USDA/APHIS/BRS Petition # 09-183-01pFebruary 4, 2010Monsanto Company 09-SY-200U Page 3 of 3AttachmentClarifications and Corrections to Petition #09-183-01p for the Determination of Non-regulated Status for MON 87769Page, Section(changes)Existing Text or InformationRevised Text or InformationPage 108,Table VII-2(Removedduplicated numbersfollowingsubheading on the4throw from bottom)Seed Fatty Acid (% Total FA) 12.31 122.51 0.024 [12.24-12.39]Seed Fatty Acid (% Total FA)Page 110,Table VII-2(Subtitle inserted inbetween the 4thand5throws from thebottom)Genistein Site OHSeed Amino Acid (% DW)Aspartic Acid Site OHGlutamic Acid Site OHHistidine Site OHGenistein Site OHStatistical Differences Observed in One SiteSeed Amino Acid (% DW)Aspartic Acid Site OHGlutamic Acid Site OHHistidine Site OHUSDA/APHIS/BRS Petition # 09-183-01p February 4, 2010

Monsanto Company 09-SY-200U Page 1 of 20 Response to Review for Completene ss and Acceptability of Monsanto Petition Number 09-183-01p for a Determ ination of Non-regulated Status for MON 87769 Soybean

This response document provides clarification and responses to questions on the Petition for Determination of Nonregulated Status of MON 87769, Petition #09-183-01p, as outlined in the November 6, 2009 letter from USDA-APHIS to Monsanto. This response contains confidential business information (CBI) that Monsan to is requesting be held confidential. The CBI information is provided in Appendix 1 to this response, with the appendix provided as CBI copy and CBI-deleted versions.

USDA Question #1 (General Issue): Please provide an alignment of the sequence data for insertion and the plasmid to support your conclusions that the insertion is intact except for the right border region.

Monsanto Response: To confirm the organization and ex tent of genetic elements in MON 87769, the reported sequence of the insert in MON 87769 was aligned to the sequence of the transformation vector PV-GMPQ1972 usi ng MegAlign in the Lasergene program. As described on page 81 of the petition, the M ON 87769 insert is 7367 base pairs long, beginning at base 9387 of PV-GMPQ1972 and ending at base 288 of the vector (see Appendix 1). This analysis showed that 43 base pairs in the insert (base pairs 93 87 through 9429, corresponding to base pairs 934-976 of the insert) were from th e Right Border region that is present in the transformation vector. This analysis also c onfirmed that the sequence of the insert in

MON 87769 is 100% identical to the co rresponding sequences in PV-GMPQ1972.

The MON 87769 insert, beginning at base 934 an d ending at base 8300, matches the sequence of PV-GMPQ1972 between bases 9387 and 288 (see Appendix 1). The sequence of the insert

in MON 87769 is 100% identical to the corr esponding sequences in PV-GMPQ1972.

USDA Question #2 (General Issue): Please supply study data or ot her information related to the stability of the fatty acids in MON 87769 soyb ean during storage. Please compare this to the stability of fatty acids in commodity s oybeans (without altered fa tty acid profiles).

Monsanto Response: MON 87769 harvested seed was stor ed over a 20 month period at room temperature and the fatty acid composition of the seed was monitored periodically (0, 3, 6, 9, 14 and 20 months). The data from the anal ysis are presented in Table 1. The data confirm that the fatty acid composition of MON 87769 harvested seed was stable over 20 months storage at room temperature. Tabl e 2 shows the fatty acid composition from the periodic analysis (0, 1, 2, 4, 8, and 12 months) of conventional soybean stored over 12 months reported in the literature (Kermasha and Alli, 199 3). From the data presented in Table 1 and Table 2, it is concluded that th e storage stability of MON 87769 soybean is comparable to the storage stability of conventional soybean.USDA/APHIS/BRS Petition # 09-183-01pFebruary 4, 2010Monsanto Company 09-SY-200U Page 2 of 20Table 1. Fatty acid analysis of MON 87769 harvestedseed stored at room temperate over 20 monthsFatty acid composition (%of total fatty acids)0Month3Months6Months9Months14Months20MonthsC16:0 (Palmitic) 12.55 12.48 12.91 11.45 11.67 11.98C18:0 (Stearic) 4.13 4.15 3.89 4.10 4.18 4.28C18:1 (Octadecenoic) 18.45 18.19 17.64 18.70 18.34 18.66C18:2n6 (Linoleic) 28.14 27.84 28.14 28.12 28.42 28.43C18:3n6 (Gamma linolenic) 5.27 5.37 5.71 5.23 5.54 5.25C18:3n3 (Alpha linolenic) 11.20 11.30 11.28 11.61 11.17 11.16C18:4n3 (Stearidonic) 18.69 19.02 19.30 19.19 18.90 18.41C20:0 (Arachidic) 0.32 0.33 0.28 0.36 0.36 0.49C20:1n9 (Eicosenoic) 0.22 0.26 0.18 0.25 0.22 0.24C22:0 (Behenic) 0.28 0.30 0.24 0.32 0.33 0.33USDA/APHIS/BRS Petition # 09-183-01pFebruary 4, 2010Monsanto Company 09-SY-200U Page 3 of 20Table 2. Fatty acid analysis of conventional soybean harvested seed stored at 20°Cover 12 months1Fatty acid composition(% of total fatty acids)0Month1Month2Months4Months8Months12MonthsC16:0 (Palmitic) 11.0 11.1 11.3 11.7 12.3 12.7C18:0 (Stearic) 4.1 5.2 6.9 8.9 13.8 16.1C18:1 (Octadecenoic) 25.2 24.9 24.7 24.3 23.0 22.3C18:2n6 (Linoleic) 49.5 48.7 48.0 46.9 43.9 41.0C18:3 (Linolenic) 8.9 8.3 7.1 6.5 5.7 4.9C20:0 (Arachidic) 0.1 0.05 ND ND ND NDC20:1n9 (Eicosenoic) 0.1ND ND ND ND NDND denotes “not detected”.1Kermasha and Alli (1993).USDA/APHIS/BRS Petition # 09-183-01p February 4, 2010

Monsanto Company 09-SY-200U Page 4 of 20 Reference: Kermasha, S., and I. Alli.1993. Changes in li pase and lipoxygenase ac tivities and fatty acid profile during the storage of unprocessed and processed full-fat soybeans. Pages 1021-1035 in Food Flavors Ingredients and Composition. Proceedings of the 7 th Internatinal Flavor Conference, Pythagorion, Samos, Greece, 24-26 June 1992. George Charakanbous (ed.). Elsevier Science Publishers, Amsterdam- London-New York-Tokyo.

USDA Question #3 (General Issue): Please submit all final field test reports for those

notifications that are c ited in the petition. While we recogni ze that some of these are not due until December, we cannot complete our review until all are received.

Monsanto Response: Our records show that all final field test reports for the notifications that are cited in the petition, 09-SY- 200U, have been submitted to USDA, except for one. No field report was filed for notification, 06-045-12n, be cause it was cancelled and a letter of cancellation was sent to the USDA on April 11, 2006. If USDA/APHIS r ecords indicate other field report(s) are missing, pleas e let us know so that we can provide a copy of any missing field report(s).

USDA Question #4 (General Issue): Standard deviation (or st andard error) values are presented for some analyses in the petition, but not others. Those sta tistics are useful for reviewers to make inferences about data presen ted in tables or to cross check petitioner’s interpretation of data. Please provide SD (or SE ) values for all mean values in tables. Also, provide sample sizes for all measures of central tendency presen ted in the petition.

Monsanto Response: Standard error (SE) values have been added to Tables VIII-3, VIII-5,
VIII-6, VIII-7, VIII-8, VIII-9, G3, G 5, H-1, I-7, I-8, I-9, and I-10.

SE values have not been added to Table VII-2, because this table is a summarization from Tables E-1 through E-12 (see Appendix E in Pe tition 09-SY-200U) to show the significant differences among analytes between MON 87769 and the conventiona l soybean control A3525. Tables E-1 - E-12 and E15 – E18, with th e associated footnotes, provide the standard error. The sample size for Tables E-1 through E12 is described in Appendix E and for Tables E-15 through E-18 in Section VII.C. The de tailed analytical methodology and statistical analysis for all measures of central tendency can be found in Appendix E.

USDA Question #1 (Specific Issue): On Page 120, 3rd Para graph, it states:

“No statistically significant differences in percent normal germinated and abnormal germinated seeds were detected between MON 87769 and control. However, the germination

means of both MON 87769 and control were outside the range of refere nce varieties included in the study for the AOSA temperature regime (20/ 30 oC). The lack of significant differences in germination between MON 87769 and conventi onal soybean control de monstrate that the

observed values of MON 87769 are w ithin the range of values expected of soybean. Thus, the

data are indicative of no increase d plant pest potential of MON 87769.”USDA/APHIS/BRS Petition # 09-183-01p February 4, 2010

Monsanto Company 09-SY-200U Page 5 of 20 Please explain the rationale of the third senten ce. How is it that th e lack of significant

differences between MON 87769 and the control demonstrate that the observed values of MON 87769 are within the range of values expect ed of soybean? The sentence before states that both MON 87769 and the control had germina tion means outside of the range of reference varieties included in the study.

Monsanto Response : To address the question, paragr aphs 3 and 4 on page 120 of USDA petition, 09-SY-200U, have been revised as follows:

In the AOSA temperature regime of 20/30 oC , the germination mean s of both MON 87769 and

the conventional control were outsi de the range of the nine refere nce varieties in cluded (Table VIII-3). However, no statistically significant differences in percent normal germinated or abnormal germinated seeds were detected betw een MON 87769 and the c onventional control. Furthermore, the germination of both M ON 87769 (89.1%) and the conventional control (92.1%) at 20/30 oC exceeded the accepted standard of 80% minimum germination for certified soybean seed recommended by the official seed certifying agencies of North America (AOSCA, 2003). Thus, the germination values of MON 87769 were not different from the A3525 conventional control, an established commer cial soybean variety, and were within the range of accepted germination values for certified soybean seed.

The biological characteristics evaluated were us ed to characterize MON 87769 in the context of a plant pest risk assessme nt. The results demonstrate that the assessed dormancy and germination characteristics of MON 87769 were not different from those of the conventional control. The observed dormancy and germinati on values of MON 87769 we re within the range of accepted values for soybean. The results, in par ticular the lack of change in hard seed, are indicative of no increased plant pest or we ediness potential of MON 87769 relative to conventional soybean.

USDA Question #2 (Specific Issue): On Page 357 under Characte rization of Materials it

states:

“The presence or absence of MON 87769 in the te st, control, and reference seed samples was verified by event-specific polymerase chain reac tion. The results of these analyses were as expected with a few exceptions. Two out of nine control seed samples and five out of 36 reference seed samples across the three seed production site s contained between 0 and 1.84% MON 87769. In addition, one of the reference seed samples from the IN site contained between 0 and 5.65% MON 87769. Nevertheless, it was determined that the levels of

MON 87769 in the control and reference seed sa mples from the isolated plots were low and did not negatively affect the quality of th e study or interpretation of the results.”

Please explain how MON 87769 ma y have contaminated the reference seed samples.USDA/APHIS/BRS Petition # 09-183-01p February 4, 2010

Monsanto Company 09-SY-200U Page 6 of 20 Monsanto Response: To address this question

, Sections H.1. Materials and H.2. Characterization of the Materials, in Appendix H on page 357 of the USDA petition, 09-SY-200U, have been revised as follows:

H.1. Materials

Dormancy and germination characteristic s were assessed on seed of MON 87769, the conventional soybean control, and the referenc e soybean varieties produced at the Jefferson County, IA; Stark County, IL; and Boone C ounty, IN sites in the 2006 field trials (Appendix G). The field trial at each site wa s established in a randomized complete block design with three replications. The seed of MON 87769, the cont rol, and reference varieties were harvested from all three replicated plots at each of the three fi eld sites and pooled to produce one seed lot of MON 87769, the control, and each reference variety from each field site for dormancy and germination testing.

Material

Type 1 Seed Materials Produced at Each Site

IA IL IN

Test MON 87769 MON 87769 MON 87769

Control A3525 A3525 A3525

Reference A3244 AG3505 AG3505

Reference ST3600 CST3461 CST3461

Reference Stewart ST3300 ST3300

Reference DKB34-51 CST37002 Lewis 372

1 The test, control, and reference seed used to assess dormancy and germination characteristics were all produced from replicated field trials conducted in 2006 to assess phenotypic characteristics. None of

the seed were obtained from commercial sources. Th e references were conventional soybean varieties with the exception of DKB34-51and AG3505, which were Roundup Ready soybean 40-3-2 varieties.

H.2. Characterization of the Materials

Prior to pooling seed, the presence or abse nce of MON 87769 in th e test, control, and

reference seed harvested from each of the three replicated plots among the three field sites was verified by event-specific polymerase chain reac tion. The results of these analyses confirmed the presence of MON 87769 in th e test seed and the absence of MON 87769 in the control and reference seed with a few exceptions. Seed samp les collected from two out of the nine control plots and from five out of a total of 36 refere nce plots across the three field trials contained between 0 and 1.84% MON 87769. In addition, s eed samples collected from one of the reference plots at the IN site contained between 0 and 5.65% MON 87769. Since the seed of

the conventional control and the reference va rieties were all produced from the same replicated field trials as M ON 87769, a low level of outcrossing can be expected between plots in close proximity. It was determined that the levels of MON 87769 in the control and reference seed samples from the isolated plot s were low and did not negatively affect the quality of the study or inte rpretation of the results.USDA/APHIS/BRS Petition # 09-183-01p February 4, 2010

Monsanto Company 09-SY-200U Page 7 of 20 USDA Question #3 (Specific Issue): On Page 361 the table indicates the germination for the

control line A3525 and M ON 87769. For the 20/30 0 C group, please include data for reference samples by site in addition to the control and MON 87769 material. Also include the protocol used to assess seed germination, the number of seeds tested of each genotype from each site, and the number of replicates from each site.

Monsanto Response: To address this question, Sections H.3. Performing Facility and Experimental Methods and H.5. Individual Site Seed Dormancy and Germination Results in Appendix H on pages 357-359 of USDA petition, 09- SY-200U, have been revised to include the protocol used to assess seed germination, the number of seeds tested of each genotype from each site, and the number of replicates from each site. Table H-1 on page 360-361 has been split into Table H-1 and Table-H2. Table H-1 shows the 20/30 o C data for reference samples by site in addition to the control and MON 87769.

H.3. Performing Facility and Experimental Methods

Dormancy and germination evaluations were c onducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigator was certified to conduct seed dormancy and germination testing consistent with the standards establishe d by the Association of Official Seed Analysts (AOSA), an internationally recognized seed testing organization (AOS A, 2000; AOSA, 2006).

Seed materials of MON 87769, the control, and f our reference varieties were produced from

each of three sites and tested under six different temperature regimes. Thus, a total of 18 different seed lots (i.e., 6 soybean material s/production site × 3 pr oduction sites) were

tested. Each of six germination cham bers used in the study were maintained dark under one of the following six temperature regimes: c onstant temperature of approximately 10, 20, or

30 o C or alternating temperatures of approximately 10/20, 10/30, or 20/30 o C. The alternating temperature regimes were maintained at th e lower temperature for 16 h and the higher temperature for 8 h. The temperature inside each germination chamber was monitored and recorded throughout the duration of the study. For each seed lot, four replicated germination towels (each containing a target of 100 seeds) were prepared per facility SOPs for each temperature regime. The types of data collect ed depended on the temperature regime. Each rolled germination towel in the AOSA-recomme nded temperature regime (i.e., 20/30 °C) was assessed periodically during the study for nor mal germinated, abnormal germinated, hard (viable and nonviable), dead, and firm-swollen (viable and nonviable) seed as defined by AOSA guidelines (AOSA, 2006). Each rolled germ ination towel in the additional temperature regimes (i.e., 10, 20, 30, 10/20, and 10/30 o C) was assessed periodi cally during the study for germinated, hard (viable and nonviable), dea d, and firm-swollen (viable and nonviable) seed.

H.5. Individual Site Seed Dorman cy and Germination Results

MON 87769, the control, and reference seed mate rials were produced at three sites to assess germination characteristics of seed grown under various environmental conditions. The

individual site data in Tabl es H-1 and H-2 indicate that germination of MON 87769 and the control seed was greater than 99% across all production sites and temperature regimes.

