replications at each of the three field sites and pooled to produce one seed lot of M ON 87712, the conventional control A3525, and each commercial reference va riety from each field site for dormancy and germination testing (Table G-1).

G.2. Characterization of the Materials

For the MON 87712, conventional control A 3525, 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 Facili ty and Experimental Methods

Seed dormancy and germination evaluations we re conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigat or was qualified to conduct seed dormancy and germination testing consistent with the st andards established by the Association of Official Seed Analysts, a seed trad e association (AOS A, 2000; 2006; 2007).

Seed lots of MON 87712, the conventiona l control A3525, and four commercial reference varieties were produced from each of three sites and tested under six different temperature regimes. Six germination cham bers were maintained under dark conditions with one the of the following temperatur e 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 temper ature for eight hours. The temperature inside each germination chamber was m onitored and recorded every 15 minutes throughout the duration of the assessment. Fo r each seed lot, four replicated paper germination towels were prepared per facil ity 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 pl anting system. A second water-moistened germination towel was placed on top of the s eed. 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 germ ination 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 characterist ic 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); howev er, additional temperature regimes were included to test a range of te mperature 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, d ead, and viable firm-swollen seed. Emergence and/or development of essential structures of seedlings that otherwise would be categorized as “normal germinated” under optim al temperature conditions may not be so at non-optimal temperatures. Therefore, fo r the additional temperature regimes, no

distinction was made between norma l 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 wa s 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 ch aracteristics analy zed included percent germinated (categorized as percent normal ge rminated and percent abnormal germinated for the AOSA temperature regime), percent vi able hard seed, per cent dead, and percent

viable firm-swollen seed. The level of stat istical significance was predetermined to be 5% ( α =0.05). MON 87712 was not statistically compared to the reference substances, nor were comparisons made across temperat ure regimes. The minimum and maximum mean values (reference range) were determined from the reference substances across the seed production sites. The following is a summ ary 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 significan t differences were detected

between MON 87712 and the conventional control A3525 (Table G-2). Four statistically significant differences were detected be tween MON 87712 and the conventional control A3525 for seed produced at the ILWY a nd 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 co ntrol 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 cont rol 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

Site 1 

### Material Type Material Name 2 

### Phenotype Sample ID

MOAN Control A3525 Conventional 11262199 MOAN Test MON 87712 Soybean Intrinsic Yield 11262197

MOAN Reference SB3369R Glyphosate-Tolerant 3 

### 11262200

MOAN Reference Pioneer 93M14 Conventional 11262201

MOAN Reference NutriPri de 8339 Glyphosate-Tolerant 3 

### 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-Tolerant 3 

### 11262059

MOFI Reference NK S30- D4 Glyphosate-Tolerant 3 

### 11262061

MOFI Reference LG-C3540 Conventional 11262063

ILWY Control A3525 Conventional 11262070 ILWY Test MON 87712 Soybean Intrinsic Yield 11262065 ILWY Reference SB3369R Glyphosate-Tolerant 3 

### 11262072

ILWY Reference Pioneer 93M11 Glyphosate-Tolerant 3 

### 11262075

ILWY Reference Stine 3300-0 Conventional 11262077

ILWY Reference SB3819 Conventional 11262081

1 

### Site = Site where seed lot was produ ced; 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 = Commercia lly-available Roundup Ready soybean.Monsanto Company 11-SY-217U 343 of 467Table G-2. Dormancy and Germination Characteristics of MON 87712 and the Conventional Control A3525 Seed Producedat each of the Three Field SitesILWY2MOAN2MOFI2Mean %(S.E.)3Mean %(S.E.)3Mean % (S.E.)3Temperature RegimeGermination Category1MON 87712ControlMON 87712ControlMON 87712Control10 °CGerminated99.8 (0.3)99.5 (0.3)100.0 (0.0)99.8 (0.3)99.5 (0.3)99.5 (0.3)Viable Hard†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Dead0.3 (0.3)0.5 (0.3)0.0 (0.0)0.3 (0.3)0.5 (0.3)0.5 (0.3)Viable Firm Swollen0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)20 °CGerminated99.8 (0.3)100.0 (0.0)99.8 (0.3)99.3 (0.5)98.8 (0.5)98.8 (0.3)Viable Hard†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Dead0.3 (0.3)0.0 (0.0)0.3 (0.3)0.8 (0.5)1.3 (0.5)1.3 (0.3)Viable Firm Swollen†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)30 °CGerminated100.0 (0.0)100.0 (0.0)99.0 (0.4)99.5 (0.3)99.0 (0.7)99.0 (0.4)Viable Hard†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Dead0.0 (0.0)0.0 (0.0)1.0 (0.4)0.5 (0.3)1.0 (0.7)1.0 (0.4)Viable Firm Swollen†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Monsanto Company 11-SY-217U 344 of 467Table G-2. Dormancy and Germination Characteristics of MON 87712 and the Conventional Control A3525 Seed Producedat each of the Three Field Sites (continued)ILWY2MOAN2MOFI2Mean % (S.E.)3Mean % (S.E.)3Mean % (S.E.)3Temperature RegimeGermination Category1MON 87712ControlMON 87712ControlMON 87712Control10/20 °CGerminated100.0 (0.0)*99.3 (0.3)99.8 (0.3)100.0 (0.0)99.8 (0.3)99.3 (0.5)Viable Hard†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Dead0.0 (0.0)*0.8 (0.3)0.3 (0.3)0.0 (0.0)0.3 (0.3)0.8 (0.5)Viable Firm Swollen†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)10/30 °CGerminated99.8 (0.3)100.0 (0.0)100.0 (0.0)99.5 (0.3)98.8 (0.5)99.0 (0.6)Viable Hard†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Dead0.3 (0.3)0.0 (0.0)0.0 (0.0)0.5 (0.3)1.3 (0.5)1.0 (0.6)Viable Firm Swollen†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)20/30 °CNormal Germinated96.5 (1.0)97.8 (1.0)99.8 (0.3)*97.8 (1.1)94.0 (0.8)94.3 (1.3)(AOSA)Abnormal Germinated2.5 (0.9)2.0 (0.8)0.0 (0.0)*2.0 (0.9)5.3 (0.8)5.0 (1.2)Viable Hard†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)Dead1.0 (0.4)0.3 (0.3)0.3 (0.3)0.3 (0.3)0.8 (0.5)0.8 (0.5)Viable Firm Swollen†0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)0.0 (0.0)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 A3525.† No statistical comparisons could be made due to lack of variability in the data.1Dormancy and germination characteristics were analyzed using an analysis of variance (ANOVA) model.2Site codes are as follows: ILWY = Stark County, IL; MOAN = Macon County, MO; and MOFI = Butler County, MO3Mean based on n = 4. S.E. = Standard Error. In some instances, the total percentage ofboth MON 87712 and the conventionalcontrol A3525 did not equalexactly 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. Contribu tion no. 29. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2006. Seedling evaluation handbook. Contribu tion no. 35. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2007. Tetrazolium testing handbook - Fa mily: Brassicaceae I. Association of Official Seed Analys ts, Lincoln, Nebraska.Monsanto Company 11-SY-217U 346 of 467 

### Appendix H: Materials, Methods, and I ndividual-Site Results from Phenotypic, Agronomic, and Environmental Intera ction 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 soyb ean control A3525, and 18 commercial reference varieties. The list of the soybean ma terials 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 conventiona l 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 s ites provided a range of environmental and agronomic conditions representative of majo r 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 designa ted for the collection of phenotypic data. Rows # 4 and 5 were designated for the colle ction 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 alleyway s 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 en sure collection of more robust arthropod

abundance data within the test area.

Each plot at the IAJA, IARL, ILCY, IL MS, 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 ro ws. The entire plot ar ea was surrounded by a border of a commercial soybean variety appr oximately 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 respectiveMonsanto 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 348 of 467Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental Interaction AssessmentNote: The study also included two additional experimental materials that were outside of the scope ofthe objectives of this evaluation of MON 87712.1T = Test, C = Control, and R = reference2Phenotypic abbreviations: SIY = soybean intrinsic yield, GT = glyphosate-tolerant, Conventional = conventional commercial3Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILMS = Effingham County, IL;ILSE = Champaign County, IL; ILWY = StarkCounty, IL; INRC = Parke County, IN; INSH= Boone County, IN; KSLA = Pawnee County, KS; MOAN = Macon County, MO;MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County, MO; MOSL = St. Louis County, MO; MOWR = Lincoln County, MO; NEYO = YorkCounty, NE; PAGR = Berks County, PA, site1; PAHM = Berks County, PA, site 2.MaterialMaterialType1Phenotype2Monsanto LotNumber Sites3MON 87712 T SIY 11223539 AllA3525 C Conventional 11223542 AllSB3369R R GT 11226924 ILWY, MOAN, MOSL, NEYOSB3579R R GT 11226925 ILCY, MOCB, MOKISB3888R R GT 11226926 ARNE, INRC, KSLANuPride 8339 R GT 11226939 ILCY, ILMS, MOAN, MOKINC+ 2A95 R GT 11226838 IARL, ILMS, KSLA, MOCB, PAHMPioneer 93M43 R GT 11226839 IAJA, ILSE, INSH, MOWR, PAHMPioneer 93M11 R GT 11226840 ARNE, ILWY, MOWR, NEYONK S28-B4 R GT 11226842 IARL, INRC, INSH, MOSL, PAGRNK S30-D4 R GT 11226843 IAJA, ILSE, MOLP, MOFI, PAGRMidland 363 R Conventional 11226698 IAJA, INRC, KSLA, MOCBStine 3300-0 R Conventional 10001134 ILCY, ILWY, MOCB, MOFI, NEYOPioneer 9382 R Conventional 11226581 IAJA, ILSE, MOWR, PAHMPioneer 93M62 R Conventional 11226582 ILMS, MOAN, MOWR, PAGRSB3819 R Conventional 11226928 ILWY, INSH, MOKI, NEYOLG 3211 R Conventional 11226860 IARL, ILCY, INSH, MOSL, PAHMLG C3540 R Conventional 11226858 IARL, INRC, MOFI, PAGRPioneer 93M14 R Conventional 11226720 ARNE, ILMS, MOAN, MOSLNuPride 3202 R Conventional 11226938 ARNE, ILSE, KSLA, MOKIMonsanto Company 11-SY-217U 349 of 467Table H-2. Field andPlanting Information1Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILMS = Effingham County, IL;ILSE = Champaign County, IL; ILWY = StarkCounty, IL; INRC = Parke County, IN; INSH= Boone County, IN; KSLA = Pawnee County, KS; MOAN = Macon County, MO;MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County, MO; MOSL = St. Louis County, MO; MOWR = Lincoln County, MO; NEYO = YorkCounty, NE; PAGR = Berks County, PA, site1; PAHM = Berks County, PA, site 2.2Month-day-year.3Width × length. Sites with arthropod collection (ARNE, ILSE, MOCB, and MOSL) had larger plot areas.Site1PlantingDate2Plantingrate(seeds/ft)Plantingdepth(in)Plotsize(ft)3Rows/plot Soil series, organic matter, pHCropping History2007 2008ARNE 6-9-09 9 1.0 30 × 30 12 Bosket sandy loam; 1.0%; 6.0 Soybean CornIAJA 6-5-09 9 1.5 10 × 20 4 Clarion loam; 2.5%; 5.6 Soybean CornIARL 6-5-09 9 1.0 10 × 20 4 Mahaska silty clay loam; 3.54%; 7.01 Corn / Wheat Sorghum /SoybeanILCY 6-30-09 9 1.4 10 × 20 4 Hoyleton-Darmstadt complex / silt loam;2.6%; 7.3Soybean MiloILMS 6-30-09 9 1.0 10 × 20 4 Bluford silt loam; 2.0%; 6.3 Corn SoybeanILSE 6-24-09 9 1.5 30 × 30 12 Flaragan silt loam; 5.0%; 6.5 Sweet corn CornILWY 6-6-09 9 2.0 10 × 20 4 Plano silt loam; 3.4%; 6.3 Soybean CornINRC 6-25-09 9 1.0 10 × 20 4 Reesville silt clay loam; 1.4%; 7.0 Soybean Wheat / FallowINSH 6-8-09 9 1.5 10 × 20 4 Crosby silt loam; 2.1%; 5.8 Corn CornKSLA 6-8-09 9 2.0 10 × 20 4 Silt loam; 2.6%; 7.6 Sunflower /Sorghum / Fallow /CornSunflowerMOAN 6-29-09 9 1.0 10 × 20 4 Mexico silt loam; 2.1%; 7.2 Soybean WheatMOCB 6-25-09 9 1.0 30 × 30 12 Putnam silt loam; 2.5%; 6.8 Soybean CornMOFI 6-12-09 9 0.5 10 × 20 4 Amagon silt loam; 1.8%; 5.0 Soybean RiceMOKI 6-25-09 9 1.25 10 × 20 4 Mexico silt loam; 3.1%; 6.4 Soybean CornMOSL 6-5-09 9 1.0 30 × 30 12 Peers silty clay; 2.9%; 7.5 Soybean CornMOWR6-6-09 9 1.0 10 × 20 4 Hatton silt loam; 2.9%; 6.5 Soybean FallowNEYO 6-5-09 9 1.0 10 × 20 4 Hastings silt loam; 3.0%; 6.2 Soybean SorghumPAGR 6-8-09 9 1.5 10 × 20 4 Bedington-Berks complex clay loam; 1.4%;5.2Corn CornPAHM 6-25-09 9 1.5 10 × 20 4 Philo / Atkins loam; 1.2%; 6.4 Soybean CornMonsanto 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 cr op plants and their receiving environment) were used to characteri ze MON 87712 by evaluating plant response to abiotic stressors, disease damage, arthropod -related damage, and pest and beneficial arthropod abundance in the pl ots 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 conventiona l control A3525 were evalua ted at all 19 sites for differences in plant response to abiotic stre ssors, disease damage, and arthropod-related damage. Three abiotic stressors, three diseas es, and three arthropod pests were evaluated four times during the growing seas on 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 c hose abiotic stressors, di seased, and arthropod pests that were either actively causing plant injury in the study area or were likely to occur in soybean during the gi ven observation period. Ther efore, abiotic stressors, diseases, and arthropod pests assessed often varied between observations at a site and

between sites.

Abiotic stressors and disease damage observati ons 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 th e arthropod-specific 0 – 5 rating scales of increasing severity listed below.Monsanto Company 11-SY-217U 351 of 467 

### 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 plan t; 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 stuntingMonsanto Company 11-SY-217U 352 of 467 

### 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 usi ng a vertical beat sheet samp ling method (Drees and Rice, 1985). The beat sheet was approximately 36×36 in ch sheet constructed of a stiff material with a collecting trough at the bottom. Th e sheet was placed between row 7 and row 8 and the collecting trough was positi oned near the base of the plan ts 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 fr om the same row, approximately 8 ft from the first sub-sample. Two sub-samples were co llected from row 9 in the same manner. A total of four sub-samples were collected inthis 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 pr eselected pest and beneficial ar thropods (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 arthro pods, were enumerated at all

sites for each collection time. Additi onally, for each individual collection ( e.g., Collection 1, ARNE site), four non-systematic ally selected sample s 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 va ried between collections from a site and between sites due to differences in tempor al activity and geographical distribution of arthropod taxa.Monsanto Company 11-SY-217U 353 of 467 

### H.9. Environmental Interact ions Evaluation Criteria

For the assessments of abiotic stress re sponse and disease damage, MON 87712 and the

conventional control A3525 were co nsidered different in suscep tibility or tolerance to an abiotic stressor or disease on a particular obser vation 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 statis tical analysis, as appropriate.