MON 87769 and control seed from the IA and IL sites had relatively hi gher percent abnormalUSDA/APHIS/BRS Petition # 09-183-01p February 4, 2010

Monsanto Company 09-SY-200U Page 8 of 20 germination at the 20/30 o C temperature regime than MON 87769 and control seed from the IN site (Table H-1). The values, however, are not uncommon for soybean. A total of two

viable hard seeds were observed in this st udy (i.e., one in each of the 10 °C and 10/20 °C temperature regimes for MON 87769 produced from the IL site) (Table H-2). In soybean, it is

not uncommon to observe low levels of hard s eed (Mullin and Xu, 2001; Potts et al., 1978). Thus, the observance of two hard seed was not unexpected. In the analysis of the data, no seed production site × seed material interactions were detected for any characteristic in any temperature regime. Therefore, MON 87769 was comparable to the control material across sites (Section VIII. D.1., Table VIII-3).USDA/APHIS/BRS Petition # 09-183-01pFebruary 4, 2010Monsanto Company 09-SY-200U Page 9 of 20Table H-1. Germination Characteristicsof MON 87769, the Conventional Control, and the Reference Soybean Variety SeedProduced from each of Three Field Sites and Tested in the AOSA-Recommended Temperature RegimeMean % (S.E.)1IA IL INTemp.Regime2GerminationCategory3MON 87769 ControlRef.Range4MON 87769 ControlRef.Range4MON 87769 ControlRef.Range420/30 °C(AOSA)NormalGerminated81.5 (3.4) 82.3 (2.9) 93.8-96.5 88.8 (3.4) 94.5 (2.5) 92.8-98.3 97.0 (0.6) 99.5 (0.5) 98.3-99.5AbnormalGerminated18.0 (3.3) 17.5 (2.9) 3.5-6.0 11.3 (3.4) 5.0 (2.0) 1.8-7.3 2.8 (0.8) 0.5 (0.5) 0.5-1.5Viable 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 0.0 (0.0) 0.0 (0.0) 0.0-0.0Dead 0.5 (0.5) 0.3 (0.3) 0.0-0.5 0.0 (0.0) 0.3 (0.3) 0.0-0.8 0.3 (0.3) 0.0 (0.0) 0.0-0.3Viable Firmswollen0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0) 0.3 (0.3) 0.0-0.0 0.0 (0.0) 0.0 (0.0) 0.0-0.0Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed was arranged in germinationchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete blockand the sub-plot treatment wasseed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No production site ×seed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional control (A3525) acrosssites.1Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equaled 100.1% due tonumerical rounding of the means. S.E. = Standard Error.2Temperature Regime. In the alternating 20/30 °C temperature regime, the lower temperature was maintained for 16 hours and thehigher temperature for eighthours.3Germinated seed in the AOSA-recommended temperature regime were categorized as either normal germinatedor abnormal germinatedseed.4Minimum and maximum mean values from among four commercial reference soybean varieties produced from replicated field trials at each site.USDA/APHIS/BRS Petition # 09-183-01pFebruary 4, 2010Monsanto Company 09-SY-200U Page 10 of 20Table H-2. Germination Characteristicsof MON 87769 and the Conventional ControlSeed Produced from each of ThreeField Sites and Tested in theAdditional Temperature RegimesTemperatureRegimeGermination CategoryMean % (S.E.)1IA ILINMON 87769 Control MON 87769 Control MON 87769 Control10 °C Germinated 100.0 (0.0) 100.0 (0.0) 99.5 (0.3) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (0.0)0.0 (0.0)0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead0.0 (0.0)0.0 (0.0)0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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 °C Germinated100.0 (0.0)100.0 (0.0)100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (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)0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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)30 °C Germinated 99.8 (0.3) 99.3 (0.5) 99.8 (0.3) 99.8 (0.3) 99.5 (0.3) 99.8 (0.3)Viable Hard0.0 (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) 0.8 (0.5)0.3 (0.3) 0.3 (0.3) 0.5 (0.3) 0.3 (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)Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed was arranged in germinationchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete blockand the sub-plot treatment wasseed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No production site ×seed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional control (A3525) acrosssites.1Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equaled 100.1% due tonumerical rounding of the means. S.E. = Standard Error.USDA/APHIS/BRS Petition # 09-183-01pFebruary 4, 2010Monsanto Company 09-SY-200U Page 11 of 20Table H-2 (continued). Germination Characteristics of MON 87769 and the Conventional Control SeedProduced from each of ThreeField Sites and Tested in the Additional Temperature RegimesTemperatureRegime1Germination CategoryMean % (S.E.)2IA IL INMON 87769 Control MON 87769 Control MON 87769 Control10/20 °C Germinated 99.5 (0.5) 99.5 (0.3) 99.8 (0.3) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (0.0) 0.0 (0.0)0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead0.5 (0.5) 0.5 (0.3)0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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)10/30 °C Germinated100.0 (0.0)100.0 (0.0)100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (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)0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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)Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed was arranged in germinationchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete blockand the sub-plot treatment wasseed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No production site ×seed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional control (A3525) acrosssites.1In the alternating temperature regimes,the lower temperature was maintained for 16hours and the higher temperature for eighthours.2Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equaled 100.1% due tonumerical rounding of the means. S.E. = Standard Error.USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 12 of 20 

### Appendix 1

Alignment of the MON 87769 Insert Sequence to the PV-GMPQ1972 Transformation Vector Sequence. The figure shows an alignment of MON 87769 insert sequence (top sequence) to the T-DNA I from the PV-GMPQ1 972 transformation vector sequence (bottom sequence). The MON 87769 in sert sequence represents base pairs 934-8300. The PV-GMPQ1972 sequence represents base pair s 9387-288. Base pairs 1-933 consist of genomic DNA flanking the 5’ end of the insert that did not align w ith sequences in the

transformation vector.

[

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 13 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 14 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 15 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 16 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 17 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 18 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 19 of 20 [

CBI DELETED

]USDA/APHIS/BRS Petition # 09-183-01p CBI-Deleted February 4, 2010

Monsanto Company 09-SY-200U Page 20 of 20 [

CBI DELETED

]Soybean MON 87769

Monsanto Document #09-SY-200U Page 1 of 4 Petition for Determination of Nonregulated Status for Soybean MON 87769

CLAIM OF DATA CONFIDENTIALITY

Monsanto is claiming the DNA sequence of plasmid PV-GMPQ1972 and the T-DNA

insert of MON 87769 as Confidential Business Information (CBI). This information is located in Monsanto’s response document (d ated February 4, 2010) as Appendix 1. The following justification is made for this CBI claim.

i . Legal Background

The Freedom of Information Act (FOIA), 5 U.S.C. § 552, specifically exempts from

release “trade secrets and commercial or fi nancial information obtained from a person as privileged or confidential” (“Exemption 4”) 5 U.S.C. § 552(b)(4). Exemption 4 applies

where the disclosure of information would be likely to cause substantial harm to the competitive position of the owner, National Parks & Conservation Association v. Morton

, 498 F.2d 765, 770 (D.C. Cir. 1974); Gulf & Western Industries, Inc. v. U.S. , 615 F.2d 527, 530 (D.C. Cir. 1979), or where, in the case of information voluntarily submitted to the government, the information is not customarily made avai lable to the public by the provider. Critical Ma ss Energy Project v. NRC, 975 F.2d 871 (D.C. Cir. 1992) (“financial or commercial information provi ded to the Government on a voluntary basis is ‘confidential’ for the purpose of exemption 4 if it is of a kind th at would customarily

not be released to the public by the person from whom is was obtained.”).

A party seeking to demonstrate “substantial competitive harm” need not show actual competitive harm, but must only demonstrate the presence of competition and the likelihood of substantial competitive injury. Id. at 530; National Parks & Conservation Association v. Kleppe , 547 F.2d 673, 679 (D.C. Cir. 1976); Miami Herald Pub. Co. v.

U.S. Small Business Administration , 670 F.2d 610, 614 (5th Cir. Unit B 1982).

For the purposes of FOIA, courts have defined the term “trade secret” to mean a “secret, commercially valuable plan, formula, process, or device that is used for the making, preparing, compounding, or processing of trade commodities and that ca n be said to be the end product of either innova tion or substantial effort.” Public Citizen Health Research Group v. FDA , 704 F.2d 1280, 1288 (D.C. Cir. 1983); Anderson v. Dept. of

Health & Human Services , 907 F.2d 936, 943-44 (l0th Cir. 1990).

Where, as in the case of Monsanto’s product subject to an FOIA request, the development time and costs of the products have been s ubstantial and the information can only be obtained by competitors at considerable cost , disclosure is prohibited. Greenberg v. Food and Drug Administration , 803 F.2d at 1213, 1216-1218 (D.C. Cir. 1986); Worthington Compressors, Inc. v. Costle , 622 F.2d 45, 51-52 (D.C. Cir. 1981).

Information on commercial development falls s quarely within this definition, and is the type of information accorded trade secret protec tion by the courts under Exemption 4 ofSoybean MON 87769

Monsanto Document #09-SY-200U Page 2 of 4 the FOIA request. The courts have been ve ry clear in finding commercial development information covered by Exemption 4 where the release of such information could allow competitors to procure a clear understanding of a company’s business practices and allow a competitor to cause harm to a company’s competitive standing. See, e.g., Braintree Electric Light Dept. v. Dept. of Energy , 494 F. Supp. 287, 289-291 (D. D.C. 1980).

The U.S. Department of Agriculture’s APHIS has defined “Confidential Business Information” for the purposes of biotec hnology submissions with in the boundaries of these statutory and court inte rpretations of Exemption 4. “Policy Statement on the Protection of Privileged or Confidential Business Information,” (the CBI Policy Statement), 50 Fed. Reg. 38561 (Sept. 23, 1985). The CBI Policy Statement defines CBI

to consist of “Trade Secret s” and “Commercial or Financial Information.” “Trade Secrets” are, in turn, defined as: “informa tion relating to the production process. This includes production data, formulas, and processe s, and quality control tests and data, as well as research methodology and data generated in the development of the production process. Such information must be (1) commercially valuable, (2) used in one’s business and (3) maintained in secrecy.”

The CBI Policy Statement states that “Commerc ial or Financial Information will also be

deemed confidential if review establishes th at substantial competitive harm would result from disclosure.” Information such as the sequence of the inserted DNA or flanking regions appears to fall squarely within this description. See e.g., USDA-APHIS

B IOTECHNOLOGY U SER ’ S G UIDE

, G ENERAL D OCUMENT P REPARATION G UIDELINES FOR
S UBMISSION TO BRS, 9 (February 5, 2008). .

ii. Justification

The DNA sequence of plasmid PV-GMPQ1972 and the T-DNA insert in MON 87769

fall within the well-established boundaries of CBI as recognized by the federal courts and by APHIS. This information is either pr otected because it was voluntarily submitted by Monsanto and Monsanto has not released this information to the public, 1 and/or this information is protected because it constitutes Monsanto’s trade secrets or commercial or financial information, as APHIS and the courts have defined those terms. 2 As discussed more fully below, this information comprises the results of extensive research and intellectual property required both for the commercial viability and regulatory authorization of this product. This information would be worth millions of dollars to one of Monsanto’s competitors in this field, and should be accorded the protections due such

confidential and valuab le information.

Monsanto is at the leading edge in the development of biotec hnology products in a rapidly growing and highly competitive industry. Monsanto faces a number of strong, multinational competitors in this field, in cluding BASF, Bayer, Dow AgroSciences, DuPont and Syngenta. Monsanto’s competito rs, both domestic and international, have the expertise not only to replicate Monsanto ’s products, but also to use Monsanto’s

1 See, e.g., Critical Mass Energy, 975 F.2d at 878.

2 See, e.g., National Parks, 498 F.2d at 770.Soybean MON 87769

Monsanto Document #09-SY-200U Page 3 of 4 technology to develop other, competing products, thereby saving millions of dollars and years of development efforts.

Monsanto has been working on the devel opment of biotechnology-derived crops since the early 1980s, and has become a leader in the field through the expenditure of several billion dollars in research and testing cost s. Monsanto can document the development and testing costs by means of monthly summar ies of the worker ho urs devoted to these projects, budgetary documents, field te st agreements and project documents.

Presently, Monsanto’s competitors cannot dup licate Monsanto’s commercially valuable products from information in the public domain without going through the same painstaking trial and error development an d testing that Monsan to has undertaken. Although certain information regarding Monsan to products has been made available, e.g., in the context of patent applications, this information is voluminous and general in nature, and does not identify information Monsanto has found most effective for a particular product. A competitor cannot dete rmine from the patent applications which particular combination of genes and transgen ic products will prove to be commercially

valuable.

The release of the DNA sequence of plas mid PV-GMPQ1972 and the T-DNA insert in MON 87769 could provide competitors with co mmercially valuable knowledge regarding the characteristics of particul ar products Monsanto is planning to commercialize and the likely time frame for commercialization. This information would be extremely helpful to these companies in devel oping their own marketing stra tegies and development plans in a highly competitive market.

The commercial value of the information re garding the sequence of the inserted DNA has been recognized by Congress in its enactment of FIFRA and the FFDCA. Section 3 of FIFRA sets up an elaborate system of protecti ons for these types of data, protecting them from any use by other manufacturers for a period of ten years, and requiring compensation for the use of these data by compet itors after that initial ten year period. In 1996, Congress amended the FFDCA to provide both di sclosure protections and compensation equivalent to that provided by FIFRA for health and safety data submitted to support pesticide residue tolerance and to lerance exemption appl ications. FFDCA § 408(i). APHIS should recogniz e the Congressional action to protect the commercial nature of these types of documents. APHIS’s failure to do so could result in the loss of millions of dollars to Monsanto in data use and compensation rights.

In addition to the compensation provisions fo r these types of data set forth by FIFRA and FFDCA, each statute contains independent prov isions for the protection from disclosure of this information. FIFRA § 10(g); FFDCA § 408(i). FOIA prohibits the disclosure of information specifically protected by statutes such as these. 5 U.S.C. § 552(b)(3). This prohibition provides additional justification for the protection of these data.

In summary, the DNA sequence of plasmid PV-GMPQ1972 and the T-DNA insert in MON 87769 provided in Monsanto’s response document (dated February 4, 2010) asSoybean MON 87769

Monsanto Document #09-SY-200U Page 4 of 4 Appendix 1 are required in order for Monsan to to obtain nonregulated status of MON 87769 and, thereby, commercial approval for this product. This information could save such competitors millions of dollars in research. Monsanto has demonstrated, and Congress has recognized, the commercial value and confidential nature of these data. The DNA sequence of plasmid PV-GMPQ1972 and the T-DNA insert in MON 87769 are an integral part of Monsanto’s busine ss and should be protected as such.Monsanto Company 09-SY-200U Page 2 of 395 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, confidentiality, and/or competitive concerns. Monsanto expects that no information that has been identified as CBI (confidential businessinformation), will be provided to any third party. Monsanto understands that a CBIdeleted copy of this information may be made available to the public in a reading room and by individual request, as part of a public comment period. Except in accordance with the foregoing, Monsanto does not authorize the release, publi cation or other distribution of this information (including website pos ting) without Monsanto's prior notice and

consent.

© 2010 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 this 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 09-SY-200U Page 4 of 395 EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Serv ice (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility unde r the Plant Protection Act (7 USC § 7701- 7772) to prevent the introduction and dissemination of plant pest s into the United States. APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longer should be regulated . If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing

unrestricted introduction of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status in whole for the ne w biotechnology-derived soybean product, MON 87769, any progeny derived from cr osses between MON 87769 and conventional soybean, and any progeny derived from crosses of MON 87769 with other biotechnology-derived soybean that has been granted nonregulated status under 7 CFR

Part 340.

Product Description

Monsanto has developed biotechnology-de rived soybean MON 87769 that contains stearidonic acid (SDA; 18:4), a su stainable alternate source of an omega-3 fatty acid to help meet the needed dietary intake of long chain omega-3 fatty acids. The production of SDA in MON 87769 is accomplished through the introduction of two desaturase genes, Primula juliae Δ 6 desaturase ( Pj.D6D) and Neurospora crassa Δ 15 desaturase ( Nc.Fad3) into conventional soybean, A3525. The tw o introduced genes encode for the Pj Δ 6D and

Nc Δ 15D proteins, respectively. SDA is an 18-carbon fatty acid with four double bonds (18:4) and is found in plants, fish and fish /algal oil products. In mammals, SDA is a metabolic intermediate in the production of eicosapentaenoic ac id (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) from al pha linolenic acid (ALA; 18:3), a common dietary constituent. In addition to S DA, MON 87769 contains approximately 7% γ

linolenic acid (GLA; 18:3), which is an in vivo metabolite in the conversion of linoleic acid (LA: 18:2) to arachidoni c acid (20:4) in mammals.

Refined oil produced from MON 87769 contains approximately 20 to 30% SDA (wt% of total fatty acids) 1 , and can be used for the produc tion of margarine, mayonnaise, shortenings, salad dressings, r eady-to-eat foods, and other food products. Since SDA has fewer double bonds than the omega-3 fatty acids, EPA (20:5) or DHA (22:6), SDA soybean oil is more stable to oxidation (i.e., less prone to fishy or ra ncid odors and taste) than fish oils, thereby expanding the potenti al formulation options for food companies and food products for consumers. Fish and plan t oils rich in omega-3 fatty acids also are used in feed applications such as aquacultu re and poultry feeds. SDA soybean oil from MON 87769 may eventually be used in aqu aculture and feed a pplications as an alternative to fish oil and other omega-3 rich feed components. The meal derived from

1 Referred to as SDA soybean oil.Monsanto Company 09-SY-200U Page 5 of 395 MON 87769 is compositionally similar to othe r commodity soybean meal and will be

used in a manner similar to conventional soybean meal.