H.10. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summariza tion, and analysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence 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 st atistical 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 flow ering, days to 50% senescence, days to physiol ogical maturity, plant height , lodging, pod shattering, final stand count, seed moisture, 100 seed weight, and yield. Growth stage, flower color, abiotic stress response, and di sease damage data were cate gorical and not statistically analyzed. Arthropod-related damage and pest and beneficial arth ropod abundance data were statistically analyzed only within indi vidual observations/colle ctions and sites due to the variation in temporal activity a nd 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 maximu m 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 354 of 467Table H-3. Data Missing orExcluded from AnalysisSite1Material nameMaterial typePlotsCharacteristicsReason for exclusionAllAllAllAllAnimal damageAnimal damage is a non-uniformenvironmental stressor.AllAllAllAllHerbicide injuryHerbicide injury is a non-uniformenvironmental stressor.ILCYAllAllAllBrown stem rot,observation 4Assessment of brown stem rot was notperformed at all plots.ILCYAllAllAllPlant heightData were not collected.IAJAAllAllAllGrowth stageMonitoringGrowth stage monitoring data collected atsenescence indicated the observations weremade after harvest which could not be possible.Therefore, growth stage monitoring datacollected at senescence were excluded.IAJAAllAllAllArthropod damageData were not collected.IAJAAllAllAllDisease andabiotic stressors,observations 2 & 3Data were not collected.IAJAMON 87712A3525Pioneer 93B82TestControlReference209, 305;303;208Final stand countData were not collected.Monsanto Company 11-SY-217U 355 of 467Table H-3. Data Missing or Excluded from Analysis (continued)Site1Material nameMaterial typePlotsCharacteristicsReason for exclusionIAJEAllAllAllAllSite was dropped due to plant damage fromfrost.IARLMON 87712Test405Flower colorData was not collected.IARLAllAllAllDisease andabiotic stressors,observation 2Inconsistent use of stressors.IARLNC+ 2A95Reference408Drought,observation 4Data were not collected.ILMSPioneer 93M14Reference103, 207, 301, 404AllThe wrong number of seed was counted andplaced into the planting envelopes. As a result,excessive number of seed was planted.ILSEAllAllAllPlant heightData were not collected.ILSEMON 87712Test408Days to 50% endof floweringOriginal data in paper notebook was crossedout but no explanation was provided. As aresult, the electronic data could not be verified.ILSEMON 87712Test408Days to 50% endof senescenceOriginal data in paper notebook was crossedout but no explanation was provided. As aresult, the electronic data could not be verified.Monsanto Company 11-SY-217U 356 of 467Table H-3. Data Missing or Excluded from Analysis (continued)Site1Material nameMaterial typePlotsCharacteristicsReason for exclusionILSEAllAllAllDisease and abioticstressors, observation 2,3, and 4Data were not collected.ILSEAllAllAllGrowth stageMonitoring after R4Data were not collected.ILSEAllAllAllArthropod damageIncorrect rating scale was used.INSHLG 3211Reference206YieldOnly one row of the plot was harvested due toequipment malfunction.INSHAllAllAllDisease and abioticstressors, observation 2Data were not collected.INRCAllAllAllArthropod damageData were not collected.INRCAllAllAllSeedling vigorData were not collected.INRCAllAllAllDisease and abioticstressors, observation 2Data were not collected.KSLAAllAllAllDays to 50% end offloweringData were not collected.Monsanto Company 11-SY-217U 357 of 467Table H-3. Data Missing or Excluded from Analysis (continued)Site1Material nameMaterial typePlotsCharacteristicsReason for exclusionKSLAAllAllAllEyespotEyespot is a corn disease and inappropriate tobe evaluated as a soybean disease stressor.KSLAAllAllAllFrost damageAssessment of frost damage was not performedon all plots.MOANPioneer 93M14Reference110, 205, 309, 402AllThe wrong number of seed was counted andplaced into the planting envelopes. As a result,excessive number of seed was planted.MOANMON 87712Test407Days tophysiologicalmaturityData was not collected.MOCBStine 3300-0Reference401Days to 50% endof floweringData was not collected.MOCBSB3579RReference110Days tophysiologicalmaturityData was not collected.MOLPAllAllAllAllSite was dropped due to poor plant stand fromflood.Monsanto Company 11-SY-217U 358 of 467Table H-3. Data Missing or Excluded from Analysis (continued)Site1Material nameMaterial typePlotsCharacteristicsReason for exclusionMOKIA3525Control108Early stand countData was recorded on a date when the vastmajority of the seedlings had not yet emerged.MOKIAllAllAllSeedling vigorData were not collected.MOKIAllAllAllDisease andabiotic stressors,observations 1 & 2Data were not collected.MOSLSB3369RReference304Sudden death,observation 4Data were not collected.MOSLAllAllAllArthropodabundanceData were not collectedMOSLAllAllAllBiomass andphotosyntheticdataData were of poor quality due to the lack ofavailable sites.MOWRPioneer 93M62Reference406Arthropodstressors,observation 2Data from two of the three arthropod stressorswere not collected.MOWRPioneer 93M43Reference110Disease andabiotic stressors,observation 3Data were not collected.Monsanto Company 11-SY-217U 359 of 467Table H-3. Data Missing or Excluded from Analysis (continued)Site1Material nameMaterial typePlotsCharacteristicsReason for exclusionPAGRLG 3211Reference101Alternaria,observation 1Data were not collected.PAGRMON 87712MON 87712(-)A3525A3525(-)Pioneer 93M62TestControlControlControlReference102, 203, 304, 404;302, 401;204, 306;205, 305, 405;101, 206, 308, 409Plant heightData were not collected.PAHMLG C3540Reference409Bacterial blight,observation 2Data were not collected1Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IAJE = Greene County, IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL;ILMS = Effingham County, IL; ILSE = Champaign County, IL; ILWY = StarkCounty, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = Pawnee County, KS;MOAN = Macon County, MO; MOCB = Shelby County, MO; MOFI = Butler County, MO; MOLP = Adair County, MO; MOKI = Callaway County, MO; MOSL = St. LouisCounty, MO; MOWR = Lincoln County, MO; NEYO =York County, NE; PAHM = Berks County, PA,site 1; PAGR = Berks County, PA, site 2.Monsanto Company 11-SY-217U 360 of 467 

### H.12. Individual Field Site Plant Growth and Developmen t 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). Th ese 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 ge nerated due to lack of variability. The nineteen flower color comparisons were categ orical and were not statistically analyzed; however, at each site, all plants of M ON 87712 and the conventional control A3525 had purple flowers as expected (Table H-4) . MON 87712 and the conve ntional control A3525 were within the same range of plant growthstage observations among the sites (Table H5).

Differences in stand count were observed between MON 87712 and the conventional

control A3525 at the individua l 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 lo wer 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 pl ants/plot), the ILSE site (260.3 vs. 232.3 plants/plot), the INSH site (327.5 vs. 305.8 pl ants/plot), the KSLA site (300.5 vs. 277.8 plants/plot), the MOWR s ite (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 A 3525 at individual sites. Plants of

MON 87712 flowered earlier than the conventional c ontrol 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 contro l 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 earli er 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 afte r 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 thr ee sites (ILWY, INRC, and INSH ). Plants of MON 87712 reached physiological maturity significantly la ter than the conventional control A3525 at 16 sites. MON 87712 had significantly more lodging than the c onventional control

A3525 at the KSLA site (3.0 vs. 1.8 ranting).

Pod shattering was significantly lower fo r 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%) an d 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 diffe rences in yield were observed between MON 87712 and the

conventional control A3525 at the individual sites. Yi eld was higher for MON 87712

than the conventional contro l 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 de tected 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 dete cted in the combined -site analysis, this suggests these differences were not indicative of a consiste nt 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 de tected for early stand count at five sites, days to 50% senescence at sixteen sites, days to physiologi cal maturity at sixteen sites, final stand count at six sites, and yield at six sites, and statistical diffe rences were detected for these phenotypic characteristics in the combined-sit e analysis, the assess ed phenotypic values of MON 87712 were within their respec tive variation of the study references.

H.13 Individual Field Site Environmenta l Interaction Results and Discussion

In an individual site assessment, no statistically significant differences ( α = 0.05) were

observed between MON 87712 and the conventi onal 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 dama ge, no statistically si gnificant 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 comp arisons. The bean leaf beetle damage was higher in MON 87712 than the conventiona l control A3525 in Ob servation 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 th an 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 conv entional control A352 5 from JapaneseMonsanto Company 11-SY-217U 362 of 467 

### beetles in Observation 1 at the ILMS s ite (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 rati ng). MON 87712 had less dama ge than the conventional control A3525 from soybean loopers in Ob servation 2 at the MOAN site (0.55 vs. 0.90 rating) and Observation 3 at the MOFI s ite (0.45 vs. 0.85 rating). MON 87712 had less damage than the conventi onal control A3525 from velv etbean caterpillars in Observation 3 at the MOFT site (0.45 vs. 0.85 rating). The mean damage ratings for

these detected differences were outside thei r respective reference range. However, the differences detected were small in magnitude and these differences were not consistent

across observations or sites. Thus, these detected diffe rences in arthropod-related damage were not indicative of a consistent re sponse associated with the trait and are not considered biologically meani ngful in terms of an advers e environmental impact of MON 87712 compared to the conventional control A3525.

In an assessment of pest and beneficial ar thropod abundance, no stat istically significant differences were detected ( α = 0.05) between MON 87712 a nd the conventional control A3525 for 106 out of 115 comparisons, incl uding 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, incl uding six pest arthropod comparisons and two beneficial arthropod comparisons, for whic h p-values could not be generated due to lack of variability in the data. A total of nine statistically signifi cant differences were

detected between MON 87712 and the conve ntional control A 3525 for arthropod abundance, including five differences for pe st arthropods and fo ur differences for beneficial arthropods.

The five differences detected out of the 59 pest arthropod compar isons involved aphids, bean leaf beetles, green cloverworms, and stink bugs (T able H-8). MON 87712 had higher aphid abundance than the conventiona l 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 A 3525 in Collectio n 2 at the ILSE site (1.3 vs. 0.3 per plot). MON 87712 had lower green cloverworm ab undance 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 conventi onal 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 a phids 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 Collecti on 3 at the MOCB site were outside their respective reference range. However, the differences detected for these taxa were not consistently detected acro ss 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 consider ed biologically meaningf ul 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 a bundance than the co nventional control A3525 in Collection 1 at the ARNE site ( 4.3 vs. 1.8 per plot). MON 87712 had higher ladybird beetle abundance thanthe 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 Co llection 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 th e 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 Collectio n 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 re sponse 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 364 of 467Table H-4. Individual-Site Phenotypic Comparison of MON 87712 to Conventional Control A3525Phenotypic Characteristic (units)Early stand count (# of plants/plot)Seedling vigor (1-9 scale)Days after 1 Jan 2009 to50% flowering3Site1MON 87712 (SE)2Control (SE)MON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)ARNE301.5 (2.7)299.0 (7.4)4.8 (0.6)4.5 (0.7)193.0 (0.0)*190.8 (0.5)IAJA321.3 (4.4)*303.3 (16.4)2.0 (0.0)2.3 (0.3)204.3 (0.5)*207.3 (0.3)IARL333.3 (4.9)*349.0 (2.5)1.3 (0.3)1.3 (0.3)205.0 (0.0)204.5 (0.3)ILCY325.5 (9.6)319.5 (6.9)1.0 (0.0)1.3 (0.3)214.8 (0.3)215.8 (0.5)ILMS310.3 (2.1)320.3 (6.9)2.5 (0.3)2.5 (0.3)218.5 (0.9)219.3 (0.6)ILSE234.8 (17.7)224.3 (9.6)7.3 (0.5)7.3 (0.3)218.3 (0.3)218.0 (0.4)ILWY336.8 (9.6)347.3 (3.1)2.0 (0.0)†2.0 (0.0)206.3 (0.3)*205.3 (0.3)INRC273.3 (11.4)269.5 (10.9)——218.0 (0.0)*216.5 (0.5)INSH321.5 (9.1)315.0 (6.8)5.3 (0.3)5.5 (0.5)205.8 (0.6)205.8 (0.6)KSLA312.8 (3.8)*293.8 (10.8)3.0 (0.0)3.3 (0.3)201.0 (0.0)200.8 (0.3)MOAN330.8 (1.1)322.3 (5.5)3.0 (0.0)3.0 (0.0)219.3 (0.3)219.5 (0.3)MOCB308.0 (6.7)278.2 (33.7)2.5 (0.3)3.5 (0.5)216.8 (0.3)217.5 (0.3)MOFI289.3 (2.8)*266.5 (7.6)3.0 (0.0)3.0 (0.4)201.0 (0.4)200.3 (0.6)MOKI153.5 (12.2)139.3 (28.6)——217.5 (0.5)217.0 (0.7)MOSL349.5 (2.7)351.0 (3.2)1.0 (0.0)†1.0 (0.0)196.0 (0.4)195.8 (0.3)MOWR354.0 (0.9)*343.3 (1.4)1.0 (0.0)†1.0 (0.0)199.3 (0.3)198.5 (0.5)NEYO338.3 (4.6)314.5 (8.4)3.0 (0.0)†3.0 (0.0)206.3 (0.3)*205.0 (0.0)PAGR244.5 (5.7)238.8 (14.2)4.0 (0.6)4.3 (0.5)209.3 (0.3)*210.5 (0.3)PAHM312.7 (7.4)309.5 (4.4)2.3 (0.5)2.8 (0.3)220.0 (0.4)220.5 (0.3)Monsanto Company 11-SY-217U 365 of 467Table H-4. Individual-Site Phenotypic Comparisonof MON 87712 to Conventional Control A3525 (continued)Phenotypic Characteristic (units)Days after 1 Jan 2009 to50% end of flowering4Days after 1 Jan 2009 to50% senescence5Days after 1 Jan 2009 tophysiological maturity6Site1MON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)ARNE222.0 (0.0)220.3 (0.5)256.3 (0.3)*253.0 (0.0)268.0 (0.0)*263.8 (0.5)IAJA230.8 (0.3)231.5 (0.5)266.8 (0.3)*264.8 (0.3)287.3 (0.5)285.8 (0.3)IARL225.8 (0.5)226.0 (0.0)268.3 (0.3)*264.8 (0.3)290.5 (0.3)*281.5 (0.3)ILCY234.0 (0.6)*232.8 (0.3)267.0 (0.0)*264.5 (0.7)285.0 (0.4)*280.5 (0.3)ILMS237.0 (0.4)237.5 (0.5)269.3 (0.3)*268.0 (0.4)282.8 (0.5)*277.5 (0.3)ILSE241.0 (1.0)239.8 (0.9)280.3 (0.7)*275.0 (0.7)284.8 (0.3)*280.8 (0.3)ILWY225.5 (0.5)226.5 (0.5)269.8 (1.0)268.5 (0.9)279.0 (0.0)†279.0 (0.0)INRC254.0 (0.0)*255.0 (0.0)272.5 (0.3)272.0 (0.7)283.0 (0.0)†283.0 (0.0)INSH228.0 (0.0)228.5 (0.3)265.3 (0.9)264.0 (1.0)274.3 (1.0)*271.0 (1.0)KSLA——265.0 (0.0)*259.5 (0.3)273.0 (0.0)*266.5 (0.3)MOAN244.3 (0.3)243.3 (1.1)275.0 (0.7)*271.3 (0.8)290.7 (0.3)*287.0 (0.4)MOCB240.5 (1.2)240.0 (1.4)277.0 (0.0)*271.8 (1.4)291.8 (0.3)*287.0 (1.5)MOFI221.5 (0.3)*223.3 (0.3)255.8 (0.3)*252.0 (0.4)283.8 (0.3)*282.0 (0.0)MOKI243.0 (0.0)*239.3 (0.6)276.8 (0.3)*273.3 (0.3)276.8 (0.3)*273.0 (0.0)MOSL219.0 (0.0)218.0 (0.0)259.5 (0.5)*255.8 (0.5)267.3 (0.5)*263.0 (0.4)MOWR219.5 (0.3)219.8 (0.3)260.0 (0.0)*257.0 (1.0)268.0 (0.0)*262.8 (0.3)NEYO224.5 (0.3)224.8 (0.3)267.0 (0.0)*264.8 (0.3)278.0 (0.0)*277.3 (0.3)PAGR226.8 (0.5)*225.5(0.3)266.0 (0.0)*264.8(0.3)280.5 (0.3)*278.5 (0.3)PAHM235.8 (0.3)235.5 (0.3)272.8 (0.3)*270.5 (0.7)294.0 (0.6)*291.5 (0.5)Monsanto Company 11-SY-217U 366 of 467Table H-4. Individual-Site Phenotypic Comparisonof MON 87712 to Conventional Control A3525 (continued)Phenotypic Characteristic (units)Flower color7Plant height (in)Lodging (1-9 scale)Site1MON 87712ControlMON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)ARNEpurplepurple38.3 (0.7)37.1 (0.7)4.8 (0.3)4.0 (0.0)IAJApurplepurple41.9 (0.5)40.9 (0.2)1.0 (0.0)†1.0 (0.0)IARLpurplepurple36.0 (0.6)35.9 (1.1)4.5 (0.3)3.8 (0.5)ILCYpurplepurple——1.0 (0.0)†1.0 (0.0)ILMSpurplepurple21.0 (0.7)21.0 (0.5)1.0 (0.0)†1.0 (0.0)ILSEpurplepurple——3.8 (0.3)3.8 (0.3)ILWYpurplepurple35.7 (0.5)35.1 (1.8)3.3 (0.3)3.3 (0.3)INRCpurplepurple24.3 (0.6)24.1 (0.7)1.0 (0.0)†1.0 (0.0)INSHpurplepurple27.1 (0.8)26.4 (0.4)1.0 (0.0)†1.0 (0.0)KSLApurplepurple41.2 (1.2)38.9 (1.1)3.0 (0.0)*1.8 (0.3)MOANpurplepurple26.7 (0.5)26.5 (0.4)1.0 (0.0)1.0 (0.0)MOCBpurplepurple26.0 (0.4)26.0 (0.9)4.0 (0.4)3.3 (0.3)MOFIpurplepurple25.8 (0.5)22.8 (1.7)1.0 (0.0)†1.0 (0.0)MOKIpurplepurple25.2 (0.6)26.7 (0.7)1.0 (0.0)†1.0 (0.0)MOSLpurplepurple34.3 (0.8)32.8 (0.7)1.0 (0.0)†1.0 (0.0)MOWRpurplepurple31.8 (0.6)30.1 (0.9)1.0 (0.0)†1.0 (0.0)NEYOpurplepurple36.0 (0.5)35.8 (0.4)1.0 (0.0)†1.0 (0.0)PAGRpurplepurple—36.7 (1.9)4.3 (0.5)4.0 (0.4)PAHMpurplepurple35.1 (0.5)34.5 (0.1)3.3 (1.0)4.0 (0.7)Monsanto Company 11-SY-217U 367 of 467Table H-4. Individual-Site Phenotypic Comparisonof MON 87712 to Conventional Control A3525 (continued)Phenotypic Characteristic (units)Pod Shattering (1-9 scale)Final stand count (# ofplants/plot)Grain moisture (%)Site1MON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)ARNE1.0 (0.0)†1.0 (0.0)293.5 (4.4)284.3 (5.0)12.3 (0.1)12.6 (0.1)IAJA1.0 (0.0)†1.0 (0.0)310.0 (7.0)*249.0 (10.1)13.1 (0.4)12.6 (0.1)IARL1.0 (0.0)†1.0 (0.0)316.0 (7.3)318.5 (8.1)13.0 (0.5)*12.0 (0.1)ILCY1.0 (0.0)†1.0 (0.0)287.0 (22.8)294.5 (8.7)10.1 (0.1)10.2 (0.1)ILMS1.0 (0.0)†1.0 (0.0)284.3 (7.1)276.8 (7.6)13.4 (0.1)13.4 (0.0)ILSE1.0 (0.0)1.3 (0.3)260.3 (9.9)*232.3 (9.1)13.7 (0.1)13.8 (0.2)ILWY1.0 (0.0)†1.0 (0.0)317.0 (3.9)320.5 (5.6)12.8 (0.0)12.7 (0.1)INRC1.0 (0.0)†1.0 (0.0)273.3 (11.4)269.5 (10.9)11.7 (0.2)12.1 (0.2)INSH1.0 (0.0)†1.0 (0.0)327.5 (3.6)*305.8 (5.8)12.8 (0.1)13.1 (0.1)KSLA2.5 (0.3)4.0 (0.8)300.5 (3.3)*277.8 (12.4)11.7 (0.2)12.3 (0.4)MOAN1.0 (0.0)†1.0 (0.0)314.5 (4.7)314.0 (9.0)11.2 (0.1)16.8 (0.1)MOCB1.0 (0.0)†1.0 (0.0)278.7 (4.7)260.0 (28.7)14.2 (0.3)14.0 (0.5)MOFI1.0 (0.0)†1.0 (0.0)274.5 (2.0)259.3 (7.6)11.6 (0.1)*11.9 (0.1)MOKI1.0 (0.0)†1.0 (0.0)188.8 (12.1)206.8 (29.9)11.9 (0.3)12.5 (0.4)MOSL1.0 (0.0)†1.0 (0.0)337.5 (2.7)336.8 (2.5)16.7 (0.4)17.1 (0.4)MOWR1.0 (0.0)†1.0 (0.0)347.8 (1.4)*338.3(0.8)13.1 (0.5)12.7 (0.1)NEYO1.0 (0.0)*1.8 (0.3)343.5 (1.4)*307.8 (6.4)11.1 (0.0)11.1 (0.5)PAGR1.0 (0.0)1.5 (0.5)266.3 (10.2)261.8 (6.6)16.0 (0.3)*15.7 (0.3)PAHM2.5 (0.3)2.8 (0.5)325.3(2.3)319.3 (0.0)14.9 (0.5)14.8 (0.4)Monsanto Company 11-SY-217U 368 of 467Table H-4. Individual-Site Phenotypic Comparisonof MON 87712 to Conventional Control A3525 (continued)Phenotypic Characteristic (units)100 seed weight (g)Yield (bu/ac)Site1MON 87712 (SE)Control (SE)MON 87712 (SE)Control (SE)ARNE16.4 (0.2)16.1 (0.3)65.0 (1.1)62.6 (1.5)IAJA15.9 (0.2)16.0 (0.2)64.1 (3.2)61.7 (0.3)IARL16.0 (0.4)15.0 (0.0)48.3 (2.1)45.8 (2.2)ILCY15.8 (0.2)15.5 (0.5)66.0 (1.4)58.4 (4.0)ILMS14.0 (0.1)13.2 (0.3)45.6 (2.2)40.9 (3.6)ILSE15.3 (0.1)15.6 (0.5)33.7 (2.1)29.9 (2.9)ILWY15.8 (0.3)15.8 (0.2)33.0 (1.9)34.6 (2.0)INRC14.7 (0.3)*13.7 (0.5)37.2 (2.9)33.1 (1.3)INSH15.7 (0.2)15.8 (0.2)54.5 (1.5)*47.9 (1.7)KSLA17.5 (0.3)18.3 (0.3)68.9 (2.9)*60.0 (2.3)MOAN14.0 (0.7)13.5 (0.3)48.3 (0.9)47.7(1.5)MOCB14.8 (0.2)14.5 (0.1)54.3 (1.4)50.7 (3.5)MOFI17.0 (0.2)16.6 (0.3)44.1(1.6)43.7 (2.9)MOKI20.5 (1.0)*17.5 (0.7)42.3 (1.5)46.9 (2.6)MOSL19.5 (0.3)19.0 (0.0)55.2 (1.8)52.5 (1.3)MOWR17.9 (0.5)17.3 (0.1)65.1 (1.7)*58.3 (0.9)NEYO15.3 (0.3)14.8 (0.3)51.7 (3.3)*46.9 (0.2)PAGR17.7 (0.1)16.8 (0.3)69.3 (1.6)*61.4 (0.8)PAHM16.3 (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.1Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILMS =Effingham County, IL; ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = PawneeCounty, KS; MOAN = Macon County, MO; MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County, MO; MOSL = St.LouisCounty, MO; MOWR = Lincoln County, MO; NEYO = York County, NE; PAGR = Berks County, PA, site 1; PAHM = Berks County, PA, site 2.Monsanto Company 11-SY-217U 369 of 467Table H-4. Individual-Site Phenotypic Comparisonof MON 87712 to Conventional Control (continued)2Mean based on n = 4 for MON 87712 and the conventional control A3525 for all data characteristics except as follows. For Early Stand County n = 3 for theconventional control A3525 at the MOKI site. For Days to 50% end of flowering n = 3 for MON 87712 at the ILSE site. For Daysto 50% senescence n = 3 forthe conventional control A3525 at the ILSE site. For Days to physiological maturity n = 3 for MON 87712 at the MOAN site. ForPlant height n = 2 for theconventional control A3525 at the PAGR site. For Final stand count n = 3 for the conventional control A3525 at the IAJA site.SE = Standard error.3Calendar day number (days after 1 Jan 2009) when50% of the plants in each plot were flowering.4Calendar day number (days after 1 Jan 2009) when 50% of the marked plants in each plothave stopped flowering.5Calendar day number (days after 1 Jan 2009) when 50% ofthe marked plants in each plot reached 50% senescence.6Calendar day number (days after 1 Jan 2009) when 50% of themarked plants in each plot reached physiological maturity.7Flower color data were categoricaland were not statistically analyzed.— Dashes indicate information not collected.Monsanto Company 11-SY-217U 370 of 467Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercial Reference VarietiesAssessment Date and Rangeof Growth Stages Observed1Site2SubstanceObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9ARNE 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 10/5/2009MON 87712 V2 V2 V6-V7 R2 R3 R5 R6 R6 R7Control V2 V2 V5-V6 R2 R3-R4 R5 R6 R6 R7References 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 371 of 467Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercial Reference Varieties(continued)Assessment Date and Rangeof Growth Stages Observed1Site2MaterialObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9ILSE 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 V5R1-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 372 of 467Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercial Reference Varieties(continued)Assessment Date and Range ofGrowth Stages Observed1Site2Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9MOAN 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 373 of 467Table H-5. Growth Stage Monitoring of MON 87712, Conventional Control A3525, and the Commercial Reference Varieties(continued)Assessment Date and Range ofGrowth Stages Observed1Site2Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9MOWR 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 number1Month-day-year.2Site codes are as follows: ARNE = Jackson County, AR; IAJA = Guthrie County, IA; IA; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILMS =Effingham County, IL; ILSE = Champaign County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = PawneeCounty, KS; MOAN = Macon County, MO; MOCB = Shelby County, MO; MOFI = Butler County, MO; MOKI = Callaway County, MO; MOSL = St.LouisCounty, MO; MOWR = Lincoln County, MO; NEYO = York County, NE; PAGR = Berks County, PA, site 1; PAHM = Berks County, PA, site 2.— Dashes indicate information not provided.Monsanto Company 11-SY-217U 374 of 467 

### Table H-6. Abiotic Stress Response E valuations 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 Compaction 1 

### 12 12 Drought 2 

### 45 45

Flood 3 

### 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 Evaluation s 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 blight 1 