Data and Information Presented to Assess Plant Pest Potential of MON 87769

The data and information presented in th is petition demonstrate that MON 87769 is agronomically, phenotypically, and biochemica lly comparable to conventional soybean and that MON 87769 is not likely to pose an increased plant pest potential, including weediness or adverse environmental impact , compared to conventional soybean. The data presented in this petition to support the ove rall safety and plant pest risk potential of MON 87769 include the following:

1. A detailed molecular characterization of the inserted DNA, that confirmed the presence of intact Pj.D6D and Nc.Fad3 gene cassettes stably integrated at a single locus of the soybean genome,

2. An extensive set of biochemical characterizat ions that demonstrate the identity of the Pj Δ 6D and Nc Δ 15D proteins produced in MON 87769,

3. A comprehensive safety assessment of the Pj Δ 6D and Nc Δ 15D proteins, that shows no allergenic potential, acute toxicity or dietary risk to humans and animals,

4. A detailed compositional and nutritional asse ssment demonstrating that MON 87769 is equivalent in composition to conventional soybean, except for the intended fatty acid change (i.e., the presen ce of SDA and GLA),

5. The confirmation by a scientific panel that SDA soybean oil from MON 87769 containing approximately 20-30% SDA and 7% GLA (wt% of total fatty acids), is generally recognized as safe (GRAS),

6. The expected lack of adve rse environmental impact from exposure to SDA and GLA produced in MON 87769 due to the presence of these fatty acids in a number of other plant species,

7. An extensive evaluation of phenotypic and agronomic characteristics and

environmental interactions that demons trate MON 87769 is not likely to have increased plant pest potential comp ared to conventional soybean,

8. An assessment of the potential impact to non-target organisms (NTOs) and endangered species concludes that MON 87769 is unlik ely to have adverse effects on these organisms under normal agricu ltural practices, and 9. An evaluation of the impact on agronomic practices and land use patterns demonstrating that MON 87769 is not likely to change existing cultiv ation practices or impact land use patterns.

Molecular Characterization of Inserted DNA

The development of MON 87769 involved the introduction of two de saturase genes, Primula juliae Δ 6 desaturase ( Pj.D6D) and Neurospora crassa Δ 15 desaturase ( Nc.Fad3) that encode for the Pj Δ 6D and Nc Δ 15D proteins. The ge netic modification in MON 87769 was extensively characterize d. The results confirm that MON 87769 contains a single insert with the intended sequence, where the insert is stably maintained over multiple generations, and that the insert will not result in unintended gene products with similarity to known allergens or toxi ns. The methodology used to characterize theMonsanto Company 09-SY-200U Page 6 of 395 genetic modification included: 1) Southern blot analyses to assay the entire soybean genome for the presence of DNA derived from the transformation plasmid, PV-GMPQ1972, to confirm that a single copy was in serted at a single site in the genome and that the insert was stably inherited; 2) DNA sequencing analyses to determine the exact sequence of the inserted DNA and a llow a comparison to the transfer DNA (T-DNA) sequence of the transformation vect or to confirm that only the expected sequences were integrated; and 3) a segregatio n analysis to confirm that the introduced trait is inherited according to Mendelian laws of genetics. Taken together, the

characterization data demonstrate that a single copy of the T-DNA was inserted at a single locus in the genome of MON 87769. The stability of the integrated DNA and absence of the backbone sequences in multiple generations of MON 87769 was also

confirmed. These results are consistent with a single site of insertion that segregates in subsequent progeny according to th e Mendelian laws of genetics.

Characterization of the Pj Δ 6D and Nc Δ 15D Proteins

A multistep approach was used to characterize the Pj Δ 6D and Nc Δ 15D proteins expressed in MON 87769 resulting from the genetic modification. This detailed

characterization confirms that both proteins are safe for hu man and animal consumption. The assessment involved: 1) characterizing th e physicochemical and functional properties of both proteins; 2) quantifying protein levels in plant tissues; 3) examining the similarity of each of these proteins to known allergens, toxins and other biologically active proteins known to have adverse effects on mammals; 4) evaluating the digestibility of both proteins in simulated gastrointestinal fl uids; 5) documenting th e history of safe consumption of these proteins or their structural/functional homologues that lack

documented adverse affects on human or anim al health; and 6) i nvestigating potential mammalian toxicity through an acut e oral gavage assay. The Pj Δ 6D and Nc Δ 15D proteins share sequence similarity to severa l proteins that are ubi quitous in the human diet and are directly consumed in many common foods. The Pj Δ 6D and Nc Δ 15D proteins are expressed at low levels in th e seed and not detectab le in other tissues analysed. Both proteins lack biologically relevant amino acid sequence similarities to known allergens, toxins and antinutritional proteins known to have adverse effects on mammals. Additionally, both pr oteins are rapidly digested in simulated gastric and

intestinal fluids and do not e xhibit any signs of toxicity wh en administered to mice via oral gavage. The safety assessment supports the conclusion that dietary exposure to the Pj Δ 6D and Nc Δ 15D proteins derived from MON 87769 poses no meaningful risk to

human or animal health.

Composition and Nutrition of Forage and Grain

Detailed compositional and nutritional comparisons of MON 87769, a conventional soybean control, and ten commercially avai lable soybean varieties were conducted. These compositional comparisons were made by analyzing the seed and forage harvested from five replicated field sites across th e United States during the 2006 field season. The analysis conducted in accordance with OECD guidelines, included protein, fat, carbohydrates, fiber, ash, moisture, amino acids, fatty acids, vitamin E, and antinutrients. The compositional analyses confirmed that MON 87769 had the intended change in fattyMonsanto Company 09-SY-200U Page 7 of 395 acid composition, while the other co mponents analyzed in MON 87769 were compositionally equivalent to conventi onal soybean. As intended, MON 87769 seed contained SDA (26%) and GLA (7.1%) and low levels of two minor fatty acids, trans-SDA (0.18 %) and trans-ALA (0.44%). Statistical analysis was conducted on the composition data for other components for ev idence of biologically-re levant changes. Statistical analysis was conducte d on data from a combination of all sites (combined-site)

and data from each of five individual sites (individual-site). Considering LA is the starting material from which SDA and GLA are produced, the LA levels were significantly different in MON 87769 compared to conventional soybean. As anticipated,

the LA values were also outside the 99% tolerance interval for the population of conventional references as well as the range of values found in th e published literature

and the International Life Science Institute Crop Composition Database (ILSI-CCD). In addition to LA, combined-site statistical differences were found in five other fatty acid levels (palmitic, oleic, linolenic, arachidic, and behenic acid). The difference in the level of each of these five fatty acids was relativ ely small in absolute magnitude (<4 wt% of total fatty acids) and/or their mean valu es and ranges in MON 87769 seed were within

the 99% tolerance interval fo r the population of the conventi onal reference varieties. Given the intended shift in the fatty acid metabolism toward an increase in SDA content in MON 87769, differences in fatty acid levels were expected.

Statistical comparisons between MON 87769 and the conventional control for the presence of other components showed that 22 non-fatty acid analytes in soybean seed were significantly different (p<0.05) in the combined-site analysis. For the 22 non-fatty acid analytes, most of the statistical di fferences between MON 87769 and conventional soybean control were seen in amino acids (1 7 out of the 22 signifi cant differences) and the magnitude of these differences were sma ll (<10%). The mean and range values of

these amino acids were within the calculated 99% tolerance interval for the population of commercial conventional soybean varieties and also within the range of values found in the published literature and the ILSI-CCD. The mean level of three isoflavones, genistein, daidzein, and glycit ein, were lower in MON 87769 harvested seed compared to

the values in the conventional soybean control. However, th e mean and range of values for these three analytes in MON 87769 were w ithin the 99% tolerance interval for the population of conventional reference varieties, and were also within the range of values found in the published literature and the ILSI-CCD. It is well documented that the soybean isoflavone levels are greatly in fluenced by many factors, ranging from environmental conditions, variety, and agronomic practices. The remaining two

statistical differences observ ed were for carbohydrates and protein, and their magnitude

of differences between MON 87769 and conventi onal soybean control were small (<6%). Furthermore, the mean and range values of carbohydrates and protein were within the calculated 99% tolerance interval for the popul ation of commercial conventional soybean

varieties and also with in the range of values found in the published lite rature and the ILSI-CCD. Therefore, these differences we re not considered to be biologically meaningful from a food and feed safety or nutritional perspec tive. Combined-site analysis of forage showed no significant differences (p>0.05) between MON 87769 and the conventional soybean control.Monsanto Company 09-SY-200U Page 8 of 395 In addition to the compositional analysis of seed and forage, four soybean processed fractions (oil, meal, lecithi n, and protein isolate) were produced from MON 87769 seed and subjected to compositional analysis in accordance with OECD guidelines. As expected, apart from the intended fatty ac id change, the composition of the soybean processed fractions from MON 87769 is e quivalent to the composition of soybean processed fractions from the conventional soybe an control. Thus, the processed fractions

from MON 87769 are concluded to be as safe and nutritious as the processed fractions prepared from conventional soybean.

Collectively, the compositional data suppor t the conclusion that MON 87769, with the exception of the expected changes in fatty acid composition, does not have biologically meaningful differences from conventional soybean from a food/feed safety and/or nutritional perspective.

History of Environmental Exposure to SDA and GLA

Environmental exposure to SDA and GLA from MON 87769 is expected to be limited primarily to soybean pests and animals that feed on seed. These fatty acids are already present in the environment. SDA, GLA, and other polyunsaturated fatty acids (PUFAs) exist in many sources in the environment without known adverse ec ological effects. Many fish species, particularly marine fish, contain SDA. SDA and GLA are produced in many plant seed oils. More than 230 species from among 17 plant families are documented to produce SDA in seed oil at con centrations greater than 1% of their total fatty acid composition. Similarly, GLA is presen t in a number of plant species at levels greater than 1% of their tota l fatty acid composition. As na tural components of the plant and animal world, SDA, GLA a nd other PUFAs are not expect ed to accumulate, persist

or be detrimental to the environment.

Phenotypic and Agronomic Characteristics and Environmental Interactions

The commercial soybean species cultivated in the U.S., Glycine max L., does not exhibit weedy characteristics and is not effective in invading established ecosystems. Soybean is

not listed as a weed in scient ific literature, nor is it pres ent on the lists of noxious weed species compiled by the Fede ral government. Soybean does not possess any of the attributes commonly associated with weeds, such as long pers istence of seed in the soil, the ability to disperse, invade, and become a dominant species in new or diverse landscapes, or the ability to compete well with native vege tation. Due to a pronounced lack of dormancy, soybean seed can germinate quickly under adequate temperature and moisture and potentially can gr ow as a volunteer plant. Ho wever, a volunteer plant likely would be killed by frost during au tumn or winter of the year it was produced. If it did become established, a volunteer plant woul d not compete well with the succeeding crop,

and could be controlled readil y by either mechanical or ch emical means. In addition, since wild populations of Glycine species are not known to exis t in the U.S., the potential

does not exist for MON 87769 to outcross to wild or weedy relatives and alter their weediness potential.Monsanto Company 09-SY-200U Page 9 of 395 A phenotypic and agronomic assessment was conducted to determine whether MON 87769 has altered soybean characteristics that may result in increased weediness or have impact on cultivation practices. Additionally, the environmental interactions of

MON 87769 were assessed relative to conve ntional soybean. The overall assessment indicates that MON 87769 is not fundamentally different from conventional soybean and is unlikely to have an increased plant pest risk. An important element in assessing plant pest potential and the envi ronmental impact of MON 87769 is to compare MON 87769 to conventional soybean. The assessment is ba sed initially on the c oncept of familiarity, which USDA recognizes, plays an important ro le in these assessments. Familiarity is based on the fact that the biotechnology-deri ved plant is developed from a conventional plant variety whose biologica l properties and plant pest potential are well known. Familiarity considers the biology of the crop, the introduced tr ait, the receiving environment and the interactions among th ese factors, and provides a basis for comparative risk assessment between a bi otechnology-derived plant and its conventional counterpart. The MON 87769 characteristics assessed included: seed dormancy and germination, pollen morphology, symbiont inter actions conducted in the laboratory and greenhouse, and plant phenotypi c and environmental interact ion evaluations conducted in

the field.

Seed dormancy and germination characterization indicated that MON 87769 seed had germination characteristics similar to that of the conventional soybean control. In particular, the lack of increased hard see d, a well-accepted characte ristic of weediness affecting seed germination rate and viability, supports a conclusion of no increased weediness potential of MON 87769 compared to conventional soybean for germination and dormancy characteristics. In an assessmen t of pollen characteristics, there were no significant differences (p>0.05) observed for an y of the parameters measured, including pollen viability and diameter. Furthermore, MON 87769 was not altered in its symbiotic relationship with Bradyrhizobium japonicum compared to conventional soybean based on assessments of nodule number, s hoot nitrogen, and biomass of nodules, shoot material, or root material. Collectively, these results support the conclusion that MON 87769 is not likely to exhibit increased weed potentia l compared to conventional soybean.

The field evaluation of phenotypic, agrono mic, and ecological characteristics of MON 87769 also supports the conclusion that MON 87769 is not likely to pose an increased plant pest potential compared to c onventional soybean. These evaluations were

conducted at 21 field locations across U.S. soybean production re gions during the 2006 and 2007 growing seasons. Fourteen phenotypic characteristics were assessed including: early stand count, seedling vigor, plant growth stage, days to 50% flowering, flower color, plant pubescence, plant height , lodging, pod shattering, final stand count, seed moisture, 100 seed weight, test weight , and yield. The observed phe notypic characteristics of

MON 87769 were not different from the conve ntional soybean control. No significant differences (p<0.05) were observed in either ye ar in a combined-site analysis of the data for any of the phenotypic characteristics m easured. The results from the 2006 and 2007

field trials demonstrate that the assessed phenotypic and agronomic characteristics of MON 87769 were not altered, co mpared to the conventional c ontrol, as a result of the introduced genes ( Pj.D6D and Nc.Fad3 ), the proteins produ ced from the introduced genes (Pj Δ 6D and Nc Δ 15D) and the intended fatty acid change. These results areMonsanto Company 09-SY-200U Page 10 of 395 indicative of no increased plan t pest or weediness potentia l of MON 87769 compared to conventional soybean.

Similarly, an assessment of the plant response to abiotic stressors (e.g., drought, nutrient deficiency, wind, etc.), disease damage, a nd arthropod damage, indicates no increased plant pest potential or a dverse ecological interactions of MON 87769 relative to conventional soybean. No biologically meani ngful differences were detected between

MON 87769 and the conventional control for the 703 comparative observations of plant response to abiotic stre ssors, disease damage, and arthropod damage and 260 comparative observations of the abundance of various pest and bene ficial arthropods in field trials over two consecutive years.

Nontarget Organisms and Threatened or Endangered Species

An environmental assessment of MON 87769 indicates that MON 87769 would not have a significant impact (either positive or negative) on NTOs , including threatened or endangered species under normal agricultural practices. The assessment considered the expected differences between MON 87769 an d conventional soybean, including the presence of the inserted gene s, the expression of the Pj Δ 6D and Nc Δ 15D proteins, and the production of the expect ed fatty acids (SDA and GLA) in MON 87769 seed. Since MON 87769 is a source of omega-3 enriched so ybean oil with no pesticidal activity, all exposed organisms are considered to be NT Os. The assessment considered pertinent information from product characterization an d protein safety assessment, history of environmental exposure to the additional fatty acids in MON 87769 seed, and results from the ecological interaction assessment. Based on the weight of evidence of this information, there is no indication that MON 87769 would have an adverse impact on NTOs including threatened or endangered sp ecies under normal agricultural practices.

The potential for MON 87769 to ou tcross with sexually compa tible species in the U.S., including threatened or e ndangered plant species, is unlikely, sinc e no known wild Glycine species related to cultivated soybean ar e known to be present in North America. In those world areas where sexually compa tible species do exist, the potential for outcrossing is low because s oybean is a highly self-pol linated species, with crosspollination to adjacent plants of other soybean varieties occurring at very low frequencies (0.3 to 3.62%) ( Beard and Knowles, 1971) . Furthermore, in the rare event when crosspollination may occur, MON 87769 and its proge ny are not expected to have a significant environmental impact. The evaluations have shown that the introduced genes ( Pj.D6D

and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change is not likel y to enhance the weediness or plant pest potential of MON 87769. Theref ore, the environmental cons equence of pollen transfer

from MON 87769 to other Glycine species is considered negligible.

Soybean Agronomic Practices and Land Use

Soybean is one of the largest U.S. crops in terms of acreage plante d and quantity of seed harvested. In 2008, soybean was planted on 75.7 million acres in the U.S., where the harvested soybean seed had an average yield of 36.9 bushels per acre and total productivity was 2.96 billion bushels, resulting in a net value greater than $27.4 billion.Monsanto Company 09-SY-200U Page 11 of 395 Specialty soybean varieties are produced on approximately 12% of the U.S. soybean acreage and according to the Midwest Shippers Association this acreage could grow to over 25% of the crop acreage in certain st ates within the next decade.

Soybean fields are typically hi ghly managed agricultural areas th at can be expected to be dedicated to crop production for many years. No significant impact would be expected following the introduction of MON 87769 on cu rrent cultivation and management practices for soybean. MON 87769 has been sh own to be no different from conventional soybean in its agronomic, phenotypic, and eco logical characteristic s and has the same levels of resistance to insects and diseases as current commercial soybean. Cultivation of MON 87769 would not be expected to differ from typical speciality soybean cultivation. Based on these considerations, there is no a pparent potential for significant impact on land use or agronomic practices.