### 19 19 Bacterial leaf spot 3 3

Brown stem rot 6 6 Cercospora 2 

### 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

Phytophtora 4 

### 13 13 Powdery mildew 16 16 Pythium 3 3

Rhizoctonia 1 1 Seedling blight 1 1 Septoria brown spot 5 

### 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 convent ional control A3525 during any observation for damage caused by an y 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 followi ng 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 376 of 467Table H-8. Arthropod-Related Damage Evaluations of MON87712 and Conventional Control A3525 Using an ObservationalSeverity ScaleArthropodNumber ofobservationsacross sites1Number of observationswhere no differenceswere detected betweenMON 87712 and theconventional controlA35252Statistically Significant Differences4Site3ObservationNumberArthropod Damage Rating (0-5 scale)MON 87712Mean (S.E.)ControlMean (S.E.)ReferenceRangeAphids5(Aphididae) 20 20−−−−−Armyworms6(Spodopteraspp.)10 10−−−−−Bean leaf beetles (Cerotomatrifurcata)35 33 ILCY 1 0.53(0.05) 0.23(0.06) 0.33-0.40MOAN 2 0.65(0.12) 0.93(0.09) 0.75-0.92Blister beetles(Meloidae)5 5−−−−−Cabbage loopers(Trichoplusia ni)1 1−−−−−Grasshoppers(Acrididae)29 29−−−−−Green cloverworms(Plathypena scabra)16 15MOWR4 0.60(0.04) 0.48 (0.05) 0.48-0.55Japanese beetles(Popillia japonica)21 20 ILMS 1 0.50(0.04) 0.30(0.04) 0.33-0.40Leafhoppers7(Cicadellidae)6 5 PAHM 1 0.55(0.12) 0.15(0.06) 0.30-0.50Silver spotted skippers(Epargyreus clarus)1 1−−−−−Monsanto Company 11-SY-217U 377 of 467Table H-8. Arthropod-Related Damage Evaluations of MON87712 and Conventional Control A3525 Using an ObservationalSeverity Scale (continued)ArthropodNumber ofobservationsacross sites1Number of observationswhere no differenceswere detected betweenMON 87712 and theconventional controlA35252Statistically Significant Differences4Site3ObservationNumberArthropod Damage Rating (0-5 scale)MON 87712Mean (S.E.)ControlMean (S.E.)ReferenceRangeSoybean loopers(Pseudoplusia includens)11 9 MOAN 2 0.55(0.10) 0.90(0.09) 0.70-0.86MOFI 3 0.45(0.05) 0.85(0.05) 0.80-0.93Stink bugs (Pentatomidae) 22 22−−−−−Three-cornered alfalfahoppers (Spissistilusf i)4 4−−−−−Thrips (Thripidae) 2 2−−−−−Velvetbean caterpillars(Anticarsiagemmatalis)4 3 MOFI 3 0.45(0.05) 0.85(0.05) 0.80-0.93Note: The experimental design was a randomized complete block withfour replications. Observations were conducted at four crop developmental stages: Observation1 = V3-V5,Observation2 = R1-R2, Observation3 = R3-R5, and Observation4 = R6-R8.1A total of 192 arthropod damage observationswere made across sites. Lack of variability in the data precluded statistical comparisons for 55 of the observations. Statisticalcomparisons could be made between MON 87712 and conventional control A3525 for 137 of the observations.2No statistically significant differences were detected (α=0.05) or numerical differences between MON 87712 and the conventional control A3525 where p-values could not begenerated due to lack of variability in the data.3Site codes are as follows: ILCY = Clinton County, IL; ILMS = Effingham County, IL;MOAN = Macon County, MO; MOFI = Butler County, MO; MOWR = Lincoln County,MO; PAHM = Berks County, PA site 2.4Means, standard errors (S.E.), and reference ranges are reported for a statistically significant difference that was detected (α=0.05) between MON 87712 and the conventionalcontrol A3525. Reference range = minimum and maximum mean valuesamong the commercial reference varieties.5Aphid includes soybean aphid.6Armyworm includes fall armyworm and beet armyworm.7Leafhopper includes potato leafhopper.Dash (−) indicates that there were no statistically significant differences were detected (α=0.05) between MON 87712 and the conventional control A3525.Monsanto Company 11-SY-217U 378 of 467Table H-9. Abundance of Pest Arthropods in SamplesCollected from MON 87712, Conventional Control A3525, and theCommercial Reference VarietiesPest Arthropod1Aphids (Aphididae)Bean leaf beetles(Cerotoma trifurcata)Corn earworms (Heliothis zea)Coll. Site3MON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)Referencerange1 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 (0.3) 0.3 - 0.8ILSE 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 379 of 467Table H-9. Abundance of Pest Arthropods in SamplesCollected from MON 87712, Conventional Control A3525, and theCommercial Reference Varieties (continued)Pest Arthropod1Green cloverworms(Plathypena scabra)Garden flea hoppers(Halticus bractacus)Leafhoppers2(Cicadellidae)Coll. Site3MON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)Referencerange1 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 (0.0) 0.3 - 2.32 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 380 of 467Table H-9. Abundance of Pest Arthropods in SamplesCollected from MON 87712, Conventional Control A3525, and theCommercial Reference Varieties (continued)Pest Arthropod1Soybean loopers(Pseudoplusia includens)Stink bugs(Pentatomidae)Three-cornered alfalfa hoppers(Spissistilus festinus)Coll. Site3MON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)Referencerange1 ARNE−−−0.8 (0.5) 0.5 (0.5) 0.0 - 2.5 3.5 (0.9) 6.3 (3.4) 0.0 - 3.3ILSE 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 (1.2) 0.3 - 3.0ILSE−−−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 (0.3) 0.5 - 1.5ILSE−−−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 (2.9) 1.0 - 2.5ILSE−−−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 381 of 467Table H-9. Abundance of Pest Arthropods in SamplesCollected from MON 87712, Conventional Control A3525, and theCommercial Reference Varieties (continued)Pest Arthropod1Tarnished plant bugs(Lygus lineolaris)Yellow-striped armyworms(Spodoptera ornithogalli)Yellow woollybear caterpillars(Spilosoma virginica)Coll. Site3MON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean (SE)Referencerange1 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 (0.0) 0.0 - 0.3MOCB−−−−−−−−−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 (0.0) 0.0 - 0.3ILSE 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 fourreplications. Arthropod collection 1 was made at R1 - R2 growth stage and the three subsequentcollections 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.1MON 87712 and conventional control A3525 values represent mean number of arthropodsfrom four replications. A dash (−) indicates arthropod not evaluated.2Leafhopper includes potato leafhopper.3Site codes are as follows: ARNE = Jackson County, AR;ILSE = Champaign County, IL; MOCB = Shelby County, MO.Monsanto Company 11-SY-217U 382 of 467Table H-10. Abundance of Beneficial Arthropods in Samples Collected from MON 87712, Conventional Control A3525, andthe Commercial Reference VarietiesBeneficial Arthropod1Spiders(Araneae)Big-eyed bugs(Geocorisspp.)Carabid beetles(Carabidae)Coll. Site2MON 87712Mean (SE)ControlMean(SE)ReferencerangeMON 87712Mean (SE)ControlMean(SE)ReferencerangeMON 87712Mean (SE)ControlMean(SE)Referencerange1 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) 0.0 (0.0) 0.0 - 0.8MOCB 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 383 of 467Table H-10. Abundance of Beneficial Arthropods in Samples Collected from MON 87712, Conventional Control A3525, andthe Commercial Reference Varieties (continued)Beneficial Arthropod1Lacewings(Chrysopidae)Ladybird beetles(Coccinellidae)Parasitic Wasps(Micro-parasitic hymenoptera)Coll. Site2MON 87712Mean (SE)ControlMean(SE)ReferencerangeMON 87712Mean (SE)ControlMean(SE)ReferencerangeMON 87712Mean (SE)ControlMean(SE)Referencerange1 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) 0.0 (0.0) 0.0 - 0.0ILSE 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) 3.0 (1.3) 1.0 - 1.8MOCB 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)* 0.8 (0.5) 1.3 - 2.3Monsanto Company 11-SY-217U 384 of 467Table H-10. Abundance of Beneficial Arthropods in Samples Collected from MON 87712, Conventional Control A3525, andthe Commercial Reference Varieties (continued)Beneficial Arthropod1Damsel bugs(Nabisspp.)Oriusspp.Coll. Site2MON 87712Mean (SE)ControlMean (SE)ReferencerangeMON 87712Mean (SE)ControlMean(SE)Referencerange1 ARNE 0.3 (0.3) 0.3 (0.3) 0.0 - 1.3 0.0 (0.0) 0.8 (0.5) 0.0 - 1.0ILSE 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3 0.0 (0.0)* 1.0 (0.4) 0.0 - 0.8MOCB 0.3 (0.3) 0.0 (0.0) 0.0 - 0.5 5.0 (2.6) 2.8 (1.0) 1.3 - 4.52 ARNE 2.3 (1.3) 2.5 (1.0) 0.5 - 4.3 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0ILSE 0.0 (0.0) 0.0 (0.0) 0.0 - 1.3 2.8 (0.5) 1.3 (0.8) 1.0 - 2.5MOCB 0.5 (0.5) 0.0 (0.0) 0.0 - 0.5 0.3 (0.3) 0.0 (0.0) 0.5 - 2.33 ARNE 1.5 (0.6) 1.0 (1.0) 0.3 - 2.0 0.0 (0.0)†0.0 (0.0) 0.0 - 0.0ILSE 0.3 (0.3) 0.8 (0.5) 0.5 - 1.5 17.3 (6.3) 6.3 (1.3) 4.3 - 11.5MOCB 0.8 (0.3) 0.5 (0.5) 0.0 - 0.5 0.8 (0.8) 3.5 (2.4) 6.0 - 11.54 ARNE 1.0 (0.6) 0.3 (0.3) 0.8 - 2.0 0.0 (0.0)†0.0 (0.0) 0.0 - 0.0ILSE 0.8 (0.5) 0.5 (0.5) 0.5 - 2.0 19.5 (4.9) 16.3 (8.1) 4.5 - 11.0MOCB 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 statisticallysignificant 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 growth stage and the threesubsequent collections at approximately twoweek intervals thereafter.Numbers represent sample means with standard error (SE)in parentheses.†No p-values were generated due to lack of variability in the data.1MON 87712 and conventional control A3525 values represent mean number of arthropods from four replications. A dash (−) indicates arthropodnot evaluated.2Site 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 vertic al 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 replica tions. 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 fl ower 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 w ith 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 barr ier pen. Tweezers and a dissecting needle were used to open flowers from each plot a nd to brush the pollen in to 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 repli cate were stained and evaluated on the same day.

I.4. Data Collection

Pollen characteristics were assessed by vi ewing 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 runni ng Microsoft Windows

2000 Professional and installed with asso ciated DP Controll er c 1.2.1.108 and DP Manager V1.2.1.107 camera software and Imag e-Pro Plus v6.2.1.491 imaging software.

I.4.1. Pollen Viability

When exposed to the stain solu tion, viable pollen gr ains stained red to purple due to the presence of living cytoplasmic content. N onviable 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 theMonsanto Company 11-SY-217U 387 of 467 

### microscope. Dense clusters of pollen or polle n grains adhering to fl ower parts were not counted because they may not have ab sorbed 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 gr ain 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 fr om digital images of MON 87712, the conventional control A3525, and commercial refere nce varieties that also were used for pollen diameter measurements.

I.5. Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS ® 

### (2008). The level of statistical si gnificance 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 th e minimum and maximum mean values from among the references. General pollen mo rphology was qualitativ e; therefore, no statistical analysis was c onducted on these observations.Monsanto Company 11-SY-217U 388 of 467 

### References for Appendix I

Alexander, M.P. 1980. A versatile stain fo r pollen fungi, yeast and bacteria. Stain Technology 55:13-18.Monsanto Company 11-SY-217U 389 of 467 

### Appendix J: Materials and Met hods for Symbiont Assessment

J.1. Materials

The soybean materials for the symbiont in teraction assessment included MON 87712, the conventional control A3525, and six commercia l references varieties (Table J-1). Nodule, root tissue, and shoot tissue collected from MON 87712, the conventional control A3525, and the commercial refe rence varieties were evaluated.

Table J-1. Starting Seed of MON 87712, Conventional Control, and Commercial Reference Varieties Used in the Symbiont Assessment

T/C/R 1 Starting Seed Name 2 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 conventiona l 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, a nd the commercial re ference varieties starting seed were planted in 6-inch pots containing nitrogen-deficient potting medium (Sunshine ® 

### Mix #3 Basic/LB2) composed of primar ily 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 repli cate 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 wa s inoculated with approximately 1 × 10 7 

### cells of Bradyrhizobium japonicum (VAULT ® 

### NP, Becker, Underwood, Ames, IA) in phosphatebuffered saline. Pots were arranged in eigh t 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 rep licates 7, 8, and 9 were planted on July 29, 2010. In all cases, replicate pots had a minimu m of one plant emerge within one week. A solution of nitrogen-free nutrient soluti on (approximately 250 ml) was added weekly after plant emergence.

J.4. Plant Harvesting/Data Collection

Six weeks after emergence, plants were exci sed at the surface of the potting medium and shoot and root plus nodule mate rial 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 washi ng 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 harv ested, 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 fr esh weight were determined for each plant. Nodules as well as root and shoot material were pl aced in a drying oven on the same day as collected. The plant material was dried fo r at least 72 hours at approximately 65°C to determine dry weight (dwt). The shoot ti ssue was ground using a Harbil 5G high-speed paint shaker prior to total nitrogen analysis. Shoot to tal nitrogen was determined by combustion using a nitrogen analyzer (R apid N Cube, Elementar Americas, Inc).Monsanto Company 11-SY-217U 391 of 467 

### J.5. Statistical Analysis

The data consisted of six measurements ta ken at the six week sampling period: nodule number, nodule dwt (g), shoot dwt (g), root dwt (g), shoot percen t total nitrogen, and shoot total nitrogen (g). An analysis of variance was conducte d 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 amon g 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. SA S 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 poten tial 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 mate rial was determined in the laboratory (BioDiagnostics Inc.) by conducting warm germ ination testing of each seed lot. The viability of MON 87712 and the conventi onal control A3525 from all sites was ≥ 90%. The viability of the commercial refere nce varieties from among the sites was ≥ 80% with two exceptions: two commercial reference vari eties harvested at the Jackson County, MO (ARNE) site had germination rates of 42 a nd 31% respectively. Based upon the viability data, it was determined that the seed were acceptable for evaluating volunteer potential.Monsanto Company 11-SY-217U 394 of 467Table K-1. Starting Seed forVolunteer Potential AssessmentSubstanceSubstance Type Phenotype/Genotype Site1Orion ID #Rep 1 Rep 2 Rep 3 Rep 4MON 87712 Test Soybean Intrinsic Yield ARAU 11259962-002 11259970-002 11259979-002 11259987-002MON 87712 Test Soybean Intrinsic Yield ARNE 11260951-002 11260927-002 11260953-002 11260942-002MON 87712 Test Soybean Intrinsic Yield ILWY 11259999-002 11260022-002 11260014-002 11260007-002MON 87712 Test Soybean Intrinsic Yield MOAN 11259953-002 11259931-002 11259939-002 11259945-002A3525 Control Conventional ARAU 11259967-002 11259974-002 11259990-002 11259983-002A3525 Control Conventional ARNE 11260932-002 11260949-002 11260947-002 11260940-002A3525 Control Conventional ILWY 11260003-002 11260010-002 11260018-002 11259995-002A3525 Control Conventional MOAN 11259958-002 11259943-002 11259934-002 11259950-002SB3888R Reference Conventional ARAU 11259986-002 11259978-002 11259971-002 11259963-002NK S30-D4 Reference Conventional ARAU 11259984-002 11259977-002 11259968-002 11259961-002LG-C3540 Reference Conventional ARAU 11259982-002 11259966-002 11259975-002 11259991-002Stine 3300-0 Reference Conventional ARAU 11259989-002 11259980-002 11259973-002 11259964-002NuPride 3202 Reference Conventional ARNE 11260946-002 11260956-002 11260931-002 11260939-002Pioneer 93M14 Reference Conventional ARNE 11260944-002 11260954-002 11260929-002 11260937-002Pioneer 93M11 Reference Conventional ARNE 11260933-002 11260938-002 11260948-002 11260955-002SB3888R Reference Conventional ARNE 11260935-002 11260930-002 11260945-002 11260952-002Stine 3300-0 Reference Conventional ILWY 11260011-002 11260002-002 11259994-002 11260019-002Pioneer 93M11 Reference Conventional ILWY 11260006-002 11260015-002 11259998-002 11260023-002SB3819 Reference Conventional ILWY 11260009-002 11260000-002 11259992-002 11260017-002SB3369R Reference Conventional ILWY 11260005-002 11260012-002 11260021-002 11259996-002SB3369R Reference Conventional MOAN 11259936-002 11259944-002 11259952-002 11259925-002NutriPride 8339 Reference Conventional MOAN 11259937-002 11259946-002 11259955-002 11259929-002Pioneer 93M62 Reference Conventional MOAN 11259942-002 11259951-002 11259935-002 11259959-002Pioneer 93M14 Reference Conventional MOAN 11259948-002 11259940-002 11259933-002 11259957-0021ARAU = 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 conven tional control A3525 were verified by event-specific polymerase chain reaction analyses.

K.3. Field Sites and Plot Designs

Data were collected from fi eld sites conducted in 2009-2010 gr owing season at four sites within the U.S. soybean production regions . These four sites provided a range of environmental and agronomic conditions repres entative 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 s eason. 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 comple te 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 c onfined seeding area was established within each plot that consisted of the center area of th e 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 396 of 467Table K-2. Seeding Information, Soil Description, and Cropping History Information forVolunteer Potential AssessmentSite1SeedingDate2TargetSeedingRate(seed/plot)ApproximateSeedIncorporationDepth (in)Plot Size(ft)ActualSeedingArea (ft) RepsSoil Series Description;Organic Matter (%); and pHPrevious Cropping History2007 2008ARAU 11/23/2009 400 1-3 8 x 20 6 x 18 4 Wiville fine sandy loam; 1.25 %; 6.2 Fallow FallowARNE 11/23/2009 400 1-3 8 x 20 6 x 18 4 Bosket fine sandy loam; 2.0 %; 5.8 Soybeans FallowILWY 11/23/2009 400 1-3 5 x 20 3 x 18 4 Plano silt loam; 3.6 %; 6.1 Corn FallowMOAN 12/02/2009 400 0.5-2 5 x 20 3 x 18 4 Mexico silt loam; 2.7 %; 6.0 Corn Fallow1ARAU = Woodruff County, AR; ARNE = Jackson County, AR; ILWY = Stark County, IL; MOAN = Shelby County, MO.2month/day/year.Monsanto Company 11-SY-217U 397 of 467 

### K.4. Planting and Field Operations

Each plot was hand-seeded by uniformly s cattering 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 mainta in 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 s eed 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 appr oximately 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 emer gence. Counts continued approximately every two weeks until mid-June 2010. A tota l of seven germination and emergence

observations were conducted at each site throughout the study.

K.6. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all stages of da ta collection, summarization, an d analysis. Study personnel ensured that measurements were taken properly, data were consistent with expectations based on experience with the cr op, 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 dur ing the study that would impact the study objectives were noted by the Lead Scientist.

K.7. Statistical Analysis

There were few volunteers observed in th e study. Due to insufficient number of volunteer plants and lack of va riability, 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, therefor e 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 s ites for MON 87712, the conventional control

A3525 or commercial referen ce 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 c ontrol A3525 where within the range ofMonsanto Company 11-SY-217U 398 of 467 

### the number of volunteers observed for the commer cial reference varieties. However, no statistical analysis was performed due to insufficient number of volunteers observed and lack of variabilityMonsanto Company 11-SY-217U 399 of 467 

### Appendix L: Materials and Methods fo r Persistence Outside of Cultivation

Assessment

L.1. Materials

The soybean materials for the persistence outsi de of cultivation assessment in the field included MON 87712, the conventional cont rol 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 Persiste nce Outside of Cultivation Assessment

Material

Name 1 Material Type Phenotype Monsanto

Lot Number Sites 2

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, NEMonsanto Company 11-SY-217U 400 of 467 

### L.2. Characterization of the Materials

Identities of MON 87712 and the conventiona l 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 conducte d in 2009 at four sites within the U.S. soybean production regions. The study sites we re 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 f our sites in a randomized complete block design with three replications. E ach plot was approximately 100 ft 2 

### 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 st ands in all plots at the MOCB site, the experiment was

continued for a second season. The site wa s established in January, 2010 and continued through July, 2010. Each entry of every repli cation was established in exactly the same plot area in which that entr y had previously been locate d 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 fl ag 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 seed s by animals. The mesh was secured on the edges of all plots. An animal exclusi on electric fence was constructed around the perimeter of the study area. A 15 foot isol ation border was established around the outside of the fenced study area. This isolation borde r 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 an d 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 we re 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 ar e 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 perime ter 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 wasMonsanto Company 11-SY-217U 401 of 467 

### a frequently mowed area near the farm hous e 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 wa s a well drained level field margin adjacent to a field under cultivation and contained mostly pere nnial 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 year s. Weather conditions at the sites during 2009 and 2010 were typical for their resp ective regions and there were no unusual

weather events.Monsanto Company 11-SY-217U 402 of 467Table L-2. Seeding Information, Soil Description, and Cropping History Information for Persistence Outside of CultivationAssessmentSite1Planting Date(mm/dd/yyyy)Planting Rate(seeds/plot)PlantingDepth (in) Plot Size (ft) RepsSoil Series; Organic Matter; pHINSH 06/05/2009 400 1.5 10 × 10 3 Crosby silt loam; 2.5%; 6.0MOCB 06/23/2009 400 1.0 10 × 10 3 Mexico silt loam; 2.7%; 7.2MOFI 06/12/2009 400 0.5 10 × 10 3 Wiville loamy fine sand; 1.3%; 6.6NEYO 06/03/2009 400 0.75 10 × 10 3 Hastings silt loam; 3.0%; 6.5MOCB 01/25/2010 varies by plot surface10 × 10 3 Mexico silt loam; 2.7%; 7.21Sites: INSH = Boone County, IN; MOCB = Shelby County,MO; MOFI = Butler County, MO; and NEYO = York County, NETable L-3. Study Area Planting Information and Soil Description for 2010 Extended Season at MOCB SiteSite1Planting Date(mm/dd/yyyy)Planting Rate(seeds/plot)PlantingDepth (in) Plot Size (ft)Reps Soil Series; Organic Matter; pHMOCB 01/25/2010 varies by plot surface10 × 10 3 Mexico silt loam; 2.7%; 7.21Sites: MOCB = Shelby County, MOMonsanto Company 11-SY-217U 403 of 467 

### L.5. Data Collection

The characteristics evaluated and the timing oftheir 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 va lue of less than one means that fewer seeds were produced than sown. This is indi cative of a population th at 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 plotMonsanto Company 11-SY-217U 404 of 467 

### L.6. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summariza tion, and analysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence with the crop, and the experiment was carefully monitored. Prior to analysis , the overall dataset was eval uated by the Study Director for evidence of biologically relevant changes a nd 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 compar ed to the conventiona l 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 maximu m mean values of the references.

L.8. Individual-Site Persistence Outside of Cultivation Resu lts 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 va lues for stand count for MON 87712 and the

conventional control A3525 at the INSH site were outside the refe rence range (29-81 plants/plot), the difference was not observe d 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 th e 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 pl ot, 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 site s upon harvest in 2009. The replacement values of 2.72 for MON 87712 and 2.63 for the c onventional 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 wa s zero. This indicates that the populations were all in decline, and did not increa se over time in the unmanaged conditions.Monsanto Company 11-SY-217U 406 of 467Table L-5. Growth Stage Monitoring of MON 87712, the Conventional Control A3525, and the Commercial ReferenceVarieties in 2009Assessment Date and Range of Growth Stages Observed2Site1MaterialObs. 1Obs. 2Obs. 3Obs. 4Obs. 5Obs. 6Obs. 7Obs. 8Obs. 9Obs. 10INSH6-24-097-8-097-21-098-12-098-24-099-11-099-28-0910-14-09−−MON 87712V1V2V3V2V1-R3R3R5-R6R7−−A3525V1V2V3V2R1-R3R3-R4R6R7−−ReferencesV1V2V3V2V2-R3V1-R3R6R7−−MOFI6-26-097-10-097-27-098-8-098-21-099-4-099-19-0910-5-0910-21-0911-5-09MON 87712V1V2-V3V2-V4R1R3-R4R5R7R7-R8R8R8A3525V1V2V2-V3R1R3R5R7R8R8R8ReferencesV1V2-V3V2-V4R1-R2R3-R4R5-R6R7-R8R7-R8R8R8MOCB7-7-097-21-098-4-098-19-098-31-099-16-099-28-0910-13-0910-29-09−MON 87712VCV2V4R3-R4R5R5R6R7R8−A3525VCV2V4R4R5R5R6R7R8−ReferencesVCV2V4-R1R4R5R5R6-R7R7R8−NEYO6-18-097-1-097-17-09−−−−−−−MON 87712VCV2V3−−−−−−−A3525VCV2V3−−−−−−−ReferencesVCV2V3−−−−−−−Obs. = Observation number— Dashes indicate information not provided due to no plants remaining in the field.1Sites: INSH = Boone County, IN; MOCB = Shelby County,MO; MOFI = Butler County, MO; and NEYO = York County, NE2Month-day-year.Monsanto Company 11-SY-217U 407 of 467Table L-6. Stand Counts of MON 87712, the Conventional Control A3525, and the CommercialReference Varieties in 2009Means and Ranges of Stand Counts ObservedSite1MaterialObs. 1Obs. 2Obs. 3Obs. 4Obs. 5Obs. 6Obs. 7Obs. 8Obs. 9Obs. 10INSHMON 87712124.082.0*1.34.70.30.70.70.7−−A352599.028.33.33.71.00.70.30.3−−References89-14029-812-92-90-20-10-10-1−−MOFIMON 87712187.3121.092.767.362.762.314.012.711.310.7A3525170.397.045.336.027.322.77.06.05.35.3References151-24393-20750-17945-11836-8432-7010-2310-209-208-21MOCBMON 87712330.0342.3328.3321.3327.0312.3296.3278.3246.7−A3525341.3345.7340.3335.3332.7321.0295.7285.3266.3−References221-331250-344241-345261-323253-342249-339219-339211-330216-335−NEYOMON 87712193.7110.311.0−−−−−−−A3525188.0103.351.3−−−−−−−References140-20044-1393-77−−−−−−−Note: The experimental design at each site was a randomized complete block with three replications. Values for MON 87712 andtheconventional control A3525 are means. References are a range ofthe minimum and maximum mean values of the four commercial referencevarieties 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).1Sites: INSH = Boone County, IN; MOCB = Shelby County,MO; MOFI = Butler County, MO; and NEYO = York County, NEMonsanto Company 11-SY-217U 408 of 467Table L-7. Plant Vigor Monitoring of MON 87712, the Conventional Control A3525, and theCommercial Reference Varietiesin 2009Means and Ranges of Vigor Ratings Observed2Site1MaterialObs. 1Obs. 2Obs. 3Obs. 4Obs. 5Obs. 6Obs. 7Obs. 8Obs. 9Obs. 10INSHMON 877122.02.09.09.0†3.0−−−−−A35252.02.08.09.03.0−−−−−References1.3-2.02.0-2.77.3-8.79.0-9.00.0-6.0−−−−−MOFIMON 877125.06.77.78.7−−−−−−A35255.37.38.39.0−−−−−−References3.7-5.74.7-7.05.7-7.78.0-9.0−−−−−−MOCBMON 877122.03.0NA2.04.75.0*5.03.31.7−A35252.73.0NA1.73.74.75.73.01.7−References2.0-4.03.0-4.0NA1.3-2.74.3-5.74.0-5.75.3-7.02.7-3.31.7-2.3−NEYOMON 877125.08.08.7−−−−−−−A35255.37.78.3−−−−−−−References5.0-5.77.0-9.07.7-9.0−−−−−−−Note: The experimental design at each site was a randomized complete block with three replications. Values for MON 87712 andthe conventional controlA3525 are means. References are a range of the minimum and maximum mean values of the four commercial reference varieties at each site.1Sites: INSH = Boone County, IN; MOCB = Shelby County, MO; MOFI = Butler County, MO; and NEYO = York County, NE2Vigor 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 R1 growth stage.— Dashes indicate information not provided due to no plants remaining in the field or data was not required. NA = not available.* Indicates a statisticallysignificant 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 MO N 87712 to the Control within Each Site

in 2009

Number of plants producing pods per plot

Means (SE) Reference Range 2

Site 1 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 deve lopment. Iowa State University, Ames,

Iowa.

SAS. 2008. SAS/STAT software version 9.2. SA S 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 de rived 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 M ON 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 da y/night processes. Research data indicate this change in day/night pro cesses results in incr eased availability of carbon and nitrogen assimilates in the plant lead ing to higher yield.

This environmental report (ER), which has be en prepared to support APHIS’ obligations under the National Environmental Policy Act (NEPA), evaluates two alternatives: the “deregulation in whole” altern ative and the no action alternat ive. Under the deregulation in whole alternative, MON 87712 would no l onger be a regulated article under 7 CFR Part 340 and would be widely available fo r 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 ha lf of the U.S., with a con centration 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 produc e 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 us ed 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 resistan ce, 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 yiel d determination. The

time of onset and the duration of the vari ous 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 va rieties are classified into one of ten majorMonsanto 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 histor ically used desirable traits such as yield for crop domestication and improvement by retaining plants that exhibit desirable traits for future breeding. In recent years molecu lar breeding techniques have been used to accelerate this process. As a result of conven tional 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 plan ted to some crops such as soybeans have increased over the years, the total acre of cr ops planted to cropland is the same as it was 100 years ago. Some crops such as soybean ha ve increased in acreage, and others, such as wheat, barley and sorghum ha ve 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 th roughout the soybean growing area. While field operations and ma nagement practices for seed production are similar to those for commercial soybeans, extr a care must be taken to ensure high purity and practices that ensure high germinati on rates of the harvested seed. Not all commercial seed is certified, but most comm ercial seed meets certification standards.

Organic Soybean Production :

In the 2008 Census of Agriculture, the most r ecent data source ava ilable, USDA reported that organic soybeans were ha rvested on 98,199 acres in 28 stat es, 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 pr oduction 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 el ements 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 rema in 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 aMonsanto Company 11-SY-217U 413 of 467 

### number of benefits, including in creased yields, decreased fertil izer needs, and reductions

in disease and weed losses.

Insect damage in soybeans usually does not re ach levels that caus e economic loss in the U.S.. Major diseases resulting in yield lo ss 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 fa r the greatest soybean yield losses. Selecting resistant varieties and crop rota tion 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 rota tion, narrow row spacing and planting date) and mechanical weed control practic es (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 soybean s planted are genetically modified for

herbicide tolerance.

Human and Animal Health:

Both humans and animals have consumed so ybean in various forms for thousands of years. Soybean improved with new trai ts 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 operati ons. Mammals and birds may seasonally consume seed, and invertebra tes can feed on the plant during the entire growing season. Plants growing in this adjace nt 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 herbic ides or other pesticides that may make it to streams, rivers, lakes, wetlands and other water bodies either thr ough runoff or drift. Groundwater impacts may be of potential conc ern 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 a nd Non-Agricultural Plants:

Soybean is primarily grown adjacent to other large acre crops such as corn, wheat, and alfalfa. Soybean is highly self-pollinating a nd exhibits a very low level of outcrossing.Monsanto Company 11-SY-217U 414 of 467 

### Soybean does not have any related wild relative s in the U.S. with which it can hybridize. Soybean is not found outside of cultiv ation and is not invasive or weedy.