Conclusion

Based on the data and information presented in this petition, it is concluded that MON 87769 is not likely to be a plant pest. Therefore, M onsanto Company requests a

determination from APHIS that MON 87769 and any progeny derived from crosses between MON 87769 and conventional soybean or deregulated biotechnology-derived soybean be granted nonregulated status under 7 CFR Part 340.Monsanto Company 09-SY-200U Page 12 of 395 TABLE OF CONTENTS

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

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

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

TABLE OF CONTENTS .................................................................................................. 12

LIST OF TABLES ............................................................................................................ 16

LIST OF FIGURES .......................................................................................................... 20

ABBREVIATION AND DEFINITIONS ......................................................................... 22

I. RATIONALE FOR THE DE VELOPMENT OF MON 87769 .................................... 29

I.A. Basis for the Request for a Determin ation of Nonregulated Status .......... 29

I.B. Rationale for the Development of Soybean MON 87769 ......................... 29

I.C. Submissions to Other Regulatory Agencies ............................................. 30

II. THE SOYBEAN FAMILY ......................................................................................... 32

II.A. Soybean as a Crop ..................................................................................... 32

II.B. History of Soybean ................................................................................... 33

II.C. Taxonomy and Phylogenetics of Soybean ................................................ 34

II.D. The Genetics of Soybean .......................................................................... 36

II.E. Pollination of Cultivated Soybean ............................................................ 36

II.F. Cultivated Soybean as a Volunteer ........................................................... 37

II.G. Characteristics of th e Recipient Plant ....................................................... 37

II.H. Soybean as a Test System in Product Safety Assessment ........................ 37

III. DESCRIPTION OF THE TRANSFORMATION SYSTEM .................................... 38

IV. GENETIC ELEMENTS ............................................................................................ 41

IV.A. Plasmid PV-GMPQ1972 ........................................................................... 41

IV.B. T-DNA I .................................................................................................... 41

IV.B.1. The Pj.D6D Coding Sequence and Pj ∆ 6D Protein ................................... 42

IV.B.2. The Pj.D6D Regulatory Sequences .......................................................... 42

IV.B.3. The Nc.Fad3 Coding Sequence and Nc ∆ 15D Protein .............................. 42

IV.B.4. The Nc.Fad3 Regulatory Sequences ......................................................... 42

IV.B.5. The T-DNA Borders ................................................................................. 42

IV.C. T-DNA II .................................................................................................. 43

IV.C.1. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein ................. 43

IV.C.2. The Arabidopsis thaliana EPSPS Transit Peptide .................................... 43

IV.C.3. The cp4 epsps Regulatory Sequences ....................................................... 43

IV.C.4. The T-DNA Borders ................................................................................. 43

IV.D. Genetic Elements Outside of the T-DNA Borders ................................... 43

V. GENETIC ANALYSIS ............................................................................................... 49

V.A. Insert and Copy Number Determination ................................................... 55

V.B. Confirmation of the Absence of Plasmid PV-GMPQ1972 Backbone ...... 58

V.C. Confirmation of the Absence of T-DNA II Sequence .............................. 60

V.D. Pj.D6D and Nc.Fad3 Cassette Copy Number and Integrity ..................... 63

V.D.1. Right Border/ 7S α ' Promotor ..................................................................... 63

V.D.2. Pj.D6D Coding Sequence ......................................................................... 64Monsanto Company 09-SY-200U Page 13 of 395 V.D.3. tml 3 ′ Nontranslated Sequence .................................................................. 65

V.D.4. 7S α Promoter ............................................................................................. 65

V.D.5. Nc.Fad3 Coding Sequence........................................................................ 66

V.D.6. E9 3 ′ Nontranslated Sequence/Left Border ............................................... 66

V.E. Southern Blot Analyses of MON 87769 Across Multiple Generations .... 74

V.E.1. Generational Stability of the Insert ........................................................... 74

V.E.2. Confirmation of the Absence of PV-GMPQ1972 Backbone Sequence ... 75

V.E.3. Confirmation of the Absence of T-DNA II Sequence .............................. 75

V.F. Organization of the Genetic Elements in MON 87769 ............................. 81

V.G. Inheritance of the In troduced Traits .......................................................... 81

V.H. Conclusions of Molecular Characterization .............................................. 85 VI. CHACTERIZATION OF THE INTRODUCED Pj Δ 6D AND Nc Δ 15D

PROTEINS ............................................................................................... 86

VI.A. Identity and Function of the Pj Δ 6D and Nc Δ 15D Proteins ...................... 86

VI.A.1. Pj Δ 6D Protein ........................................................................................... 87

VI.A.2. Nc Δ 15D Protein ........................................................................................ 89

VI.B. Characterization of the Pj Δ 6D and Nc Δ 15D Proteins Produced in

MON 87769 .............................................................................................. 90

VI.C. Expression of Pj Δ 6D and Nc Δ 15D Proteins in MON 87769 ................... 92

VI.C.1. Protein Expression Analysis of Tissues from the 2006 U.S. Field

Trials ......................................................................................................... 93

VI.D. Assessment of the Potential for Allergenicity of the Pj Δ 6D and

Nc Δ 15D Proteins Produced in MON 87769 ............................................. 97

VI.E. Assessment of the Potential for Toxicity of the Pj Δ 6D and Nc Δ 15D

Proteins ..................................................................................................... 97

VII. COMPOSITIONAL AND NUTRITI ONAL ASSESSMENT OF MON 87769 ...... 99

VII.A. Intended Changes to Fatty Acid Levels in MON 87769 Soybean Seed . 100

VII.B. Compositional Comparisons of Soybean Seed and Forage from

MON 87769 and the Conventional Control ............................................ 101

VII.B.1. Fatty Acid Levels in Soybean Seed ........................................................ 102

VII.B.2. Nutrient Levels Other than Fa tty Acids in Soybean Seed ...................... 103

VII.B.3. Naturally Occurring Antinutrient Levels in Soybean Seed .................... 104

VII.B.4. Proximate and Fiber Levels in Soybean Forage ..................................... 105

VII.B.5. Compositional Equivalence of MON 87769 to Conventional Soybean ................................................................................................... 105

VII.C. Compositional Comparisons of Processed Fractions from Soybean

Seed of MON 87769 and the Conventional Control ............................... 113

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

MON 87769 ............................................................................................ 113

VII.E. Safety and Nutrition Assessment Conclusion ......................................... 114 VIII. PHENOTYPIC, AGRONOMIC, AN D ENVIRONMENTAL

INTERACTIONS ASSESSMENT ......................................................... 115

VIII.A. Characteristics Measured for Assessment .............................................. 115 VIII.B. Interpretation of Phenotypic and Environmental Interaction Data ......... 117 VIII.C. Interpretation of Detected Differences Criteria ...................................... 117Monsanto Company 09-SY-200U Page 14 of 395 VIII.D. Phenotypic, Agronomic and Environmental Interactions

Characteristics ......................................................................................... 119 VIII.D.1. Seed Dormancy and Germination Characteristics .................................. 119 VIII.D.2. Field Phenotypic, Agronomi c Characteristics and Environmental

Interactions .............................................................................................. 123 VIII.D.2.1. Field Phenotypic and Agronomic Characteristics................................... 124 VIII.D.2.2. Environmental Interaction Analyses ....................................................... 128

VIII.D.3. Pollen Characteristics.............................................................................. 129 VIII.D.4. Symbiont Interactions ............................................................................. 130

VIII.E. Overall Conclusions for Phe notypic, Agronomic, and Environmental

Interactions Evaluation ........................................................................... 136

IX. U.S. AGRONOMIC PRACTICES .......................................................................... 138

IX.A. Introduction ............................................................................................. 138 IX.B. Overview of U.S. Soybean Production ................................................... 139 IX.B.1. Soybean Production ................................................................................ 139 IX.B.2 Specialty Soybean ................................................................................... 144 IX.B.3. Identity Preservation ............................................................................... 146 IX.B.4. Soybean Seed Production ....................................................................... 148 IX.C. Production Manageme nt Considerations ................................................ 151 IX.D. Weed Management ................................................................................. 156 IX.E. Management of Insects ........................................................................... 157 IX.F. Management of Diseases and Other Pests .............................................. 157 IX.G. Crop Rotation Pr actices in Soybean ....................................................... 159 IX.H. Soybean Volunteer Management ............................................................ 160 IX.I. Stewardship of MON 87769 ................................................................... 162 IX.J. Impact of the Introduction of MON 87769 on Agricultural Practices .... 163 X. ENVIRONMENTAL CONSEQUENCES AND IMPACT ON AGRONIMIC

PRACTICES ........................................................................................... 165

X.A. Plant Pest Assessment of MON 87769 ................................................... 166

X.A.1. Characteristics of the Genetic Insert ....................................................... 166 X.A.2. Mode of Action and Safety of Pj Δ 6D and Nc Δ 15D Proteins ................. 167 X.A.3. Environmental Safety of Fatty Acids Produced in MON 87769 ............ 168 X.A.3.1. Biological Role and Metabolis m of Polyunsaturated Fatty Acids .......... 168 X.A.3.2. Environmental Exposure to Fatty Acids in MON 87769 ........................ 169

X.A.3.3. Environmental Fate of Fa tty Acids Present in MON 87769 ................... 171 X.A.4. Impact on NTOs ...................................................................................... 174 X.A.4.1 Impact Due to introduced Genes and Proteins ........................................ 174

X.A.4.2. Potential Adverse Effects of MON 87769 Fatty Acids on NTOs ........... 174

X.A.4.3. Interactions with NTOs under Field Conditions ..................................... 175

X.B. Weediness Potential of MON 87769 ...................................................... 175

X.C. Potential for Pollen-mediated Gene Flow ............................................... 176

X.C.1. Vertical Gene Flow ................................................................................. 176 X.C.1.1. Hybridization with Cultivated Soybean Glycine max ............................. 176 X.C.1.2. Hybridization with the Wild Annual Species within Subgenus Soja ..... 179 X.C.1.3. Hybridizatio n with Wild Perennial Species of Subgenus Glycine .......... 179Monsanto Company 09-SY-200U Page 15 of 395 X.C.2. Transfer of Genetic information to Species with which Soybean Cannot Interbreed (Horizontal Gene Flow) ............................................ 180 X.D. Summary of Plan t Pest Assessments ...................................................... 180 XI. ADVERSE CONSEQUENCES OF INTRODUCTION ......................................... 181

REFERENCES ............................................................................................................... 182

APPENDICES ................................................................................................................ 199 Appendix A. USDA Notifications ................................................................................ 199 Appendix B. Materials and Methods Used for Molecular Analyses of MON 87769 .. 203 Appendix C. Characterization of Pj Δ 6D and Nc Δ 15D Proteins Produced in MON 87769 – Materials, Methods and the Results ................................ 206 Appendix D. Materials and Methods Used for the Determination of Protein Expression in MON 87769 Tissue .......................................................... 237 Appendix E. Materials and Methods Us ed for the Compositional Analysis of MON 87769 Soybean Seed, Forage and Processe d Fractions from the Five Replicated Field Sites in the U.S. in 2006 Growing

Season ..................................................................................................... 240 Appendix F. Safety and Nutritional Impact Resulting from the Intended Fatty Acid Change in MON 87769 ......................................................... 316 Appendix G. Materials, Me thods, and Individual-Site Resu lts of the Plant Phenotypic and Agronomic Assessment of MON 87769

from the 2006 and 2007 Field Trials....................................................... 321 Appendix H. Materials, Me thods, and Individual-Site Re sults from the Seed Dormancy and Germination Assessment of MON 87769 ...................... 338 Appendix I. Methods and Results fr om the Assessment of MON 87769 for Plant

Interactions with Abiotic Stressors , Diseases, and Arthropods under

Field Conditions in 2006 and 2007 ......................................................... 344 Appendix J. Materials and Methods fr om the Pollen Viability and Morphology

Assessment of MON 87769 .................................................................... 365 Appendix K. Materials and Methods for the Assessment of MON 87769 for Plant Interactions with Nitrogen -fixing Soil Symbionts .................................. 368 Appendix L. Petitioner’s E nvironmental Assessment ................................................. 373Monsanto Company 09-SY-200U Page 16 of 395 LIST OF TABLES

Table II-1. World Soybean Production in 2007/2008 ............................................... 32 Table II-2. List of Species in the Genus Glycine Willd., 2n Chromosome Number, Genome Symbol, a nd Distribution .......................................................... 35 Table IV-1. Summary of Genetic Elements in Plasmid Vector PV-GMPQ1972 ....... 46 Table V-1. Summary of Gene tic Elements in MON 87769 ...................................... 50 Table V-2. Summary Chart of the E xpected Sizes of DNA Fragments Using Combinations of Restriction Enzymes and Probes ................................. 54 Table V-3. Chi-square ( χ 2 ) analysis of MON 87769 Progeny ................................... 84 Table VI-1. Summary of Pj Δ 6D Protein Levels in Tissues Collected from MON 87769 Produced in the 2006 U.S. Growing Season ...................... 95 Table VI-2. Summary of Nc Δ 15D Protein Levels in Tissues Collected from MON 87769 Produced in the 2006 U.S. Growing Season ...................... 96

Table VII-1. Combined-Site Summary of SDA, GLA, trans-SDA and trans-ALA

Levels in MON 87769 Seed .................................................................. 101

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional An alysis of MON 87769 vs. the Conventional Control (A3525) and Comm ercial Reference Varieties . 106 Table VIII-1. Phenotypic, Agronomic a nd Environmental Interaction

Characteristics Evaluated in U.S. Field Trials ...................................... 116 Table VIII-2. Seed Dormancy and Ge rmination Characteristics Evaluated ................ 121

Table VIII-3. Germination Characteristics of MON 87769 and A3525 ...................... 122 Table VIII-4. Field Phenotypic Evaluation S ites for MON 87769 during 2006

and 2007 ................................................................................................ 125 Table VIII-5. Plant Growth and Developmen t Data across 17 Locations during

2006 ....................................................................................................... 126 Table VIII-6. Plant Growth and Developm ent Data across Four Locations during

2007 ....................................................................................................... 127

Table VIII-7. Pollen Grain Diam eter and Viability Analyses ..................................... 130 Table VIII-8. Symbiont Interaction Assessment of MON 87769 and the

Conventional Control at 4- and 6-Weeks after Emergence in an

Initial Assessment .................................................................................. 134 Table VIII-9. Symbiont Interaction Assessment of MON 87769 , the Conventional Control, and the Negative Isoline at 6-Weeks after Emergence in a

Refined Assessment .............................................................................. 135

Table VIII-10. Percent Difference between MON 87769 and the Conventional Control or Negative Isoline Control ...................................................... 136 Table IX-1. Soybean Production in the U.S., 1999 – 2008 ....................................... 141 Table IX-2. U.S. Soybean Produc tion by Region and State in 2008 ........................ 142 Table IX-3. U.S. Soybean Produc tion Costs and Returns in 2006 ........................... 143 Table IX-4. Ratings for Control of Vo lunteer Glyphosate-Tolerant Soybean in Labeled Rotational Crops ...................................................................... 162

Table X-1. Plants that Contain Stearidonic Acid (SDA), Gamma-Linolenic Acid (GLA), and Other Polyunsat urated Fatty Acids (PUFAs) ............ 172

Table X-2. Fish that Cont ain Stearidonic Acid (SDA) ............................................ 173Monsanto Company 09-SY-200U Page 17 of 395 Table X-3. Summary of Published Lite rature on Soybean Cross-Pollination ......... 178 Table A-1. USDA Notification Approved for M ON 87769 and Status of

Trials Conducted under These Notifications ......................................... 200

Table C-1. N-terminal Amino Acid Sequence Analysis of the MON 87769- Produced Pj Δ 6D Protein ........................................................................ 224

Table C-2. N-terminal Amino Acid Sequence Analysis of the MON 87769-

Produced Nc Δ 15D Protein .................................................................... 230 Table E-1. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Forage fr om IA-1 Site for Fiber and

Proximates ............................................................................................. 250 Table E-2. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Soybean S eed from IA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones ............................................. 251 Table E-3. Summary of the Com positional Comparison of MON 87769 vs.

the Conventional Control Fo rage from IA-2 Site for

Fiber and Proximates ............................................................................. 257 Table E-4. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Soybean S eed from IA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones ............................................. 258 Table E-5. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Forage fr om IL Site for Fiber and

Proximates ............................................................................................. 264 Table E-6. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Soybean Seed from IL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones ............................................. 265 Table E-7. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Forage from MI Site for Fiber and

Proximates ............................................................................................. 271 Table E-8. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Soybean Seed from MI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones ............................................. 272 Table E-9. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Forage fr om OH Site for Fiber and

Proximates ............................................................................................. 278 Table E-10. Summary of the Com positional Comparison of MON 87769 vs. the Conventional Control Soybean Seed from OH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones ............................................. 279 Table E-11. Statistical Summary from th e Combined-Site Compositional Analysis of MON 87769 vs. the Conve ntional Control (A3525) Forage for Fiber and Proximates ........................................................... 285Monsanto Company 09-SY-200U Page 18 of 395 Table E-12. Statistical Summary from th e Combined-Site Compositional Analysis of MON 87769 vs. the C onventional Control (A3525) Soybean Seed for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones ............................................. 286

Table E-13. Trans-ALA, GLA, trans-SDA and and SDA Levels in MON 87769 Individual-Site Summary ...................................................................... 292

Table E-14. Literature and ILSI Database Values for Components in Conventional Soybean Seed and Forage ............................................... 294 Table E-15. Summary of th e Compositional Comparison of MON 87769 vs. the Conventional Control Soybean Meal Fraction from Combined-Site for Amino Acids, Fiber, Proximates

and Antinutrients ................................................................................... 303 Table E-16. Summary of th e Compositional Comparison of MON 87769 vs. the Conventional Control Soybean RBD Oil from Combined-Site for Fatty Acids and Vitamin E ..................................... 307 Table E-17. Summary of th e Compositional Comparison of MON 87769 vs. the Conventional Control Soybean Protein Isolate

from Combined-Site for Amino Acids and Moisture ............................ 310 Table E-18. Summary of th e Compositional Comparison of

MON 87769 vs. the Conventional Cont rol Soybean Lecithin Fraction from Combined-Site for Phosphatides .................................................. 313 Table G-1. Test, Control, and Reference Materials Assessed in the 2006 Field

Trials ...................................................................................................... 324 Table G-2. Test, Control, and Reference Materials Assessed in the 2007 Field

Trials ...................................................................................................... 325 Table G-3. Phenotypic and Agronom ic Comparison of MON 87769 to the

Conventional Control in the Individu al-Site Analysis for the 2006 Field Study ............................................................................................ 326 Table G-4. Plant Growth Stages of MON 87769, the Conventional Control, and

Reference Varieties at 17 U.S. Sites in 2006 ........................................ 329 Table G-5. Phenotypic and Agronom ic Comparison of MON 87769 to the

Conventional Control in the Individu al-Site Analysis for the 2007 Field Study ............................................................................................ 333 Table G-6. Plant Growth Stages of MON 87769, the Conventional Control, and

Reference Varieties at Four U.S. Sites in 2007 ..................................... 335 Table G-7. Field Site Planting Informa tion, Soil Description, and Cropping History ................................................................................................... 336

Table H-1. Germination Characteristics of MON 87769, the Conventional Control, and the Reference Soybean Va riety Seed Produced from

each of Three Field Sites and Te sted in the AOSA-Recommended Temperature Regime ............................................................................. 341

Table H-2. Germination Characteris tics of MON 87769 and the Conventional

Control Seed Produced from each of Three Fi eld Sites and Tested in the Additional Temperature Regimes ............................................... 342Monsanto Company 09-SY-200U Page 19 of 395 Table I-1. Abiotic Stressor Evaluations Using an Observational Severity

Scale for MON 87769 and the Conventiona l Control in the 2006

Field Trials ............................................................................................ 347 Table I-2. Abiotic Stressor Evaluations Using an Observational Severity

Scale for MON 87769 and the Conventiona l Control in the 2007

Field Trials ............................................................................................ 348 Table I-3. Disease Damage Ev aluations Using an Observational Severity

Scale for MON 87769 and the Conventiona l Control in the 2006

Field Trials ............................................................................................ 349 Table I-4. Disease Damage Ev aluations Using an Observational Severity

Scale for MON 87769 and the Conventiona l Control in the 2007

Field Trials ............................................................................................ 350 Table I-5. Arthropod Damage Evaluated Using an Observational Severity

Scale for MON 87769 and the Conventiona l Control in the 2006

Field Trials ............................................................................................ 351 Table I-6. Arthropod Damage Evaluated Using an Observational Severity

Scale for MON 87769 and the Conventiona l Control in the 2007

Field Trials ............................................................................................ 352 Table I-7. Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the Conventional C ontrol, and the Reference

Varieties in U.S. Field Trials in 2006 .................................................... 353

Table I-8. Abundance of Beneficial Arthr opods in Beat Sheet Samples Collected from MON 87769, the Conventional Control, and the

Reference Varieties in U.S. Field Trials in 2006 .................................. 356 Table I-9. Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the Conventional Control, and the Reference

Varieties in U.S. Field Trials in 2007 .................................................... 358

Table I-10. Abundance of Beneficial Arthr opods in Beat Sheet Samples Collected from MON 87769, the Conventional Control, and the

Reference Varieties in U. S. Field Trials in 2007 ................................ 361 Table K-1. Comparison of Uni noculated Conventional Control (A3525-UN)

to the Inoculated Conven tional Control (A3525) for Nodule Number and Shoot Total Nitrogen ........................................................ 370 Table L-1. Organic and Conven tional Soybean Seed Sources ................................ 383 Table L-2. Deregulated Biotec hnology-derived Soybean Products ........................ 391Monsanto Company 09-SY-200U Page 20 of 395 LIST OF FIGURES

Figure III-1. Schematic of the Development of MON 87769 .................................... 40 Figure IV-1. Plasmid Map of Vector PV-GMPQ1972 ............................................... 45 Figure IV-2. Deduced Amino Acid Sequence of Primula juliae Δ 6 Desaturase from PV-GMPQ1972 ............................................................................. 48 Figure IV-3. Deduced Amino Acid Sequence of Neurospora crassa Δ 15 Desaturase from PV-GMPQ1972 .......................................................... 48 Figure V-1. Plasmid Map of Vector PV GMPQ1972 Showing Probes 1-12 ............ 51 Figure V-2. Plasmid Map of Vector PV-GMPQ1972 Showing Probes 13-18 .......... 52 Figure V-3. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87769 ..................................................................... 53 Figure V-4. Southern Blot Analysis of MON 87769 with Insert and Copy

Number Probes....................................................................................... 57 Figure V-5. Southern Blot Analysis of MON 87769 with PV-GMPQ1972

Backbone Probes .................................................................................... 59 Figure V-6. Southern Blot Analysis of MON 87769 with T-DNA II Probes ........... 62 Figure V-7. Southern Blot Analysis of MON 87769 with Right Border/P-7s α ′

Probe ...................................................................................................... 68 Figure V-8. Southern Blot An alysis of MON 87769 with CS- Pj.D6D Probe........... 69 Figure V-9. Southern Blot Analysis of MON 87769 with T -tml Probe .................... 70 Figure V-10. Southern Blot Analysis of MON 87769 with P- 7s α Probe .................... 71 Figure V-11. Southern Blot Analysis of MON 87769 with CS- Nc.Fad3 Probe ......... 72 Figure V-12. Southern Blot Analysis of MON 87769 with T- E9 /Left Border

Probe ...................................................................................................... 73 Figure V-13. MON 87769 Breeding Diagram............................................................. 77 Figure V-14. Generational Stabilit y of MON 87769 with T-DNA I Probes ............... 78 Figure V-15. Generational Stability of MON 87769 with PV-GMPQ1972

Backbone Probes .................................................................................... 79 Figure V-16. Generational Stabilit y of MON 87769 with T-DNA II Probes.............. 80 Figure V-17. Breeding Path for Genera ting Segregation Data on MON 87769 ......... 83 Figure VI-1. Deduced Amino Acid Sequence of Pj Δ 6D from PV-GMPQ1972 ........ 88 Figure VI-2. Hydropathy Plot of th e Deduced Amino Acid Sequence of Pj Δ 6D ...... 88 Figure VI-3. Deduced Amino Acid Sequence of Nc Δ 15D from PV GMPQ1972 ..... 89 Figure VI-4. Hydropathy Plot of the Deduced Amino Acid Sequence of

Nc Δ 15D ................................................................................................. 90 Figure VII-1. Fatty acid biosynthesis in plants and the introduced changes to produce MON 87769 ........................................................................... 103 Figure VIII-1. Schematic Diagram of Data Interpretation Methods ........................... 118 Figure C-1. SDS-PAGE of the MON 87769-Produced Pj Δ 6D Protein .................. 222 Figure C-2. Wester n Blot Analysis of MON 87769-Produced Pj Δ 6D Protein ....... 223 Figure C-3. MALDI-TOF MS Coverage Map of the MON 87769-Produced Pj Δ 6D Protein ...................................................................................... 224 Figure C-4. Glycosylation Anal ysis of the MON 87769-Produced Pj Δ 6D

Protein .................................................................................................. 227 Figure C-5. SDS-PAGE of the MON 87769-Produced Nc Δ 15D Protein ............... 229Monsanto Company 09-SY-200U Page 21 of 395 Figure C-6. MALDI-TOF MS Covera ge Map of the MON 87769-Produced

Nc Δ 15D Protein ................................................................................... 231 Figure C-7. Wester n Blot Analysis of MON 87769-Produced Nc Δ 15D Protein.... 232 Figure C-8. Glycosylation Anal ysis of the MON 87769-Produced Nc Δ 15D

Protein .................................................................................................. 234Monsanto Company 09-SY-200U Page 22 of 395 ABBREVIATION AND DEFINITIONS ∗

~ Approximately 2T-DNA Plasmid vector containing two separate T-DNA regions each surrounded by left and right bor ders of the Ti plasmid

35S The promoter and leader from the cauliflower mosaic virus (CaMV) 35S RNA

A3525 Conventional Soybean Control

aadA Bacterial promoter and coding sequence for an aminoglycosidemodifying enzyme, 3'(9)-O-nucle otidyltransferase from the transposon Tn7

AA Amino Acid

AAA Amino Acid Analysis

ADF Acid Detergent Fiber

AD8 Allergen Database; www.allergen online.com, released in January

2008
ALA Alpha Linolenic Acid

AN(C)OVA Analysis of (Co)Variance

ANOVA Analysis of Variance

AOAC Association of Analytical Communities

AOCS American Oil Chemists Society

AOSA Association of Offi cial Seed Analysts

AOSCA Association of Official Seed Certifying Agencies

APS Analytical Protein Standard

ASA America Soybean Association

B-Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA
B-Right Border DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA

BSA Bovine Serum Albumin

CaMV Cauliflower Mosaic Virus

CAPS N-Cyclohexyl-3-aminopropanesulfonic acid

CAST Council for Agricultural Science and Technology, USDA

CBI Confidential Business Information

CEQ The Council on Environmental Quality

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

‘Instructions to Authors’ in the Journal of Biological Chemistry

.Monsanto Company 09-SY-200U Page 23 of 395 CEW Corn Earworm [ Helicoverpa zea (Boddie)]

CFIA Canadian Food Inspection Agency

CFR Code of Federal Regulations

COA Certificate of Analysis

CPB Cartagena Protocol on Biosafety

CSFII Continuing Survey of Food Intakes by Individuals

CS- cp4 epsps Coding Sequence (codon-modified) of the aroA gene from Agrobacterium sp . strain CP4 encoding the CP4 EPSPS protein

CS- Nc.Fad3 Coding Sequence (codon-optimi zed) of the gene from Neurospora

crassa encoding Δ 15 desaturase

CS- Pj.D6D Coding sequence for the fatty acid Δ 6 desaturase from Primula juliae

CS- rop Coding Sequence for repressor of primer protein used for maintenance of plasmid copy number in E. coli

CTAB Hexadecyltrimethylammonium Bromide

CTP Chloroplast Transit Peptide

CV Coefficient of Variation

Da Dalton

DAP Days After Planting

dCTP Deoxycytidine Triphosphate

DDI Daily Dietary Intake

DHA Docosahexaenoic Acid (22:6)

DHB Dihydroxybenzoic Acid

DI De-Ionozed

DEEM-FCID Dietary Exposure Evalua tion Model-Food Commodity Intake

Database

dNTP Deoxynucleotide Triphosphate

DT Defatted and Toasted (in reference to soybean meal)

DTT Dithiothreitol

DW Dry Weight
DWCF Dry Weight Conversion Factor

dwt Dry Weight of Tissue

E. coli Escherichia coli

ECL Enhanced Chemiluminescence

EDTA Ethylenediaminetetraacetic Acid

EFSA European Food Safety Authority

ELISA Enzyme-Linked Immunosorbent Assay

ELSD Evaporative Light Scattering Detector

EPA Eicosapentaenoic AcidMonsanto Company 09-SY-200U Page 24 of 395 EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase

EU European Union

FA Fatty Acid

FASTA Algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences

FDA Food and Drug Administration

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FMV Figwort mosaic virus 35S promoter

FONSI Finding of No Significant Impact

FW Fresh Weight

fwt Fresh Weight of Tissue

GLP Good Laboratory Practice

GE Genetically Engineered

GMO Genetically Modified Organism

GLA Gamma Linolenic Acid (18:3)

GOI Gene of Interest

GRAS Generally R ecognized as Safe

HPLC High Performance Liquid Chromatography

HRP Horseradish Peroxidase

HOSFO High Oleic Sunflower Seed Oil

IA Immunoaffinity

IABW Immunoaffinity Binding and Washing

IDP Identity Preserved

IFIC International Food Information Council

IgG Immunoglobulin G
ILDIS International Legume Data base and Information Service

ILSI International Life Science Institute

ILSI-CCD International Life Sciences Institute Crop Co mposition Database

IRM Insect resistance Management

ISO International Organiza tion for Standardization

IUPAC-IUB International Union of Pure and Applied Chemistry-International

Union of Biochemists

kDa Kilo Daltons

LB Loading Buffer

Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA

LOD Limit of Detection

LOQ Limit of QuantitationMonsanto Company 09-SY-200U Page 25 of 395 LA Linoleic Acid

LC Long Chain (i.e., long chain fatty acids)

MAFF Ministry of Agriculture, Forestry and Fisheries, Japan

MALDI-TOF MS Matrix-Assisted laser De sorption/ionization Time-of-Flight Mass Spectrometry
MEEC Maximum Expected En vironmental Concentration

MES 2-(N-morpholino)ethanesulfonic Acid

MH + Protonated Mass Ion

MHLW Ministry of Health, Labor and Welfare, Japan

MMT Million Metric Tons

MOE Margin of Exposure

MW Molecular Weight

MWM Molecular Weight Marker

N/A Not Applicable

Nc Δ 15D Neurospora crassa delta-15 desaturase
NDF Neutral Detergent Fiber

NEPA National Environmental Policy Act

NFDM Non-Fat Dried Milk

NI Novel Ingredient

NIST National Institute of Standards and Technology

NLT Not Less Than

NMT Not More Than

NMWC Nominal Molecular Weight Cut-Off

NOAEL No Observable Adverse Effect Level

NOEC No Observable Effect Concentration

NOEL No Observable Effect Level

NOP National Organic Program

NTO Nontarget Organism
OECD Organization for Economic Co-operation and Development

OR Origin of replication

ORF Open Reading Frame

OR -ori-pBR322 Origin of replication from pBR322 necessary for maintenance of plasmid in E. coli

OR –ori V Origin of replication from th e broad host range plasmid RK2 necessary for maintenance of plasmid in Agrobacterium

OSL Overseason Leaf

Pj Δ 6D Primula juliae delta-6 desaturase

P-7S α ′ Promoter and leader from the Sphas1 gene of soybean

P-7S α Promoter and leader from the Sphas2 gene from soybeanMonsanto Company 09-SY-200U Page 26 of 395 PAGE Polyacrylamide Gel Electrophoresis

PBS Phosphate Buffered Saline

PBST Phosphate Buffered Saline Containing 0.05% (v/v) Tween-20

PC Phosphotidyl Choline

PCR Polymerase Chain Reaction

PE Phosphotidyl Ethanolamine

PEG Polyethylene Glycol

P-FMV Promoter for the 35S RNA from Figwort Mosaic Virus (FMV)

PMSF Phenylmethanesulfonyl fluoride

POI Protein of Interest

PPA Plant Protection Act

ppm Parts Per Million

PTH Phenylthiohydantoin

PRESS Predicted Residual Sums of Squares

PVDF Polyvinylidene Difluoride

PVPP Polyvinylpolypyrrolidone

PUFA(s) Polyunsaturated Fatty Acid(s)

PV-GMPQ1972 Plasmid Vector used to develop MON 87769

RbcS4 Promoter, leader, and 5' nontranslated region of the Arabidopsis

thaliana RbcS4 gene encoding ribulose 1,5-bisphosphate carboxylase small subunit 1A

RBD Refined, Bleached, and Deodori zed (in reference to oils)

Right Border DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA

SAS Statistical Analysis System

SBO Soybean Oil (non-SDA)
SBM Soybean Meal

SCN Soybean Cyst Nematode

SD Standard Deviation

SDA Stearidonic Acid

SDA-TAG Stearidonic aci d - Triacylglycerol

SDA-EE Stearidonic acid – Ethyl ester

SDA-FA Steridonic acid – Fatty acid

SDS Sodium Dodecyl Sulfate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE Standard Error

SGF Simulated Gastric Fluid

SIF Simulated Intestinal Fluid

SMIC Soybean Meal Information CenterMonsanto Company 09-SY-200U Page 27 of 395 SOP Standard Operating Procedure

Strep Streptavidin

TDF Total Dietary Fiber

TBST Tris-Buffered Saline with 0.1% (v/v) Tween-20

T-DNA Transfer DNA

TES T hreatened or Endangered Species
TIU Trypsin Inhibitor Unit

TFA Trans Fatty Acid

TMB 3,3’,5,5’-Tetramethylbenzidene

Tris Tris (hydroxymethyl) aminomethane

TSSP Tissue-Specific Site Pool

Tween-20 Polyoxyethylenesorbitan Monolaurate

TAG Triacylglycerol, i.e., a Triglyceride

T/C/R Test, Control and Reference

TDF Total Dietary Fiber

T-DNA I Transferred DNA I

T-DNA II Transferred DNA II

T- E9 3 ′ nontranslated region of the pea rbcS2 gene that functions to direct polyadenylation of the mRNA

TG Triglyceride

TS- CTP2 Transit peptide regions of Arabidopsis thaliana EPSPS used to direct proteins into chloroplasts

T- tml 3 ′ nontranslated region of the tml gene from Agrobacterium tumefaciens octopine-type Ti plasmid that functions to direct polyadenylation of the mRNA

USDA U.S. Department of Agriculture

USDA-APHIS United States Department of Agriculture – Animal and Plant Health Inspection Service

USDA-ARS United State Department of Agriculture – Agricultural Research

Service

USDA-ERS United States Department of Agriculture – Economic Research

Service

USDA-GRIN United State Department of Agriculture – Germplasm Resources

Information Network

USDA-NASS United States Department of Agriculture – National Agricultural

Statistics Service

USDA-NSHS United States Department of Agriculture – National Seed Health System

USFWS United States Fish and Wildlife Service

v/v Volume Per VolumeMonsanto Company 09-SY-200U Page 28 of 395 w/v Weight Per VolumeMonsanto Company 09-SY-200U Page 29 of 395 I. RATIONALE FOR THE DEVELOPMENT OF MON 87769

I.A. Basis for the Request for a Dete rmination of Nonregulated Status

The Animal and Plant Health Inspection Serv ice (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility, under the Plant Protection Act (7 U.S.C. § 7701- 7772) to prevent the introduction and disseminati on of plant pests into the U.S. APHIS regulation 7 CFR § 340.6 provide s that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and no longer should be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing unrestricted

introduction of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status in whole for the ne w biotechnology-derived soybean product, MON 87769, any progeny derived from cr osses between MON 87769 and conventional soybean, and any progeny derived from crosses of MON 87769 with other biotechnology-derived soybean that has been granted nonregulated status under 7 CFR

Part 340.