Environmental consequences

Potential environmental impacts of the no ac tion 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 decrea se 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 affectedenvironment. 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 pr oduction 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 onMonsanto 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 dere gulated 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 Environmenta l 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 biot echnology-derived soybean line MON 87712, which will be used in traditional breeding pr ograms to produce commer cial varieties with increased yield opportunity. The yield incr ease 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 tr anscription factors to regu late the plant’s day/night processes and results in increased availability of assimilates (products of plant metabolism) in MON 87712 compared to an ap propriate 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 dur ing the reproductive phase of the soybean plant, as well as by the significantly hi gher 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 ba sic charge of the U.S. Department of Agriculture's (USDA) Animal and Plant H ealth Inspection Service (APHIS). APHIS provides leadership in ensuring the health a nd 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 a nd Drug Administration (FDA), and the Environmental Protection Agency (EPA). APHIS regulates bi otechnology-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 statemen t concerning regulation of products derived from new plant varieties, including thos e biotechnology-derived, was published in the Federal Register on May 29, 1992. 9 

### Under this policy, FDA us es a consultation process

to ensure that human food and animal feed sa fety 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- 23005Monsanto Company 11-SY-217U 418 of 467 

### M.1.2.2. USDA Regulation of Biot echnology-derived Organisms

The APHIS Biotechnology Regulatory Service’s (BRS) mission is to protect America’s agriculture and environment using a dynamic and science-ba sed regulatory framework that allows for the safe development and us e of biotechnology-deriv ed 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 biotechnol ogy-derived organisms and products. A biotechnology-deri ved organism is considered a regulated article if the donor organism, recipient organi sm, vector, or vector agen t used in engineering the organism belongs to one of th e taxa listed in the regulati on (7 CFR 340.2) and is also considered a plant pest. A bi otechnology-derived organism may also be regulated under

Part 340 when APHIS has reason to believ e that the biotechnology- derived organism may be a plant pest or APHI S does not have sufficient information to determine if the biotechnology-derived organism is un likely to pose a plant pest risk.

Under 7 CFR 340.6 entitled “Petition for Determ ination of Nonregulated Status”, A person may petition the agency to evaluate s ubmitted data and determine that a particular regulated article is unlikely to pose a plant pest risk, and, th erefore, should no longer be regulated. The petitioner is required to provide informati on 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 transcripti onal accessory protein that modulates exis ting 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. A lteration of a plant’s reproduc tive 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 l eads to increased availability of assimilates and hence increased yield. Interstate movements a nd field trials of MON 87712 have been conducted under permits issued or notif ications 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 whet her the increase in yield through production of the BBX32 protein and/or the gene in sertion 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 pl ants 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-derive d organisms under the provisions of the Plant Protection Act. APHIS must respond to petitioners that request a determination of the nonregulated status of biotechnology-de rived 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 notificati ons acknowledged by APHIS are list ed in the petition in Appendix

A. Field tests conducted under APHIS overs ight allow for evalua tion 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 performa nce. These data are also valuable to APHIS for assessing the potenti al 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 reasonabl e alternatives to those acti ons, consistent with NEPA regulations 13 

### 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 safe ty assessment and nutritional assessment summary for MON 87712. The EPA regulates pl ant-incorporated pr otectants (PIPs) under the Federal Insecticide, Fungicide, an d 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 addi tion, the MON 87712 trait does not provide herbicide tolerance or otherwise impact herb icide 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]Monsanto Company 11-SY-217U 420 of 467 

### To support commercial introduction of MON 87712 in the U.S., re gulatory submissions

will be made to countries that will eventu ally commercialize or import significant quantities of soybean and/or soybean produc ts from the U.S. These will include submissions to a number of foreign governme nt regulatory authorities, including: Ministry of Agriculture, People’s Republic of China; Japan’s Minist ry of Agriculture, Forestry, and Fisheries, Minist ry of Environment, and the Mi nistry of Health, Labor, and Welfare; the Canadian Food Inspection Agen cy and Health Canada; the Intersectoral Commission for Biosafety of Genetically Mo dified Organisms, Mexico; the European Food Safety Authority, as well as to regulat ory authorities in other soybean importing countries with functioning re gulatory systems. As appr opriate, 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 an d uses are discussed in Sec tion VIII.B of the petition and summarized here; refer to th e petition for more detail.

Soybean was a relatively minor crop used mos tly for forage in the U.S. from the late

1700s to the 1920s and 1930s. When breeders de veloped cultivars with shatter-resistant

seeds that allowed for use of mechanical harvesting equipment, acreage expanded rapidly, major investments were made in br eeding in the 1930s, and today soybean is the world’s most important oilseed crop.

Soybean is grown as a commercial crop in ov er 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 soyb ean, which accounted for 44% of the world's export of whole soybean seeds (USDA FA S, 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 202 0 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), a nd the majority was used to supply the feed industry for livestock use or the food i ndustry for edible vegetable oil and soybean protein isolates.

The U.S. soybean acreage in the past 10 year s has varied from a pproximately 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 wasMonsanto Company 11-SY-217U 421 of 467 

### 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 through out much of the easter n half of the U.S. and is concentrated in the Midwest (Tab le 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 Acreage 1 

### 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 gr owers 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 vegeta tive or “R” for reproductive. Some of the vegetative stage designations are VE fo r emergence, and V1 through Vn for the appearance of the sets of three-part (tri foliate) leaves. There are eight designated reproduction stages, for example, R1 for th e 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 va rious growth stages in soybean are highly dependent on photoperiod (hours of daylight and darkness) a nd 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 tota l time from planting to maturity would be different. Also, in contrast to most other temperate-season crops, soybean is a “shortday” plant, meaning that matu rity is delayed by longer day le ngth (Major et al., 1975). In soybeans, flowering is initiated only after the night is longer (and days grow shorter) than a critical length (Hol shouser, 2010). Once flowering be gins, temperature controls the duration of flowering time (Heatherly and Elmore, 2004).Monsanto Company 11-SY-217U 422 of 467 

### M.2.1.2. Soybean Variety Development and Yield

Maturity Groups. Because soybean growth is so depe ndent on day/night length, different varieties are developed for different latitudes. In the U.S. ten geographically-designated “maturity groups” originally defined by Scott an d Aldrich (1970) are widely used (Zhang et al., 2007). These maturity groups are mappe d as bands extending from north to south, beginning with Group 00 at the far north an d ending with Group VIII in the far south. 15


### Groups II, III and IV, which extend from appr oximately 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 ma turity, a soybean cu ltivar suited to a southern maturity group would mature too la te if planted too fa r north. Conversely, a northern cultivar would mature too early if planted in the south (Heatherly and Elmore 2004).

Maturity groups are often designated by Arab ic rather than Roman numerals, so the sequence is 00 to 8, and ther e 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 improve ment through breeding has been largely achieved through selection of genes that regulate the e xpression of desirable traits, such as those associated with higher yi elds 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 ha ve developed desirable varieties by retaining for furthe r breeding those plants that po ssess the desirable traits, as determined by visual inspection or by testing. In recent years breeders have used the

more direct methods of molecu lar breeding techniques, such as marker assisted breeding, to accelerate the process of identifying breedi ng lines containing a desired set of positive traits. These techniques rely on inventories of genomic regi ons or genetic markers that have been positively associated with the de sirable 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 developm ent and testing in the field (Voosen, 2009).

Hundreds of soybean varieties ar e tested each year in perfor mance 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, matu rity date, percent lodging (plants fallen over on the ground), height, and herbicide resist ance (Tylka et al., 2010). In different parts of the country and/or in other trials, additional charac teristics may be identified, such as iron deficiency tolerance or pr otein 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.Monsanto Company 11-SY-217U 423 of 467 

### M.2.1.3. Yield Increases in U.S. Soybean Production

As a result of new varieties developed thr ough 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 annu al 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 d ecisions that positively impact yield for growers in recent years include variety select ion, management of soybean cyst nematode, early planting, narrow rows, elimination of weed competition, keeping insect pressure below economic thresholds, and crop rota tion (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 ha s 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 projec tions through 2020 show an average annual rate of in creased 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 a ffect yield increases are the expansion of soybean production into north ern and western parts of th e country, where yields are typically lower than in the core Midwestern production acr e, and a shift in some areas away from narrow rows to improve air circ ulation, which helps combat disease (USDA, 2010).Monsanto Company 11-SY-217U 424 of 467 

### 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 Tempor al 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. Yiel ds 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, wher e 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 th e 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 repres ent averages over c ounties, 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 ac re in 2010, on a farm in Missouri, where the statewide average yield in 2010 was 42 bushe ls per acre (Alsag er 2010, 2010). Thus,Monsanto Company 11-SY-217U 425 of 467 

### even with the positive management decision s that were previously discussed, observed yield is highly variable, is dependent on many factors and likel y rarely reaches the theoretical maximum yield potential.

Growers are accustomed to this regional and te mporal variability in yield, influenced by variable environmental conditions and the ge netics of the varieties they select for planting. However, growers are also accustom ed to the steady improvements of average soybean yield observed over past decades and ge nerally continue to pursue varieties that further increase productivity a nd profitability on their farm. Within this context, U.S.

farmers will understand the value of the tr ait MON 87712 that offers the opportunity to increase the yield of th eir soybean production.

M.2.1.5. Crop Storage and Transportation

Once soybean are harvested from a field, they ma y 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 importa nt role with their long term storage of the grain. Since processing facilities crush soybeans througho ut 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 f acilities 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 constructin g or renting temporary facili ties 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 ca n also compete with corn for available storage space; and while corn can be stored on the ground, soybeans rarely are (Hurburgh, 2005). University extension se rvices provide practical guidelines for temporary storage of soybeans and other crops (Harner et al., 1998; Hellevang, 1998; Dorn, 2011; Maier and Wilk e, 1998; Hurburgh, 2005).

Growers usually deliver their soybeans to the sa le point using their ow n 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 soyb ean 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 cr ops can result in gr eater shipping competition and higher shipping costs, which translates to lower pr ices offered to grower s (Ash et al., 2006).Monsanto Company 11-SY-217U 426 of 467 

### M.2.1.6. Economics

In the short term, individual growers’ decisi ons 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 profita bility (Ash et al., 2006). Managing input costs and managing the crop for yield are major co mponents to the economics of producing a soybean crop. Growers’ costs include bot h 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 associ ated with growing a particular crop in a given year, such as seed and fertilizer. A pr oducer’s cost of grow ing a particular crop includes a proportional part of the overhead of his or her entire farming operation, plus all the operating costs associat ed with that crop.

Figure M-2 shows the average per acre net valu e 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 su bsidies. Overhead costs represent well over half the total costs (up to 69%), with the “opportunity cost of la nd (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 wh en 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 So ybean 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 2005NetValue(dollarperacre)

Year Soybean

Corn

WheatMonsanto Company 11-SY-217U 427 of 467 

### While an individual grower typically makes planting decisions based on the relationship between operating costs and expected prices (Ash et al., 2006), ma ny factors influence both operating costs and expected prices. G overnment price supports can have a large effect on costs, and supply and demand govern s 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 exceed ed in 1940, 20 million acres exceeded in 1956, 30 million in 1964, 40 million in 1967, a nd 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 c onsumption (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, in creased yields and increased rotations with corn resulted in increased acreage plan ted to soybeans (Ash et al., 2006).

USDA projections to 2020 show a 2.7% incr ease in acres plan ted 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 yi eld 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 dema nd/production); soybean use for biodiesel (increases demand/production), government fa rm programs, both for soybeans and for other competitor crops (may result in incr eases or reductions); increases in overseas demand, especially in China (increases produc tion); and competition from major overseas growers, especially in Brazil and Argen tina (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 grea tly decreased. USDA attribut es 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 s oybeans adapted to more northerly climates (USDA ERS, 2010). As shown in Figure M-2, wh eat, 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 cropla nd 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 agricultura l land used for crop in the U. S. was 330 million acres - the same as it was the first year USDA began tr acking, in 1910. Peaks occurred in the early 1930s, in 1949, and in the early 1980s, when th e area of cropland used for crops reached over 380 million acres (USDA ERS, 2007).Monsanto Company 11-SY-217U 428 of 467 

### The combined acreage planted to the two larges t U.S. crops, corn and soybeans, was at an all-time high of 168.8 million acres in 2011 (U SDA NASS, 2011). This was achieved through maximization of existing cropland, re duction of acreage so wn to other grain crops and hay, and an increase in the rate of double-cropping (raisin g 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 S ection VIII.B.2 of the petition and summarized here. Standardized seed pr oduction practices are responsible for maintaining high-quality seed stocks, an essential basis for U.S. agricu lture. The value of seed quality (including genetic purity, vigor, and minimi zing presence of weed see d, seed-borne diseases, and inert materials, such as dirt) has been iden tified as a major factor in determining crop yields (Oplinger 1986, Pederson 2008a, USDA, 2000).

Soybean seed has four classes: 1) breeder, 2) foundation, 3) register ed, and 4) certified (Association of Official Seed Certifying Ag encies [AOSCA], 2009). Breeder seed is seed directly controlled by the originating or sponsoring plant br eeding 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 s eed 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 s oybean 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 th e early 1900s in the U.S. to preserve the genetic identity and variety purity of see d. The federal government passed the U.S. Federal Seed Act of 1939 to recognize seed certi fication and the establ ishment of official certifying agencies. Regulations first a dopted in 1969 under the Federal Seed Act recognize land history, field isolation, and varietal purit y standards for foundation, registered, and certified seed. Seed certificatio n services are ava ilable 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 processi ng plants at these seed companies clean, condition, and bag the harvested soybean seed; ve rify 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.Monsanto Company 11-SY-217U 429 of 467 

### 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 in cludes internal and external audits (ISO, 2009). Field inspections are conducted on seed production fields throughout the soybean growing season to evaluate variety pur ity and ensure soyb ean 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 requireme nt to prevent mechanical mixing of other soybean varieties (AOSCA, 2009). The Ameri can 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, sp ecial 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 ER S, 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 s oybeans (USDA ERS, 2010); however, the Census of Agriculture reported 98,199 harv ested 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 producti on 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 organi c soybeans are used fo r tofu and other soy products in the U.S. Some organic soybe an 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 s oybeans were $6.20 per bushel higher than non-organic, that organic yields were much lower than non-organic, and that the average price premium receive d for organic soybeans was $9.16 per bushel, which more than offset the added producti on costs (McBride and Greene, 2009). OtherMonsanto Company 11-SY-217U 430 of 467 

### researchers have found that or ganic soybean price premiums in 2009 ranged from 65 to 139% and averaged 107% (Clark and Alexander, 2010).

Organic producers must use organi c 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 di sease resistance profile , quality aspects and fit to a local growing region (USDA AMS, 2011). Several companies produce organically certified soybean s eed as well as conventional n on-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 soybe an growers, except that organic growers may not use synthetic fertilizers or pesticid es. Because synthetic herbicides are not allowed, organic growers generally need to us e more tillage to cont rol 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 organi c production operations to have distinct, defined boundaries and buffer zones to prev ent unintended contact with prohibited substances (such as pesticides and syntheti c 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 bi otechnology such as that used to produce

MON 87712 is an excluded method u nder the National Organic Program. 17



### On April 15, 2011, USDA AMS released a Po licy Memorandum regarding “Clarification of Existing Regulations Regarding the Use of Genetically Modified Organisms in Organic Production and Hand ling” (USDA AMS, 2011). This memo was intended to answer questions that have been raised concerning biotechnol ogy-derived crops and organic production and handli ng. The memorandum reiterates that organic operations must follow a set of production standards a nd 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 exclude d methods (i.e., “GMOs”) in organic operations. Therefor e, the use of biotechnology-d erived crops remains an excluded method but not a prohibited substa nce. Organic production operations must also develop and maintain an organic produc tion system plan approved by an accredited certifying agent. This plan enables the production operati on to achieve and document compliance with the National Organic Standa rds, including avoi dance of prohibited

substances and the use of excluded methods. 18



### Organic certification involves oversight by an accredited certifying ag ent of the materials and practices used to produce or handle an or ganic agricultural produc t. This oversight

16 7 CFR 205.202(c)

17 7 CFR § 205.2.

18 7 CFR Part 205.Monsanto Company 11-SY-217U 431 of 467 

### includes an annual review of the certified operation’s organic syst em 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 n ecessarily constitute a violation of the National Organic Standards (USDA AMS, 20 11). The unintentional presence of the products of excluded methods will not affect the status of an organic pr oduct or operation when the operation has not used excluded me thods and has taken reasonable steps to avoid contact with the products of excluded me thods as detailed in an approved organic system plan. Organic certification indi cates that organic production and handling processes have been followed, not that the pr oduct 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 re gulations do not allow the use of excluded methods such as biotechnology; however, the inadvertent pres ence of products of biotechnology “does not constitute a use because there was no intent on the part of the certified operator to use excl uded methods” (USDA AMS, 2011).

Organic soybean producers use production prac tices designed to prevent commingling of their crop with neighboring cr ops treated with herbicides and other pesticides (spray drift), or that may be using plant varie ties 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 (Kueppe r, 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 natu re 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 vari ety) to another. Outcrossing most commonly results from cross-pollination. Si nce soybean is highl y 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 s eed) that allow them to reasonably avoid biotechnology-derived soybean and maintain or ganic or conventional production status.

M.2.3. Agronomic Practices for Soybeans

M.2.3.1. Production Management Considerations

Production management considerations are desc ribed in detail in Section VIII.D of the petition and are summarized here. Refe r to Section VIII.D for more detail.

Pre-Season. Well in advance of planting a soyb ean 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 se ed or variety that will be planted, and soil fertility management requirements. Soybean rotation practices are summarized below. Variety selection has been discussed previous ly. Soybean requires 16 essential elements for growth and development. Deficiencies in any of these elements can reduce yieldsMonsanto Company 11-SY-217U 432 of 467 

### (Hoeft et al., 2000). The primary or majo r 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-fi xing Bradyrhizobia bacteria ( Bradyrhizobium japonicum ) that live in the nodules on its roots. Nitrogen fertilizer applications at planting generally do not improve yield and decr ease nodulation while increa sing the plant’s dependency on the soil for nitrogen (Pedersen, 2008d). Theref ore, 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 whet her any adjustments need to be made. In corn-soybean rotations in the Midwest, phospho rus and potassium fertilizers are applied prior to a corn crop in accord ance with soil test recommenda tions, but are seldom applied prior to a soybean crop.

Planting and Early Season. An understanding of the grow th stages of soybean is important for the proper timing of certain ma nagement practices, such as herbicide and insecticide applications. In addition, the im pact of certain weat her conditions, insect pests, and diseases on soybean yield is de pendent on growth stage. The system of soybean growth stages divides plant devel opment into vegetative (V) and reproductive (R) stages (Pedersen, 2004). The vegetative stages begin with VE, which designates emergence. V stages continue and are numbe red according to how many fully developed trifoliate leaves are present ( i.e. , V1, V2, etc.). The reprod uctive (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 va rious growth stages in soybean are highly dependent on photoperiod (hours of daylight and darkness) a nd 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 tota l 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 gr eatest impact. Highest yi elds are generally achieved when planting in early to mid-May. Row sp acing influences yield because it impacts canopy development and research conducted in the Midwest s hows 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 ab ility to branch and adjust the number of pods on branches to co mpensate for large differences in seeding rate. Maximum yields generally require planting rates that re sult in about 2.5 to 5 plants per square foot (Hoeft et al., 2000).

In order to maximize yields, weeds must be co ntrolled during the ear ly growth stages of soybean because weeds compete with soyb ean 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 mi d to late season stages. Ideal daytime temperatures for soybean growth are betw een 75oF and 85oF (Hoeft et al., 2000). Soybean is photoperiod sensitive, which mean s that it transitions from vegetative toMonsanto Company 11-SY-217U 433 of 467 

### flowering stage in direct re sponse to length of daylight (Scott and Aldrich, 1970). Most soybean varieties begin flowering soon after th e day length begins to shorten. Good soil moisture is most critical during the pod-f illing 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 photosynthe sis to maximize yield. High humidity and temperatures during seed development and maturity can result in poor seed quality because these conditions promote the devel opment of reproductive-stage diseases.

Harvest Season. When dry matter accumulation ends, the plant is considered to be physiologically mature. The seed moisture co ntent 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 harves ted 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 belo w 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). Time ly 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.Monsanto Company 11-SY-217U 434 of 467 

### M.2.3.2. Crop Rotation

The use of crop rotation in soybean productio n 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 fe rtilizer 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 insecticid es (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 ro tation 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 substant ial, the grower must make cropping decisions by evaluating both the ag ronomic 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 stat e 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 fo llows 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 pract ice as a way to reduce the risk of damage

from diseases and nematodes (Hoeft et al., 2000; Al-Kai si et al., 2003). Corn and soybean occupy more than 80% of the farmla nd 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 af ter 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 indicateMonsanto Company 11-SY-217U 435 of 467 

### 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 gr owing soybean are very similar to corn. The soils and climate in the Midwestern, East ern 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 requireme nt 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 dete rmining 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 rate d as less problematic than weeds in U.S. soybean production, management of insect pe sts during the growth and development of soybean is important for protec ting the yield of soybean (Are f and Pike, 1998). Insect injury can impact yield, plant ma turity, or seed quality. Ins ect injury in soybean seldom

reaches levels to cause an economic loss in the primary soybean production areas, as indicated by the low percenta ge (16%) of soybean acreage that receives an insecticide treatment (USDA NASS, 2007).

Characterizing soybean responses to insect in jury is essential in establishing economic injury levels (Higley and Boethel, 1994). Most often, soybean insects pests are categorized or defined by the plant parts th ey injure, namely r oot-feeding, stem-feeding, leaf-feeding, or pod-feeding in sects. The root- and stem-f eeding insect groups are often the hardest to scout and typically are not de tected 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 damagi ng 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 signif icant 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, defoliati on 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 ot her growth stages. Re search indicates that the soybean plant can sustain a 35% leaf lo ss prior to the pre-bloom period without lowering yield (NDSU, 2002). However, from p od-set to maturity, the plant can tolerate only a 20% defoliation level before yield is imp acted. Insect damage occurring in the full pod stage (R4) or seed-filli ng stage (R5) would have the most impact on yield (McWilliams et al 2004).Monsanto Company 11-SY-217U 436 of 467 

### M.2.3.5. Management of Di seases 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 importa nce (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 pl ant development and health when infection occurs, the seve rity of the disease on individu al plants, and the number of plants affected (Heathe rly 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 impor tance. 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 dise ases and nematodes varied by region.Monsanto Company 11-SY-217U 437 of 467 

### Figure M-3. Soybean Yield Losses in th e U.S. Averaged Over the Period 1996 to

2009 Attributable to Pests and Diseases
Source: Wrather and Koenning, 2010



### Selection of varieties that ar e 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 manageme nt by reducing initial inoculums or reducing the rate of disease developmen t (Heatherly and Hodges, 1999).

Preplant tillage can bury crop residue, wh ich encourages the decomposition of fungalresting structures. Crop rotation is ofte n recommended as a disease-management strategy. Rotating crops in terrupts the disease cycle and allows time for the decomposition of inoculums. One exception is Rhizoctonia , a soil-inhab itant pathogen that grows on a wide variety of crops and can survive sufficiently in the soil to make crop rotation an impractical means of control ling this pest. Row spacing, plant population, and planting date can also be ch anged 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). Th e 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, pl anting resistant varieties is regarded as the best and most effective management practi ce to prevent losses from this pest. 100000 600000 1100000 1600000 2100000SoybeanYieldLoss(ThousandBushel)Monsanto Company 11-SY-217U 438 of 467 

### High-quality seed is essential for control ling 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 dise ase that typically infests soybean during reproductive stages of development and can cause defoliation and reduce yields significantly in geographies su ch 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 ti me, foliar application of fungicides is the standard disease-management practice to lim it yield losses due to soybean rust.

M.2.3.6. Weed Management

The management of weeds in soybean productio n is discussed in Section VIII.F. Weed control in soybean is essential to optimizi ng yields because weeds compete with soybean for light, nutrients, and soil moisture. Weed s can also harbor insects and diseases, and also can interfere with harvest, causing ex tra wear on harvest equipment (Pedersen, 2008). Approximately 98% of the soybean acreage received an herbicide application in 2006, indicating the importance of excellent w eed control in maximizing soybean yield (USDA NASS, 2007).

Herbicide-tolerant soybean was introduced to provide grower s with additional options to improve crop safety and/or improve weed c ontrol. 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 st andard weed control program in U.S. soybean production and was utili zed 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 du ration of the competition. When weeds are left to compete with soybean for the entir e 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 da te 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 compe titive ability of weeds by burying the plants, disturbing or weakening their root systems, or causing sufficient phys ical 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.Monsanto Company 11-SY-217U 439 of 467 

### M.2.4. Human Health and Worker Safety

Humans safely consume soybean and have don e 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 fibe r (OECD, 2001). The essential amino acids required by humans are contained in soyb eans (OECD, 2001). An ti-nutrients include trypsin inhibitors (interfere with digestion) , lectins (inhibit grow th), isoflavones (also referred to as phytoestrogens that may affect reproduction), stachyose and raffinose (produce gas), and phytic acid (binds mine ral nutrients, making them unavailable) (OECD, 2001). Soybeans also contain allergen ic 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 cons ultation process. Biotechnology-derived soybean on the market pose no unique risks re lative to other soybean developed using traditional breeding methods. Most soybeans currently grown are herbicide tolerant and

have been shown to be as safe and nutriti ous as their conventi onal 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) th at happened across all industries in 2008 (USBLS, 2011 and USDOL-OSHA, 2011). No fatalities were reported in 2008 directly associated with soybean fa rming, 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 build ings 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 fertilizer s), These hazards can be minimized, among other things, through the use of hazard r ecognition, hazard control and the use of personnel protective equipment (USDOL-OSHA, 2011). Genetically engineered soybean with new traits produced by biotechnology pos e no unique worker safety issues relative to other soybean developed using traditional breeding methods.Monsanto Company 11-SY-217U 440 of 467 

### 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 relativ ely high levels of essential amino acids that

are deficient in other common feed (OECD, 2001). Trypsin i nhibitors, lectin s and phytic acid are the primary anti-nutrients in soybeans that should be minimized in animal diets. Toasting or heating the soybeans during pr ocessing reduces the content of trypsin inhibitors and lectins (OECD, 2001). All bi otechnology-derived soybean with new traits produced by biotechnology have been revi ewed 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 deve loped using traditional breeding methods. Soybeans currently grown are la rgely 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 ar e 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 f eed primarily on soyb ean are seed-feeding insects and rodents found in ag ricultural 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 seed s. Deer may also browse in soybean fields on the forage and on seed left after ha rvest. 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 terr estrial birds; and mode rate 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 ag ricultural 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 pl ants could be in ditches, hedge rows, fence rows, wind breaks, yards, etc. These plants could be annuals, bie nnials or perennials. Regardless of the agricultura l 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 fiel ds can occur from herbicide drift, however, measures can be implemented to minimize drift.Monsanto Company 11-SY-217U 441 of 467 

### 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 discu ssed 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 likel y to increase with increased tillage. In 2009, based on the states’ water quality repo rts, EPA identified sedimentation and turbidity as two of the top 10 causes of im pairment 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 de veloped to confirm its assertion. EPA has projected conservation tillage to be “the ma jor 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’ wate r 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 tu rbidity (U.S. EPA, 2008b). Pest icides accounted for less than one percent of reported ca uses of surface water impairment in all but four of the 17 leading U.S. soybean-producing states. In thos e four states, pesticid es accounted for two to eight percent of reported causes of impairment . Of the pesticides that were reported as contributing to impairment among the 17 leadin g 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 conc ern in some areas where nitrogen levels are either approaching or have exceed ed 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 n itrogen fertilizer, and therefore does not contribute appreciably to nitr ogen levels in groundwater.

M.2.7.2. Air

Many agricultural activities, including those as sociated 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 CO 2 

### emissions attributable to human activity are derived from the combustionMonsanto Company 11-SY-217U 442 of 467 

### 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 biochemi cal processes are crit ical to maintaining soil health and quality. Soil microbial comm unities are highly complex and are often characterized by high microbial diversity (T iedje et al., 1999). The occurrence and abundance of soil microorganisms are affected by 1) soil characteristics like tilth, organic matter, nutrient content, and moisture capacit y, 2) typical physico-ch emical factors such as temperature, pH, and redox potential, and 3) soil management practices. Agricultural practices such as fertiliza tion 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 micr obial populations is expect ed in agricultural fields.

Maintaining soil pH in the range of 6.0 to 7.0 will enhance the availabi lity 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 organi c 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 abou t two-thirds of the nitrogen contained in the harvested crop (Hoeft, 2002) . Results of most studies show that ap plication of nitrogen fertilizer does not improve yields in soybeans (CAST, 2009 p. 25). Soybean removes approximately 0.85 pounds of phospha te 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-micr obe symbiosis results in the formation of root nodules, which provide an environment in which differentiate d bacteria called bacteroids are capable of re ducing or “fixing” atmospheri c nitrogen. The product of nitrogen fixation, ammonia, can then be utilized by the plant. As a result of this relationship, nitrogen inputs ar e typically not necessary fo r agricultural production of soybeans.

M.2.7.4. Adjacent Agricultural Crop s 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 theMonsanto Company 11-SY-217U 443 of 467 

### U.S. with which it can hybrid ize. Soybean is not found outsi de of cultivation and is not invasive or weedy (OECD, 2000).