I.B. Rationale for the Development of Soybean MON 87769

Monsanto has developed biotechnology-de rived soybean MON 87769 that contains stearidonic acid (SDA; 18:4), a su stainable alternate source of an omega-3 fatty acid to help meet the needed dietary intake of long chain omega-3 fatty acids. The production of SDA in MON 87769 is accomplished through the introduction of two desaturase genes, Primula juliae Δ 6 desaturase ( Pj.D6D) and Neurospora crassa Δ 15 desaturase ( Nc.Fad3) into conventional soybean, A3525. The tw o introduced genes encode for the Pj Δ 6D and

Nc Δ 15D proteins, respectively. S DA is an eighteen-carbon fa tty acid with four double bonds (18:4) and as such is found in plants, fi sh and fish/algal oil products. In mammals,

SDA is a metabolic intermedia te in the production of eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) fr om alpha linolenic acid (ALA; 18:3), a common dietary constituent. In addition to SDA, MON 87769 contains approximately 7% GLA, which is an in vivo metabolite in the conversion of linoleic acid (LA; 18:2) to arachidonic acid (20:4) in mammals (see Figure VII-1).

Although the benefits of omega-3 fatty acid consumption are widely recognized, typical Western diets contain very lit tle fish, and it is not prac tical to expect the general population to take fish oil supplements on a re gular basis. An alte rnative approach to increase omega-3 fatty acid intake is to prov ide a wider range of f oods that are enriched in omega-3 fatty acids so that people can choose foods that su it their usual dietary habits.

Human and animal studies ha ve shown that 1 g dietary SDA is approximately equivalent to 200 – 300 mg dietary EPA in terms of increasing tissue concentrations of EPA (James et al., 2003; Yamazaki et al., 1992). Thus, MO N 87769 can serve as an alternate source to help meet the needed dietary intake of long chain omega-3 fatty acids in food and feed.Monsanto Company 09-SY-200U Page 30 of 395 The oil from MON 87769 contains approximate ly 20 to 30% SDA (wt% of total fatty acids). SDA soybean oil can be used for the production of margarine, mayonnaise, shortenings, salad dressings, r eady-to-eat foods, and other food categories. The use of SDA soybean oil in selected food categorie s could provide a wide range of dietary alternatives for increasing the omega-3 fatty acid intake. Because SDA has fewer double bonds than EPA and DHA, SDA soybean oil is more stable to oxida tion (e.g., less prone to fishy or rancid odors a nd taste), thereby expanding th e potential food formulation options for food companies and food products for consumers, and serving as an

alternative to fish oils wher e they currently ar e used in food produc tion. Fish oil and plant oils rich in omega-3 fatty acids are used in feed applications, especially in aquaculture and poultry feeds. SDA soybean oil may eventually be used in aquaculture and feed applications as an alternative to fish oil and other omega-3 rich feed components.

Given the targeted commercial applications of SDA soybean oil from MON 87769 as an alternate source of omega-3 fatty acids in f ood and feed, it is anticipated that MON 87769 will be a low acreage (<5% of total U.S. soybean acreage, i.e. up to 3 – 3.5 million acres) product planned initially for production in North America. In order to derive commercial value from this product, the SDA soybean crop will be grown and processed in an identity-preserved manner (ID P) in the Northern U.S. s oybean growing region. The oil will be used in food applications and may be used in feed where omega-3 products are currently being used. The coproduct, soybean meal, has been shown to be compositionally comparable to other commodity soybean meal, and it will be used in a

manner similar to conven tional soybean meal.

I.C. Submissions to Othe r Regulatory Agencies

Under the Coordinated Framework for Regul ation of Biotechnology, the responsibility for regulatory oversight of biotechnology-derived crops falls on two federal agencies: FDA and USDA (USDA, 1986). Deregulati on of MON 87769 by USDA constitutes only one component of the overall regulatory overs ight and review of this product. As a practical matter, MON 87769 cannot be rel eased and marketed until FDA and USDA have completed their reviews and assessmen ts under their respective jurisdictions.

Submission to FDA

MON 87769 falls within the scope of th e 1992 U.S. Food and Drug Administration policy statement concerning regulation of pro ducts derived from new plant varieties, including those developed th rough biotechnology (FDA, 1992). In compliance with this policy, on March 23, 2009, Mons anto initiated consultation with the FDA on the food and feed safety and nutritional asse ssment for MON 87769 (FDA BNF 00117).

Submissions to Foreign Government Agencies

To support commercial introduction of MON 87769 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. and have establishe d regulatory approval pr ocesses in place.

These will include submissions to a number of foreign government regulatory authorities,Monsanto Company 09-SY-200U Page 31 of 395 including: Ministry of Agri culture, People’s Republic of Ch ina; Japan’s Ministry of Agriculture, Forestry, and Fi sheries (MAFF) and the Mini stry of Health, Labor, and Welfare (MHLW); the Canadian Food Inspectio n Agency (CFIA) and Health Canada; the Intersectoral Commission for Biosafety of Genetically Modified Organisms (CIBIOGEM), Mexico; the European Food Safe ty Authority (EFSA); and the regulatory authorities in other soybean importing countries with functioning regul atory systems. As appropriate, notifications of importation will be made to importing countries that do not have a formal approval process.Monsanto Company 09-SY-200U Page 32 of 395 II. THE SOYBEAN FAMILY

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 Developmen t (OECD, 2000; OECD, 2001), and 2) a summary prepared by USDA-APHIS (2006) and a biology document published by Canadian Food Inspection Agency-Pla nt Biosafety Office (CFIA, 1996),

II.A. Soybean as a Crop

Soybean is the most prevalen tly grown oilseed in the wo rld, with approximately 222.1 million metric tons of harvested seed (MMT) produced in 2007, which represented 56% of world oilseed seed production that year (ASA, 2008; Soyatech, 2008). Soybean is grown as a commercial crop in over 35 countri es. The major producers of soybean are the U.S., Brazil, Argentina, China, and Indi a, which accounted for approximately 91% of the global soybean production in 2007 (Soyatech, 2008); also see Table II-1. Approximately one-third of the 2007 world so ybean production was produced in the U.S. (Soyatech, 2008). The soybean produced in China and India are primarily for domestic use, while a significant portion of that produced in U.S., Brazil, and Argentina is traded globally in the form of soybean harvested seed , soybean meal or soybean oil. Globally, the U.S. was the largest soybean seed export country, while Argentina led the soybean meal and soybean oil export markets in 2007 (ASA, 2008; Soyatech, 2008).

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

Country Production (million metric tons)

U.S. 71.4

Brazil 61.0 Argentina 47.0

China 15.6

Other 8.9

India 7.9
Paraguay 6.2

Canada 3.1

EU 1.0 Source: Soyatech (2008).

Approximately 50% of the world soybean s eed supply was crushe d to produce soybean meal and oil in 2007 (ASA, 2008; Soyatech, 20 08), and the majority was used to supply the feed industry for livestoc k use or the food industry fo r edible vegetable oil and soybean protein isolates. A nother 34% of the world soybea n seed supply was traded to other geographies, with China, EU, Japan, a nd Mexico being the top soybean seed import geographies (ASA, 2008). The remainder of the soybean seed produced was used as certified seed, feed, or stocks.Monsanto Company 09-SY-200U Page 33 of 395 Soybean is used in various food products, including tofu, so ybean 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 30% of all the vegetable oils consumed globally, and was the second largest source of vegetable oil worldwide, slightly behind palm oil at approximately 32% share (Soyatech, 2008).

Soybean meal is used as a 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. By far, soybean meal is the world's most important protein feed, accounting for nearly 69% of world prot ein meal supplies (ASA, 2008). Industrial uses of soybean range from a carbon/nitroge n source in the production of yeasts via fermentation to the manufacture of soaps, in ks, paints, disinfectan ts, and biodiesel. Industrial uses of soybean have been summarized by Cahoon (Cahoon, 2003) and the American Soybean Association (ASA, 2008).

Global soybean plantings reached 90.8 milli on hectares in 2007/08, an 8.9% increase over the previous four years with an av erage of 82.3 million hectares planted from 2002/03 – 2007/08 (Soyatech, 2008). Soybean production has realized, on average, a 6.2% annual growth between 1995/96 to 20 06/07. Increased planting flexibility, increased yield from narrow-row seeding pr actices, a higher rate of corn-soybean rotations, and low production co sts favored expansion of soybean areas in the mid-1990s, and the expanded areas tended to be concentrat ed where soybean yields were highest.

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 only co uld be traced back to the Zhou dynasty (1027 to 221 B.C.) where the soybean was utilized as a domesticated crop in the northeastern part of China. By the first century A. D., the soybean probably reached Central and Southern China as well as pe ninsular Korea. The movement of soybean germplasms was probably associated with the development and consolidation of territories and the degeneration of Chinese dynasties (Ho, 1969; Hymowitz, 1970).

From the first century A.D. to the 15th and 16th centuries, soybean was introduced into several countries, with land r aces eventually developing in Japan, Indonesia, Philippines, Vietnam, Thailand, Malaysia, Myanmar, Nepal, and Northern India. The movement of soybean throughout this period was due to the establishment of sea and land trade routes, the migration of certain tribes from China, and the rapid acceptance of seeds as a staple food by other cultures (Hymowitz and Ne well, 1981; Hymowitz et al., 1990).

Starting in the late 16th cen tury and through the 17th cent ury, soybean was used by the Europeans, and in the 17th century, soybean sauce was a common item of trade from the

East to the West.Monsanto Company 09-SY-200U Page 34 of 395 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 seed 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 seed to dozens of growers throughout the U.S. Soybean has been cultivated extensivel y and improved through conventional breeding programs following its introduction in the U. S. and subsequently has become a key

source of nutrients for food and feed use in the U.S. (Hymowitz and Singh, 1987).

II.C. Taxonomy and Phylogenetics of Soybean

Cultivated soybean, Glycine max (L.) Merr., is a diploidize d tetraploid (2n=40), which belongs to the family Leguminosae, the subf amily Papilionoideae, the tribe Phaseoleae, the genus Glycine Willd., and the subgenus Soja (Moench) F.J. Herm.

Family: Leguminosae Subfamily: Papilionoideae

Tribe: Phaseoleae

Genus: Glycine Subgenus: Soja (Moench) F.J. Herm. Species: max

The genus Glycine Willd. is of Asian and Australian origin and is divided into two subgenera, Glycine and Soja (Moench) F.J. Herm. The subgenus Glycine consists of 22 wild perennial species, which are 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 09-SY-200U Page 35 of 395 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 09-SY-200U Page 36 of 395 Glycine soja grows wild in China, Japan, Korea, th e 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 sh ort, slender racemes. The pods are short and tawny with hirsute pubescence, producing oval- oblong seeds (Hermann, 1962).

Glycine max (L.) Merr., the cultivated soybean, is an annual that generally exhibits an erect, sparsely branched, bush-type growth 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 stra ight or slightly curved, and one to three ovoid to sub-spherical seeds are produced per pod.

A third and unofficial species named G. gracilis is also described within the context of

the Soja subgenus in addition to G. soja and G. max . The G. gracilis is known only from Northeast China, is interm ediate in morphology between G. max and G. soja , and is

sometimes considered a variant of G. max . The three species in the Soja subgenus can cross-pollinate, and the hybrid seed can germinate normally and subsequently produce fertile pollen and seed (Singh and 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 fo r outcrossing (USDA-APHIS, 2006).

II.D. The Genetics of Soybean

Glycine is the only genus in the tribe Phaseoleae where species have diploid chromosome numbers of 40 and 80, but not 20 (Lackey, 1981) . The unique chromosome number of Glycine is probably derived from diploid ancestors with a 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 extinct) 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 tetrap loid with diploidized genome (Gurley, 1979; Lee and Verma, 1984; Skorupska, 1989).

II.E. Pollination of Cultivated Soybean

Soybean is largely a self-pollinated speci es, 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 09-SY-200U Page 37 of 395 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 (0.3 to 3.62%) ( Beard and Knowles, 1971 ). Pollination typically takes place on the day the flower ope ns. 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. Th e pollen usually remains viable for two to four hours, and no viable polle n can be detected by late af ternoon. Natural or artificial cross-pollination can only take place during the short time wh en 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 they did become established, volunt eer plants would not compete well with the succeeding crop, and could be controlled readily by either mechanical or chemical means (OECD, 2000).

II.G. Characteristics of the Recipient Plant

The soybean variety used as the recipien t for the DNA insertion to create MON 87769 was A3525, a nontransgenic conventional variet y developed by Asgrow Seed Company. A3525 is a mid-maturity group I II soybean variety with very high yield potential. It has superior yields relative to lines of similar maturity and has excellent agronomic characteristics (Steffen, 2004).

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

In developing the data to support the sa fety assessment of MON 87769, A3525 was used as the nontransgenic conven tional soybean comparator. In general, the genetic background of MON 87769 was matched with that of the control so the effect of the genetic insertion and the presence of the Pj Δ 6D and Nc Δ 15D proteins could be assessed

in an unbiased manner. Since MON 87769 was derived from the A3525 conventional variety, it was deemed appropriate to us e the nontransformed A3525 as the control variety because its use would minimize the pot ential bias in subsequent comparative assessments. In addition, commercial convent ional varieties were used as reference materials to establish ranges of responses or values representative of commercial soybean

varieties. Reference varieties were select ed based on prevalence and performance of the soybean varieties at each trial location. As a general principle, vari eties that were well adapted to the local environments and c ould be used by the local producers were considered for use as reference soybean varieties.Monsanto Company 09-SY-200U Page 38 of 395 III. DESCRIPTION OF THE TRANSFORMATION SYSTEM

MON 87769 was developed through Agrobacterium -mediated transformation of soybean meristem tissue using plasmid vector PV -GMPQ1972 (Section IV.A , Figure IV-1 and Table IV-1). Agrobacterium -mediated transformation is a well-documented process for the transfer and integration of exogenous DNA into a plant’s nuclear genome (Bevan, 1984). PV-GMPQ1972 contains two separate T-DNAs (herein referred to as a 2T-DNA system). The first T-DNA, designated as T- DNA I, contains two expression cassettes:

the Pj.D6D gene expression cassette and the Nc.Fad3 gene expression cassette. The second T-DNA region (T -DNA II) contains the cp4 epsps gene expression cassette that encodes the CP4 EPSPS protei n (5-enolpyruvylshikimate-3- phosphate synthase protein

from Agrobacterium sp. strain CP4) that pr ovides tolerance to th e action of glyphosate,

which is the active ingredient in Roundup ® agricultural herbicides.

The use of the 2T-DNA system is the basis for an effective approach to generate markerfree plants. It allows for insertion of th e T-DNA with the traits of interest (e.g., T-DNA I) and the T-DNA encoding the selectable marker (e.g., cp4 epsps , T-DNA II) into two independent loci with in the genome of the plant. Following selection of the transformants that contain both T-DNAs, th e inserted T-DNA encoding the selectable marker (e.g., T-DNA II) can be segregated in progeny through subs equent selfing of plants (self-pollination) and genetic selecti on, while progeny with the T-DNA containing the trait(s) of interest are maintained (e.g., T-DNA I). This 2T-DNA binary vector approach has been successfullyused in tobacco (Komari et al., 1996), soybean (Xing et al., 2000), barley (Matthews et al ., 2001), corn (Miller et al., 2002), and rice (Breitler et al., 2004; Komari et al., 1996) . MON 87769 was developed using such 2T-DNA vector transformation and selection techniques.

The Agrobacterium -mediated soybean transformati on to produce MON 87769 was based on published methods that generate transfor med plants without u tilization of callus (Armstrong et al., 2007; Martinell et al., 2002). Agrobacterium tumefaciens strain ABI contains a disarmed Ti plasmid that is incap able of inducing tumor formation due to the deletion of the phytohormone gene s originally present in the Agrobacterium plasmid (Koncz and Schell, 1986). Th e transformation vector, PV -GMPQ1972, contains both the left and right border sequences flanking th e transfer DNA (T- DNA) to facilitate

transformation.

Embryos were excised from germinated A 3525 seed and meristem atic tissues were targeted for transformation. After co-culturing this tissue with the Agrobacterium carrying the vector, the meristems were placed on selection medium containing glyphosate to inhibit the growth of untransf ormed plant cells, and antibiotics carbenicilin

and claforan to inhibi t the growth of excess Agrobacterium , respectively. The meristems then were placed on media conducive to s hoot and root development, and only rooted

® Roundup is a registered tradem ark of Monsanto Technology LLC.Monsanto Company 09-SY-200U Page 39 of 395 plants with normal phenotypic characteristics were selected and transferred to soil for growth and further assessment.

The R0 plants generated through the aforem entioned transformation system were selfpollinated, and the subsequent R1 plants were screened for the single insert present in MON 87769 using Invader (Third Wave Technologies, Inc.) and Southern blot analysis. Invader is a non-PCR based assay that can be used to accurately qua ntify transgene copy number in plant genomes (Gupta et al., 2008) . The R1 plants containing the T-DNA I

cassette for the Pj.D6D and Nc.Fad3 genes of interest (GOI) and not having the

T-DNA II cassette with the cp4 epsps gene cassette were advanced for further evaluation and development. Each R1 plant was self- pollinated to generate a population of R2 plants that were repeatedly self-pollinated through the R6 generation. These progeny were subjected to further molecular (Sout hern blot) and phenotypic assessments.

MON 87769 was selected as the lead event based on superior phenotypic characteristics and molecular profile. Re gulatory studies on MON 87769 were initiated to further characterize the genetic inse rtion and the expressed protei ns, and to confirm the food, feed, and environmental safety relative to conventional soybean. The major steps involved in the development of MO N 87769 are depicted in Figure III-1.Monsanto Company 09-SY-200U Page 40 of 395 Figure III-1. Schematic of the Development of MON 87769 Assembled Agrobacterium binary plasmid vector PV-GMPQ1972 and transferred to Agrobacterium

tume f

aciens, strain ABI

Transformed A3525 (a conventional soybean variety) meristematic tissue with the plasmid vector PV-GMPQ1972 in Agrobacterium tumefaciens

Selected transformants and generated rooted shoots

from the transformed meristematic tissues

Introgressed MON 87769 into other germplasms and evaluated these for commercial performance Conducted characterization and safety studies Evaluated the transformed plan ts for insert integrity and SDA production, and selected homozygous plantsMonsanto Company 09-SY-200U Page 41 of 395 IV. GENETIC ELEMENTS

This section describes the vector, the donor genes and the regulatory sequences used in the development of MON 87769, the deduced amino acid sequence of the Pj Δ 6D and

Nc Δ 15D proteins produced in MON 87769, and a description of the CP4 EPSPS protein selectable marker employed to produce M ON 87769. In this section, T-DNA refers to

DNA that is transferred to th e plant during transformation. An expression cassette is comprised of a coding sequence and the regulat ory elements necessary for the expression of the coding sequence.

IV.A. Plasmid PV-GMPQ1972

This section describes the donor genes and re gulatory sequences used in the development

of MON 87769 and the deduced amino acid sequences of the Primula juliae Δ 6

desaturase (Pj ∆ 6D) and Neurospora crassa Δ 15 desaturase (Nc ∆ 15D) proteins produced

in MON 87769.

The plasmid vector PV-GMPQ1972 used to transform soybean meristems to produce MON 87769 is shown in Figure IV-1. The v ector is an approximately 16.5 kb plasmid containing two T-DNA regions, each delineated by a left a nd right border region. The first T-DNA (T-DNA I) region contai ns two expression cassettes: the Pj.D6D gene expression cassette and the Nc.Fad3 gene expression cassette. The second T-DNA region (T-DNA II) contains the cp4 epsps gene expression cassett e that was used for early event selection and wa s segregated away from T- DNA I by conventional breeding (self-pollination). The T-DNA II elements ar e therefore not present in MON 87769 other than those common elements that are also present in T-DNA I. The T-DNA present in MON 87769 (T-DNA I) is approximately 7.4 kb, and the DNA backbone and T-DNA II region that is not incorpor ated into the soybean genome is approximately 9.1 kb.

The Pj.D6D expression cassette consists of the Pj.D6D coding sequence under the regulation of the 7S α ′ promoter and the tml 3' nontranslated sequence. The Nc.Fad3 expression cassette consists of the Nc.Fad3 coding sequence under the regulation of the

7S α promoter and the E9 3' nontranslated region. The cp4 epsps expression cassette in T-DNA II, which is not present in MON 87769, consists of the cp4 epsps coding region under the regulation of the FMV promoter and the E9 3 ′ nontranslated region.

The backbone region outside of the T-DNA c ontains two origins of replication for maintenance of plasmid in bacteria (OR- ori V, OR- ori- pBR322), as well as a bacterial selectable marker gene ( aadA ). A description of the genetic elements and their prefixes (e.g., P-, L-, I-, TS-, OR-, B-, CS-, and T-) in PV-GMPQ1972 is provide d in Table IV-1.

IV.B. T-DNA I

This section describes the elements containe d on T-DNA I that were integrated into the soybean genome to produce MON 87769.Monsanto Company 09-SY-200U Page 42 of 395 IV.B.1. The Pj.D6D Coding Sequence and Pj ∆ 6D Protein

The Pj.D6D gene was isolated from Primula juliae (Primrose) and encodes a single polypeptide, designated Pj Δ 6D, of 446 amino acids (Ursin et al., 2008). The deduced full-length amino acid sequence is shown in Figure IV-2. The Pj Δ 6D protein is a Δ 6

desaturase which creates a double bond at the 6 th position from the ca rboxyl end of a fatty

acid.

IV.B.2. The Pj.D6D Regulatory Sequences

Adjacent to the right border regi on of plasmid PV-GMPQ1972 is the Pj.D6D expression

cassette. The Pj.D6D coding sequence is under the regulatory control of the P- 7S α ' transcriptional promoter. P- 7S α ' is a seed-specific promoter and leader sequence from

the Sphas1 gene encoding the alpha prime subunit of the beta-conglycinin storage protein

of Glycine max (Doyle et al., 1986). Following the Pj.D6D coding sequence is the 3' nontranslated region of the tml gene derived from the oc topine-type Ti plasmid of Agrobacterium tumefaciens (T- tml) (Kemp et al., 2000) that directs transcriptional termination and polyadenylation of the Pj.D6D mRNA.

IV.B.3. The Nc.Fad3 Coding Sequence and Nc ∆ 15D Protein

The Nc.Fad3 gene was isolated from Neurospora crassa and encodes a single polypeptide, designated Nc Δ 15D, of 429 amino acids (Ursin et al., 2006). The deduced full-length amino acid sequence is shown in Figure IV-3. The Nc Δ 15D protein is a Δ 15

desaturase which creates a double bond at the 15 th position from the carboxyl end of a fatty acid.

IV.B.4. The Nc.Fad3 Regulatory Sequences

Adjacent to the Pj.D6D expression cassette is the Nc.Fad3 expression cassette. The

Nc.Fad3 coding sequence is under the re gulatory control of the P- 7S α promoter. P- 7S α is a seed-specific promoter and leader sequence from the Sphas2 gene that encodes the alpha subunit of beta-conglycinin seed storage protein in Glycine max (Wang and Dubois, 2004). Following the Nc.Fad3 coding sequence is the 3' nontranslated region of the pea (Pisum sativum ) ribulose-1,5-bisphosphate carboxylase, small subunit ( rbcS2 ) gene (T- E9 ) (Coruzzi et al., 1984) that direct s transcriptional termination and polyadenylation of the Nc.Fad3 mRNA.

IV.B.5. The T-DNA Borders

Plasmid vector PV-GMPQ1972 contains righ t border and left border regions that

delineate the T-DNAs and are involved in thei r efficient transfer into soybean genome. These border regions (Figure IV-1; Table IV-1) were derived from Agrobacterium tumefaciens plasmids 

### ( Barker et al., 1983; De picker et al., 1982 

### )

.Monsanto Company 09-SY-200U Page 43 of 395 IV.C. T-DNA II

This section describes the elements containe d on T-DNA II that were segregated away in progeny, through subsequent selfing and genetic selection, to produce MON 87769.

IV.C.1. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein

The cp4 epsps gene from Agrobacterium sp. strain CP4, a common soil-borne bacterium, has been sequenced and shown to encode a 47.6 kDa EPSPS prot ein consisting of a single polypeptide of 455 amino acids 

### ( Padgette et al., 1996 

### )

.

IV.C.2. The Arabidopsis thaliana EPSPS Transit Peptide

The cp4 epsps coding sequence is preceded by a ch loroplast transit peptide sequence, (TS- CTP2) , derived from the Arabidopsis thaliana epsps gene (Klee et al., 1987). This transit peptide directs the transport of the CP 4 EPSPS protein to the chloroplast, which is where the plant EPSPS resides and the site of aromatic amino acid biosynthesis (Kishore et al., 1988; Klee et al., 1987). Tr ansit peptides are typically cleaved from the translated polypeptide following delivery to the pl astid (Della-Cioppa et al., 1986).

IV.C.3. The cp4 epsps Regulatory Sequences

The CTP2/ cp4 epsps gene cassette that produces the CT P/CP4 EPSPS protein consists of the promoter (P- FMV) for the 35S RNA from figwor t mosaic virus (FMV) (Rogers, 2000) that directs transcription in plant cells. Located between the P- FMV promoter and

the CTP2/cp4 epsps coding sequence is the 5' nontranslated ShkG leader sequence from

the Arabidopsis ShkG gene encoding EPSPS (Klee et al., 1987). Following the CTP2/cp4

epsps coding sequence is the T- E9 element

, the 3' nontranslated region of the pea ( Pisum

sativum ) ribulose-1,5-bisphosphate carboxyl ase, and the small subunit ( rbcS2 ) E9 gene (Coruzzi et al., 1984) that di rects transcriptional termina tion and polyadenylation of the CTP2/cp4 epsps mRNA.

The unlinked T-DNA II insertion was eliminat ed as a result of segregation during breeding. T-DNA II elements are not presen t in MON 87769 other than those common elements that are also part of T-DNA I. This has been confirmed by Southern blot analyses, which are presented in Section V.C.

IV.C.4. The T-DNA Borders

The right and left T-DNA borders used in T-DNA II are the same as those described

under Section IV.B.5.

IV.D. Genetic Elements Ou tside of the T-DNA Borders

Four genetic elements exist outside of th e T-DNA borders that are essential for the

maintenance and selection of the vector PV-GMPQ1972 in bacteria. They include:
OR-ori V, origin of replication for maintenance of plasmid in Agrobacterium (Stalker et al., 1981); CS- rop, coding sequence for r epressor o f p rimer (ROP) protein forMonsanto Company 09-SY-200U Page 44 of 395 maintenance of plasmid copy number in E. coli (Giza and Huang, 1989); OR-oripBR322

, origin of replication from pBR322 for maintenance of plasmid in E. coli (Sutcliffe, 1978); and aadA , a bacterial promoter and coding sequence of an enzyme from transposon Tn7 that confers spectinomycin and streptomycin resistance for molecular cloning and selection purposes (Fling et al., 1985).

Because these elements are outside of the bor der regions, they are not expected to be transferred into the soybean genome. Th e absence of the backbone sequence in MON 87769 has been confirmed by Southern blot analyses, which are presented in

Section V.B.Monsanto Company 09-SY-200U Page 45 of 395 Figure IV-1. Plasmid Ma p of Vector PV-GMPQ1972 Plasmid PV-GMPQ1972 was used in Agrobacterium -mediated transformation to develop MON 87769. Approximate locations of the gene tic elements (with approximate positions relative to the plasmid vector) are shown on the exterior of th e map. PV-GMPQ1972 contains two T-DNA regions design ated as T-DNA I and T-DNA II.Monsanto Company 09-SY-200U Page 46 of 395 Table IV-1. Summary of Genetic Elem ents in Plasmid Ve ctor PV-GMPQ1972

Genetic Element Position in

Plasmid Function and Source (Reference)

T-DNA I (continued from bp 16465) Intervening Sequence 1–14 Sequence used in DNA cloning

B 1 -Left Border 15-456 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of T-DNA (Barker et al., 1983)

Vector Backbone Intervening Sequence 457–1619 Sequence used in DNA cloning

CS 2

- rop 1620-2092 Coding sequence for repressor of primer protein from the ColE1 plasmid for maintenance of plasmid copy number in E. coli (Giza and Huang, 1989) Intervening Sequence 2093-2340 Sequence used in DNA cloning

OR 3

- ori-pBR322 2341-2969 Origin of replication from pBR322 for maintenance of plasmid in E. coli (Sutcliffe, 1978) Intervening Sequence 2970-3469 Sequence used in DNA cloning

aadA 3470-4358 Bacterial promoter and coding sequence for anaminoglycoside-modifying enzyme, 3’(9)-Onucleotidyltransferase from the transposon Tn7 (Fling et al., 1985) that confers spectinomycin and streptomycin resistance Intervening Sequence 4359-4494 Sequence used in DNA cloning T-DNA II (not present in MON 87769) B-Right Border 4495-4851 DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA (Depicker et al., 1982) Intervening Sequence 4852-4884 Sequence used in DNA cloning

P 4

- FMV 4885-5448 Promoter for the 35S RNA from figwort mosaic virus (FMV) (Rogers, 2000) that directs transcription in plant cells Intervening Sequence 5449-5491 Sequence used in DNA cloning

L 5

- ShkG 5492–5558 5' nontranslated leader sequence from the Arabidopsis ShkG gene encoding EPSPS (Klee et al., 1987) that helps regulate gene expression

TS 6

– CTP2 5559–5786 Transit peptide region of Arabidopsis thaliana EPSPS (Klee et al., 1987) that directs transport of the CP4 EPSPS protein to the chloroplast

CS- cp4 epsps 5787-7154 Codon modified coding sequence of the aroA gene from the Agrobacterium sp. strain CP4
encoding the CP4 EPSPS protein 

### ( Barry et al.,
1997; Padgette et al., 1996 

### )
Intervening Sequence 7155-7196 Sequence used in DNA cloning

1 B – Border; 2 CS – Coding Sequence; 3 OR – Origin of Replication; 4 P – Promoter; 5 L-Leader ;
6 TS – Targeting Sequence.Monsanto Company 09-SY-200U Page 47 of 395 Table IV-1 (continued). Summary of Geneti c Elements in Plasmid Vector PV-GMPQ1972

T-DNA II (cont.)

T 7

- E9 7197-7839 A 3' nontranslated region of the pea rbcS2 gene which functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984) Intervening Sequence 7840-7886 Sequence used in DNA cloning

B-Left Border 7887-8328 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of T-DNA (Barker et al., 1983)

Vector Backbone Intervening Sequence 8329-8414 Sequence used in DNA cloning

OR- ori V 8415-8811 Origin of replication from the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium (Stalker et al., 1981) Intervening Sequence 8812-9072 Sequence used in DNA cloning

T-DNA I B-Right Border 9073-9429 DNA area from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA (Depicker et al., 1982) Intervening Sequence 9430-9480 Sequence used in DNA cloning

P- 7S α ′ 9481-10321 Promoter and leader from the Sphas1 gene of Glycine

max encoding beta-conglycinin storage protein (alpha'- bcsp) (Doyle et al., 1986) that directs mRNA transcription in seed Intervening Sequence 10322-10337 Sequence used in DNA cloning

CS- Pj.D6D 10338-11678 Coding region for the Δ 6 desaturase from Primula juliae (Ursin et al., 2008) Intervening Sequence 11679-11686 Sequence used in DNA cloning

T- tml 11687-12636 3' nontranslated region of the tml gene from Agrobacterium tumefaciens octopine-type Ti plasmid (Kemp et al., 2000) that directs polyadenylation of

the mRNA Intervening Sequence 12637-12737 Sequence used in DNA cloning

P- 7S α 12738-14417 Promoter and leader from the Sphas2 gene fromsoybean encoding the alpha subunit of betaconglycinin (Wang and Dubois, 2004) that directs mRNA transcription in seed Intervening Sequence 14418-14445 Sequence used in DNA cloning

CS- Nc.Fad3 14446-15735 Codon optimized coding sequence for the gene from
Neurospora crassa encoding Δ 15 desaturase (Ursin et al., 2006) Intervening Sequence 15736-15787 Sequence used in DNA cloning

T-

E9 15788-16430 3' nontranslated region of the pea rbcS2 gene which functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984) Intervening Sequence 16431-16465 Sequence used in DNA cloning

7 T – Transcript Termination Sequence and polyadenylation signal sequences.Monsanto Company 09-SY-200U Page 48 of 395 1 MTKTIYITSS ELEKHNKPGD LWISIHGQVY DVSSWAALHP GGIAPLLALA GHDVTDAFLA

61 YHPPSTSRLL PPFSTNLLLE KHSVSETSSD YRKLLDSFHK MGMFRARGHT AYATFVIMIL
121 MLVSSVTGVL CSENPWVHLV CGAAMGFAWI QCGWIGHDSG HYRIMTDRKW NRFAQILSSN
181 CLQGISIGWW KWNHNAHHIA CNSLEYDPDL QYIPLLVVSP KFFNSLTSRF YDKKLNFDGV
241 SRFLVQYQHW SFYPVMCVAR LNMLAQSFIL LFSRREVANR VQEILGLAVF WLWFPLLLSC
301 LPNWGERIMF LLASYSVTGI QHVQFSLNHF SSDVYVGPPV GNDWFKKQTA GTLNISCPAW
361 MDWFHGGLQF QVEHHLFPRM PRGQFRKISP FVRDLCKKHN LTYNIASFTK ANVLTLETLR

421 NTAIEARDLS NPIPKNMVWE AVKNVG

Figure IV-2. Deduced Amino Acid Sequence of Primula juliae Δ 6 Desaturase from PV-GMPQ1972 The amino acid sequence of the Pj Δ 6D protein was deduced from the full-length Pj.D6D coding sequence present in PV-GMPQ1972.