Soybean is primarily grown adjacent to othe r large acre crops such as corn, wheat, and alfalfa. It may also be grown near vegeta bles, 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 ra tes of outcrossing. The potential for crosspollination in soybean is limited. This is re cognized 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). Numero us 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%.Monsanto Company 11-SY-217U 444 of 467 

### M.3. Alternatives

The decision-making process of deregul ation is governed by 7 CFR § 340.6 (d)(3)(i) which states that APHIS may approve the petiti on 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” alte rnative, MON 87712 would no longer be a regulated article under 7 CFR Part 340 and w ould be widely available for planting, movement and importation without prior author ization 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 increa se, the price of the seed, and other factors. The rate of adoption is expected to be gradual partly because of th e difficulty to a grower of assessing a yield increase within the inherent temporal and spatial va riability in yield due to many other factors, as discussed in A ppendix 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 in sect 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 varie ties will compete for market share with varieties that contain MON 87712. In addition, it is assu med that there will be demand for conventional, organic soybean as well as other specialty soybean and these soybean varieties will also compet e 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 coul d be grown under USDA notification or permit under confined release conditions. However, cu rrently deregulated biotechnology-derived soybean would continue to be available and w ould be expected to continue to be widely grown. It is expected that higher yiel ding soybean varietie s developed through conventional breeding would continue to be in troduced 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 cult ivation or use of MON 87712 in specificMonsanto Company 11-SY-217U 445 of 467 

### geographies or conditions to mitigate potentia l plant pest risk. For example, APHIS could impose conditions that requi re assurance of the integrity and purity of the material containing MON 87712, or condi tions requiring the implementation of stewardship practices in the use of MON 87712 that form ed 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 presen t 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 re quested a determination of non-regulated

status without conditions.

M.4. Environmental Consequences

This section describes the environmenta l 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on limite d 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 w ould be expected to continue as they are now, potentially with continued westward a nd 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 altern ative 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 ba se of soybean germplasm would be the opportunity for increased yield in commer cial soybean varie ties that contain MON 87712. As summarized in the peti tion, MON 87712 soybean is agronomically, phenotypically, and compositiona lly 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 th at are biotechnology-derived, as over 90% of soybean currently grown are biotechnology-d erived. 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on limite d acres under notification or permit. AlsoMonsanto Company 11-SY-217U 446 of 467 

### under the no action alternative, soybean vari ety 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 co mmercial 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 ba se of soybean germplasm would be the increased yield resulting from use of comm ercial soybean varieties that contain MON 87712. Data presented in the petitio n showed that MON 87712 displayed a delay in days to 50% senescence and days to physio logical maturity (Tab le VII-4). Farmers select soybean varieties for planting on thei r farm based upon the correct maturity group for their region. Leaf sene scence and physiological matu rity 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 vari ety reaches its physiological maturity.

It is not expected that the delay in physiol ogical maturity or leaf senescence observed will impact the maturity group desi gnation of soybean varieties th at are commercialized using MON 87712 for the following reasons. The majo rity of soybean vari eties on the market today are produced from a breeding process te rmed forward breeding. In this process new soybean varieties are created by crossing two existing varieties. The varieties crossed are not necessarily fr om 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 charac teristics like protein, and oil levels (Heatherly and El more, 2004). Varieties that yiel d better than the checks of the same maturity group are selected for commercialization. The entire process from

initial cross to introduction of a new variet y usually takes several years during which each new variety is repeatedly evaluated agai nst the best commercially available varieties of the same maturity group. When MON 8771 2 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, th e system that breeders use to evaluate new varieties and assign a variety to a correct ma turity group will be used to place the new variety into the correct maturity group. Br eeders consider many ch aracteristics 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 u ltimately 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 lim ited acres under notification or permit. Under the no action alternative, continued yi eld increases would be expected in soybean, at least for the foreseeable future, before a true yield plateau is reached (AppendixMonsanto 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). Th ese 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 alte rnative 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 cont ain 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 ge rmplasm 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 tec hnology 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 so ybean 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 varia tion is common. As discussed in Appendix M.2.1.2, because of the high variability in cr op 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on lim ited 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 (A ppendix M.2.1.3). Seed pr ices are likely to continue to be a major opera ting cost in soybean producti on, with overhead cost the majority of the total costs.

Deregulation in Whole Alternative . The difference between the no action and the deregulation in whole altern ative 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 at tributable to the

introduction of MON 87712 into a broad ba se of soybean germplasm would be the opportunity for increased yield from th e use of commercial MON 87712 soybean varieties, in addition to the incremental yiel d opportunity resulting from the use of other technologies or conventional breeding. The ec onomic benefit of high er yield is well understood and is compelling to growers as it helps them in crease productivity and profitability on their farm. To the U.S. economy as a whole, increased productivity (increased value of production without increas ing inputs) is of value as well as it helpsMonsanto 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 fo rm of increased profits), will vary depending on the government policies and global market condi tions, 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on limite d acres under notification or permit. Also, under the no action alternative, based on USDA projections, acr es 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 acr es, 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 expa nd 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 maxi mization of existing cropland. Other factors included decreases in other crop ac reage and double-cropping.

Deregulation in Whole Alternative . The difference between the no action and the deregulation in whole altern ative 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 ba se of soybean germplasm would be the opportunity for increased yield in commer cial soybean varie ties 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 in creases 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on lim ited acres under notification or permit. Under the no action alternative, soybean seed production would be ex pected 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 altern ative is expected to be the gradual introduction ofMonsanto 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 ba se of soybean germplasm would be the opportunity for increased yield in commer cial soybean varie ties 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 so ybean with the exception of its increased yield trait. It is expected that MON 87712 soybean gr own for seed production purposes would yield more seed. Seed growers are accu stomed to variations in seed yield due to the same factors that impact gr ain 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on lim ited acres under notification or permit.

Under the no action a lternative, organic soybean pr oduction would be expected to continue as described in Appendix M.2.2.2. a nd it is expected that biotechnology-derived soybean varieties will continue to be grow n on over 90% of the soybean acres. As described in Appendix M. 2.2.2., organic soybe an producers employ practices that allow them to reasonably avoid biotec hnology-derived soybeans.

Deregulation in Whole Alternative . The difference between the no action and the deregulation in whole altern ative 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 ba se of soybean germplasm would be the opportunity for increased yield in commer cial soybean varie ties that contain MON 87712. As summarized in the peti tion, (Section VII), MON 87712 soybean is agronomically, phenotypically, and compositiona lly comparable to conventional soybean with the exception of its yield trait. Soybean is principall y self pollinated; however, low levels of cross pollination occur. The pollen from MO N 87705 has been characterized and compared to pollen produced by conventio nal soybean. No statistically significant

differences were detected between MON 87712 and the conventional control A3525 for percent viable pollen or polle n 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 s oybean and the practices that organic farmers are using to avoid biotechnology-derived tra its 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% ofU.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 lim ited acres under notification or permit. Soybean breeders will continue to develop hi gher yielding soybean varieties and farmers will likely use these new varieties on their fa rm 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 altern ative 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 commer cial soybean varie ties that contain MON 87712. MON 87712 soybean is ag ronomically, phenotypically, and compositionally comparable to conventional so ybean with the excepti on 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 ex pected to result in change s in agronomic practices and no impacts on tillage, crop rotation, irriga tion, management of insects, disease management, weed management or herbicid e tolerant weeds are expected with the

introduction of MON 87712.

The expected yield increase associated w ith 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. Potassi um and phosphorous are rarely applied to soybean because soybean follows well fertiliz ed crops like corn a nd wheat in a rotation and residual levels of potassium and phos phorous are sufficient to produce a soybean crop. The application of fertil izers to corn has not changed substantially from the late 70’s even though higher yielding soybean varie ties 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 se ason 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 incr ease yield is not expected to change fertilization practices for soybean comp ared to the no acti on 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 lim ited 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 altern ative is expected to be the gradual introduction ofMonsanto 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 commer cial soybean varie ties that contain MON 87712. As summarized in the peti tion, (Section VI) MON 87712 soybean is compositionally comparable to conventional soybean. Detailed compositional analyses

were conducted in accordance wi th OECD guidelines to assess levels of key nutrients and anti-nutrients in MON 87712 compared to leve ls present in the parental conventional soybean control of a similar genetic bac kground as well as conventional commercial reference varieties. These compositional comp arisons 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 nut rients and anti-nutrients in conventional commercial soybean varieties have a history of safe consumption. Nutrients assessed in this study included proximates, fiber, amino aci ds, fatty acids, and vitamin E in seed, and proximates and fiber in forage. The anti-nut rients assessed in seed included raffinose, stachyose, lectin, phytic acid, trypsin inhibitors, and isoflavones..

Assessment of the analytical results confir med that the differences observed in the combined-site analysis were not meaningful to food and f eed 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 comm ercial reference varieties that were grown concurrently in the same field trial. Theref ore, it is concluded that soybean seed and forage produced from MON 87712 are compos itionally 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 comp ared 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 in cluding 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 fr action produced from M ON 87712 are expected to be as safe and nutritious as soybeans a nd 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 HealthMonsanto 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 limite d acres under notificat ion 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 s ource. Anti-nutrients of potential concern in soybean meal are trypsin inhibitors, l ectins 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 altern ative 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 commer cial soybean varie ties that contain

MON 87712. As summarized in Section VI I, MON 87712 soybean is agronomically, phenotypically, and compositiona lly 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 equivale nt to conventional soybeans in terms of both nutritional and anti-nutritional composit ion. Therefore, soybean as forage and soybean meal produced from MON 87712 are exp ected to be as safe and nutritious as soybean forage and meal produced from ex isting conventional and GE soybeans. As discussed above, the BBX32 protein is pr oduced 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 safe ty. 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 a llergenic 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 lim ited 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, outcrossi ng to native species is not a c oncern with soybean.

Deregulation in Whole Alternative . The difference between the no action and the deregulation in whole altern ative 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 commer cial soybean varie ties that contain

MON 87712. As summarized in Section VI I, MON 87712 soybean is agronomically, phenotypically, and compositiona lly 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 ch ange as a result of the introduction of MON 87712. Therefore, MON 87712 is exp ected to impact animal and plant communities the same as commodity soyb ean 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on lim ited 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 altern ative 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 variet ies that contain MON 87712. The presence of the MON 87712 trait is not exp ected to affect agronomic pr actices such as tillage or herbicide or pesticide app lication. As described in Appendix M.2.7.3., the increased yield that will occur with MON 87712, or in creases in soybean yield from any other source, may result in the removal of mo re phosphate and potash from the soil, and therefore may require the replacement of thes e nutrients at a slightly higher rate than would be needed with lower-yielding soyb eans. For the same volume of soybeans produced, however, the phosphate and potas h needs would be the same. Because agronomic practices will be negligibly a ffected by the introduc tion of MON 87712, the introduction of MON 87712 is expected to ha ve negligible impacts on surface water, groundwater or air quality, in terms of per acre impacts co mpared to existing soybean varieties. Because MON 87712 offers the pot ential 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 prod uced. 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 s lightly less than before th e 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 e xpected 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 an d deregulation in whole alternative, it is likely that crop yields will increase. The increased yiel d associated with MON 87712 is expected to allow for less land -use change compared to the no action alternat ive, and is not expected to have any direct impact on e xhaust emissions or fertilizer applications. Therefore, MON 87712 is not expe cted to have a deleterious e ffect on air quality. Of the global anthropogenic CO 2 

### emissions related to climate ch ange, ~80% are derived from

the combustion of fossil fuels and cement production, and ~20% are derived from land use change and deforestation related to agri culture (IPCC, 2007). Hi gher yielding crop varieties will likely help to meet the higher food demands in the 21 st 

### century without expanding the amount of land used in agricult ure, therefore leading to less land-use change and potentially lower anthropogenic CO 2 

### 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 alternativ e, MON 87712 would continue to be regulated and would only be grown on limite d acres under notificat ion 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 altern ative 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 commer cial soybean varie ties that contain

MON 87712. One critical interaction between s oybeans and microbes in the soil is in the development of a symbiotic re lationship between soybean a nd rhizobia. As noted in Section VII.C, measurement of nodule num ber 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 de tected (5% level of significance) between

MON 87712 and the conventional control for each measured para meter 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 observation s for plant responses to abiotic stressors, disease, and the observed phenotypic charac teristics 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, incr eases in soybean yiel d may result in the removal of more phosphate and potassium from the soil, and may re quire the replacement of these nutrients at a slight ly 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 littl e 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 conti nued to increase suggesting that higher yields in soybean are

not related to increased ferti lizer inputs. Sim ilarly, the expected yield increase in MON 87712 soybean varieties is unlikely to ch ange this trend. Rather, improved crop genetics and more precise farming methods ar e expected to provide increased yield on

the same or reduced land base usin g the same or reduced inputs.Monsanto Company 11-SY-217U 455 of 467 

### Figure M-4: Average historical U.S. ferti lizer 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 2010AverageFertilizerApplied(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 2010AverageSoybeanYield(bu/ac)AverageCornYield(bu/ac)

Year 

# Average U.S. Corn and Soybean Yield

from 1964

‐ 2010

Corn SoybeanMonsanto 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 lim ited acres under notification or permit. Adjacent agricultural crops and non-agricultur al plants are discussed in Appendix M.

2.7.4.

Deregulation in Whole Alternative . The difference between the no action and the deregulation in whole altern ative 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 commer cial soybean varie ties that contain

MON 87712. As summarized in Section VI I, MON 87712 soybean is agronomically, phenotypically, and compositiona lly 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 th e invasiveness or pers istence 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 alte r 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 im pact of the action when added to other past, present, and reasonably foreseeable future actions regardle ss 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 im pacted by the proposed action.

As described in Appendix M.4.0, the primar y impact of derecu lation 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 foreseea ble future actions , and based on the trend of incremental annual average soybean yield gains observed ov er several decades, th ese are expected to

occur whether or not MON 87712 is introduced. Note that these proj ected 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 1508.7Monsanto Company 11-SY-217U 457 of 467 

### deregulation of MON 87712; thus, there ar e 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 biotechnolo gy-derived soybean products have been deregulated or are under consideration fo r 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 Bi otechnology-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 GU 262 AgrEvo October, 1998 Phosphinothricin-tolerant A5547-127 AgrEvo April, 1998Altered Oil Profile G94-1, G94-19, G168 DuPont 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

h ttp://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 dete rmined that none of the in dividual biotechnology-derived soybean products it has previously deregu lated 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 inse rt in MON 87712 is discussed in Section

IV of and summarized here. Data have de monstrated 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 ob servations taken on soybean varietiesMonsanto Company 11-SY-217U 459 of 467 

### containing MON 87712 in Monsan to’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 a nd biotechnology-derived traits. Given that there have been no plant pest characteristics associated with MON 87712, or with any of the previously de regulated events liste d 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 estab lished 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 petiti oned 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 comp leted 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 al so 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 sa fety are expected due to the breeding of MON 87712 with these other soybean because th ese varieties have an established history

of safe use.

Furthermore, the process of conventiona l breeding to combine biotechnology-derived traits or biotechnology-deriv ed and conventional soybean to produce combined trait products is designed to identify and remove off-types (i.e., plants th at lack the intended phenotype or that do not show expected agronom ic or phenotypic char acteristics) 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 th at the traits presen t in stacked products are performing as expected and thus do not di splay novel phenotypes. Give n this evaluation it can be concluded that no significant impacts are expected through the use of MON 87712 in combination with previously deregula ted biotechnology-derived soybean products.Monsanto Company 11-SY-217U 460 of 467 

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