1 MAVTTRSHKA AAATEPEVVS TGVDAVSAAA PSSSSSSSSQ KSAEPIEYPD IKTIRDAIPD
61 HCFRPRVWIS MAYFIRDFAM AFGLGYLAWQ YIPLIASTPL RYGAWALYGY LQGLVCTGIW

121 ILAHECGHGA FSRHTWFNNV MGWIGHSFLL VPYFSWKFSH HRHHRFTGHM EKDMAFVPAT
181 EADRNQRKLA NLYMDKETAE MFEDVPIVQL VKLIAHQLAG WQMYLLFNVS AGKGSKQWET
241 GKGGMGWLRV SHFEPSSAVF RNSEAIYIAL SDLGLMIMGY ILYQAAQVVG WQMVGLLYFQ
301 QYFWVHHWLV AITYLHHTHE EVHHFDADSW TFVKGALATV DRDFGFIGKH LFHNIIDHHV
361 VHHLFPRIPF YYAEEATNSI RPMLGPLYHR DDRSFMGQLW YNFTHCKWVV PDPQVPGALI
421 WAHTVQSTQ

Figure IV-3. Deduced Amino Acid Sequence of Neurospora crassa Δ 15 Desaturase from PV-GMPQ1972
The amino acid sequence of the Nc Δ 15D protein was deduced from the full-length Nc.Fad3 coding sequence present in PV-GMPQ1972.Monsanto Company 09-SY-200U Page 49 of 395 V. GENETIC ANALYSIS

This section details the molecular analyses us ed to characterize the integrated DNA insert in MON 87769. Southern blot analysis confir med the presence of a single insertion site for each genetic element present in T-DNA I, the absence of plasmid backbone and T-DNA II elements, and the insert stability across generations. In addition, DNA sequencing analyses confirmed the nucleotide sequence and the expected organization of the insert in MON 87769. A comparison of the soybean genomic DNA flanking the insert in MON 87769 to the sequence of the insertion site in conventional soybean demonstrated that no major rearrangements occurred at the insertion site during transformation. Furthermore, insert segregati on analysis shows that the insert segregates as expected indicating that the Pj.D6D and Nc.Fad3 gene cassettes segregate according to Mendel’s laws of genetics. All these results indicate a single detectable chromosomal

insertion of T-DNA I in MON 87769.

Genomic DNA from MON 87769 was digested with appropriate restri ction enzymes and subjected to Southern blot analyses to ch aracterize the T-DNA that was integrated into the soybean genome. Genomic DNA from c onventional soybean (A3525) was used as a negative control to de termine potential nonspecific hybridiz ation signals and to establish hybridization signals to e ndogenous soybean DNA sequences since several genetic

elements in T-DNA I were derived from so ybean. There were two types of positive controls used. For all Southern blots, PV-GMPQ1972 plasmid DNA was digested with a restriction enzyme or enzyme combinati on to produce DNA banding patterns that were relevant to the molecular assessment of MON 87769. Additionally, in some analysis where multiple probes were used, probe templates were used as positive hybridization

controls for Southern blots. DNA markers were included to provide size estimation of the hybridized bands on all Southern blots. The sizes of bands present in the short run lanes were estimated using the molecular weight markers on the right side of the figures, whereas size estimations for the long run lane s were obtained using the molecular weight markers on the left side. The genetic elem ents present in MON 87769 are listed in Table V-1 starting at partial Right Bord er and ending at partial Left Border. The probes used in the Southern analyses and the map of the plasmid (PV-GMPQ1972) used in the transformation to generate MON 87769 are show n in Figures V-1 and V-2, respectively. The information and results derived from the molecular sequence analysis were used to construct a linear map of the insert in M ON 87769. This linear map depicts restriction

sites identified in the insert and the s oybean genomic DNA flanking the insert, and provides information on the expected bandi ng patterns and sizes of the DNA fragments after restriction enzyme digestions. The linear map is shown in Figure V-3. A table based on the insert linear map and the pl asmid map, summarizing the expected DNA fragments for Southern analyses, is presente d in Table V-2. The materials and methods used in the analyses are presented in Appendix B.Monsanto Company 09-SY-200U Page 50 of 395 Table V-1. Summary of Genetic Elements in MON 87769

Genetic Element 1 Location in Sequence 1 Function (Reference) Sequence flanking 5'

end of the insert 1-933 Soybean genomic DNA

2 B-Right Border 934-976 DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA (Depicker et al., 1982) Intervening Sequence 977-1027 Sequence used in DNA cloning

3 P -7S α ′ 1028-1868 Promoter and leader from the Sphas1 gene of Glycine max encoding beta-conglycinin storage protein (alpha'-bcsp) (Doyle et al., 1986) that directs mRNA transcription in seed Intervening Sequence 1869-1884 Sequence used in DNA cloning

4 CS

- Pj.D6D 1885-3225 Coding region for the fatty acid Δ 6 desaturase from Primula juliae (Ursin et al., 2008) Intervening Sequence 3226-3233 Sequence used in DNA cloning

5 T- tml 3234-4183 3 ′ nontranslated region of the tml gene from Agrobacterium tumefaciens octopine-type Ti plasmid (Kemp et al., 2000) that directs polyadenylation of the mRNA Intervening Sequence 4184-4284 Sequence used in DNA cloning

P- 7S α 4285-5964 Promoter and leader from the Sphas2 gene fromsoybean encoding the alpha subunit of betaconglycinin (Wang and Dubois, 2004) that directs mRNA transcription in seed Intervening Sequence 5965-5992 Sequence used in DNA cloning

CS- Nc

. Fad3 5993-7282 Codon optimized coding sequence for the gene from Neurospora crassa encoding delta-15 desaturase (Ursin et al., 2006) Intervening Sequence 7283-7334 Sequence used in DNA cloning

T- E9 7335-7977 3 ′ nontranslated region of the pea rbcS2 gene which functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984) Intervening Sequence 7978-8026 Sequence used in DNA cloning

B-Left Border 8027-8300 DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al., 1983) Sequence flanking 3'

end of the insert 8301-9131 Soybean genomic DNA

1 Numbering refers to the sequence that includ es the insert in MON 87769 and adjacent genomic DNA starting with 933 bp flanking the 5’ of the se quence and ending with 831 bp flanking 3’ end

of the insert.

2 B

– Border; 3 P

– Promoter; 4 CS

– Coding Sequence; 5 T – Transcript Termination Sequence and polyadenylation signal sequences.Monsanto Company 09-SY-200U Page 51 of 395 Figure V-1. Plasmid Map of Vector PV GMPQ1972 Showing Probes 1-12 PV-GMPQ1972 was used to develop MON 87769. Approximate locations of the genetic elements and in the Southern analyses (with approximate positions relative to the plasmid vector) are shown on the exterior of the map. The probes used in the Southern analyses are detailed in Table V-2. PV-GMPQ1972 contains two T-DNA re gions designated as

T-DNA I and T-DNA II.Monsanto Company 09-SY-200U Page 52 of 395 Figure V-2. Plasmid Map of Vector PV-GMPQ1972 Showing Probes 13-18 Plasmid PV-GMPQ1972 was used to develop MON 87769. Genetic elements and restriction sites for enzymes used in the S outhern analyses (with approximate positions relative to the plasmid vector) are shown on the exterior of the map. The probes used in the Southern analyses are detailed in the accompanying Table V-2. PV-GMPQ1972 contains two T-DNA regions design ated as T-DNA I and T-DNA II.Monsanto Company 09-SY-200U Page 53 of 395Figure V-3. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 87769A linear map of the insert and genomicDNA flanking the insert in MON 87769 is shown. The upper portion of the figuredisplays genetic elements within the insert(rectangular bar), as well asall restriction sites used in Southern blot analyses.The numbered designation of the restriction sites are consistent with the information presented in Table V-1. Arrowsunderneath the designated insert indicate the direction of transcription. Shown on the lower portion of the map are theexpected sizes of the DNA fragments after digestions with respective restriction enzyme or combination of enzymes. Thetwo large arrows above the designated insert indicate the end of the insert and the beginning of soybean genomic flankingsequence.BsrG I/PshA I> 9.2 kb6.8 kb 1.6 kb2.0 kb 1.9 kb 4.2 kbLguI 121BsrG I 30415' Genomic Flank DNA P- 7S α

' CS- Pj.D6D

T- tml P- 7S α T- E9

B-Right Border B-Left BorderBstX I 691292943' Genomic Flank DNAPshA I 2282PshA I 4167PshA I 8416LguILguI/BstX IBstX I 8467

CS- Nc.Fad3BsrG I/PshA I> 9.2 kb6.8 kb 1.6 kb2.0 kb 1.9 kb 4.2 kbLguI 121BsrG I 30415' Genomic Flank DNA P- 7S α

' CS- Pj.D6D

T- tml P- 7S α T- E9

B-Right Border B-Left BorderBstX I 691292943' Genomic Flank DNAPshA I 2282PshA I 4167PshA I 8416LguILguI/BstX IBstX I 8467

CS- Nc.Fad3Monsanto Company 09-SY-200U Page 54 of 395Table V-2. Summary Chart of the Expected Sizes of DNAFragments Using Combinationsof Restriction Enzymes andProbesProbes used13 - 1516 – 187891011121-3, 64 and 5Southern blotin FigureV-4a andV-14a*V-4b andV-14b*V-7V-8V-9V-10V-11V-12V-5 andIV-15*V-6 andV-16*PlasmidBamHI1.3, 1.9,2.3, 3.1,and 7.9 kb2.3, 3.1,and 7.9 kb1.3, 2.3,and 7.9kb1.3 and1.9 kb1.9 kb3.1 kb3.1 kb2.3 and7.9 kb2.3 and7.9 kb2.3 and7.9 kbProbe templates1.6, 1.7,and 2.0 kb0.8, 1.0,and 1.3 kb--1----------0.7, 0.9,1.5, and1.8 kb2.0, and1.9 kbMON 87769LguI> 9.2 kb> 9.2 kb--------------LguIandBstXI6.8 kb6.8 and1.6 kb6.8 kb6.8 kb6.8 kb6.8 kb6.8 and1.6 kb1.6 kbNE26.8 and1.6 kbBsrG IandPshA I----2.0 kb1.9 and2.0 kb1.9 and4.2 kb4.2 kb4.2 kb4.2 kbNE22.0 and4.2 kb1‘--’ indicates that the particular restriction enzyme orcombination of enzymes was not used in the analysis.2‘NE’ indicates that no DNA band was expected or detected.*Southern blots not conducted with material digested withBsrG Iand PshA I.Monsanto Company 09-SY-200U Page 55 of 395 Southern blot analysis confir med the presence of a single insertion site for each genetic element present in T-DNA I, confirmed the lack of plasmid backbone elements and absence of T-DNA II, and the insert stabil ity across generations . In addition, DNA sequencing analyses confirmed the expected organization and nucleot ide sequence of the

insert in MON 87769. Furtherm ore, insert segregation an alysis confirmed that the Pj.D6D and Nc.Fad3 gene cassettes segregate according to Mendel’s laws of genetics.

All these results indicate a si ngle detectable chromosomal insertion of the T-DNA I in

MON 87769.

V.A. Insert and Copy Number Determination

The number of T-DNA inserts (inser t number) in the MON 87769 genome was determined by Southern blot analysis using MON 87769 and conventional soybean DNA that was digested with the restriction enzyme Lgu I. Lgu I does not cleave within T-DNA I and, therefore, releas es a restriction fragment containing the entire T-DNA I and adjacent plant genomic DNA (Figure V- 3). The number of restriction fragments

detected should indicate the number of inserts present in MON 87769.

The number of copies of the T-DNA I (copy nu mber) integrated at a single locus was determined by digesting the MON 87769 DNA w ith the combination of restriction

enzymes Lgu I and Bst X I, which cleave once within th e insert and within each flank (Figure V-3). If MON 87769 contains one copy of T-DNA I, Southern blots probed with the entire T-DNA I will result in two bands, each representing a porti on of the T-DNA I, along with adjacent plant genomic DNA. The samples were subjected to Southern blot analysis using six overlapping 32 P-labeled T-DNA I probes (probes 13-18, Figure V-2) spanning the entire T-DNA I insert. This an alysis was divided between two Southern blots. Each blot was probed with a combina tion of three probes. The results of this analysis are presented in Figure V-4a for probes 13-15 and Figure V-4b for probes 16-18. The expected DNA fragments are summarized in Table V-2.

To determine the endogenous background hybrid ization of various probes to MON 87769 DNA, conventional soybean DNA was used as a control. As shown in Figure V-4, conventional soybean DNA digested with Lgu I alone (lanes 3 and 7) or a combination of Lgu I and Bst X I (lanes 1 and 5) produced several hybr idization signals. This is expected because several genetic elements within T-DNA I were derived from soybean. These hybridization signals result from the probes hybridizing to endogenous DNA sequences residing in the conventional soybean genome a nd are not specific to the inserted DNA. These signals were produced in all lanes, including those contai ning the conventional soybean DNA material and therefore, are considered to be endogenous background hybridization.

To ensure that each of the T-DNA probes hybr idized to their intended target, plasmid vector DNA and probe template spikes were used as controls on the Southern blots. Plasmid PV-GMPQ1972 DNA controls digested with Bam H I, mixed with conventional soybean DNA predigested with Lgu I and Bst X I, and analyzed using probes 13-15 (lane 9, Figure V-4a ) produced the expect ed bands at approxim ately 1.3, 1.9, 2.3, 3.1, and 7.9 kb, in addition to the endogenous b ackground hybridization produced by theMonsanto Company 09-SY-200U Page 56 of 395 conventional soybean DNA (refer to Table V-2) . The probe template controls (probes 13-15) mixed with predigested conventiona l DNA and hybridized with probes 13-15 (lanes 10 and 11) produced the expected ba nds at 1.6, 1.7, and 2.0 kb, in addition to the endogenous background hybridization produced by the conventional soybean DNA

Plasmid PV-GMPQ1972 DNA digested with Bam H I, mixed with predigested conventional soybean DNA and analyzed wi th probes 16-18 (lane 9, Figure V-4b) produced the expected bands at approximately 2.3, 3.1, and 7.9 kb, in addition to the endogenous background hybridization produced by the conventional soybean DNA (refer to Table V-2). Conventional soybean DNA spiked with probes 16-18 (lanes 10 and 11) produced expected bands at 0.8, 1.0, a nd 1.3 kb, in addition to the endogenous background hybridization. The 0.1 genomic equivale nt of the probe templates resulted in a fairly faint hybridization signal (lane 11), but is detectable on the actual autoradiography film.

MON 87769 DNA was digested with Lgu I and hybridized with overlapping T-DNA probes (probes 13-15, Figure V- 4a and probes 16-18, Figure V-4b). A single unique band of approximately 10.0 kb was detected in lanes 4 and 8 in addition to the endogenous background hybridizat ion signals produced by the conventional soybean

DNA. This result shows that MON 87769 contains one insert located on an approximately 10.0 kb Lgu I restriction fragment.

MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6, Figure V-4a) and hybridized with probes 13-15 pr oduced a single unique band of approximately 6.8 kb in addition to the endogenous background hybri dization observed in the conventional soybean controls (lanes 1 and 5). MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6, Figure V-4b) and hybridized with probes 16-18 produced two bands of approximately 1.6 and 6.8 kb in addition to the endogenous background hybridization observed in the conventional soybean control (l anes 1 and 5). These results confirm that a single copy of T-DNA I is present in MON 87769.Monsanto Company 09-SY-200U Page 57 of 395 Figure V-4. Southern Blot Analysis of MON 87769 with Insert and Copy Number

Probes Each blot was hybridized simulta neously with three overlapping 32 P-labeled T-DNA probes (probes 13-18, Figure V-2) . Figure V-4a was hybridi zed with probes 13-15 and Figure V-4b was hybridized with probes 16-18. Each lane contains ~10 μg of digested genomic DNA isolated from leaf tissue. Lane designations for each blot are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Lgu I) 4: MON 87769 ( Lgu I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean (Lgu I ) 8: MON 87769 ( Lgu I ) 9: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 10: Conventional soybean ( Lgu I and Bst X I) spiked with probe template [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with probe template [~0.1 genomic equivalent



### → Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 58 of 395 V.B. Confirmation of the Absence of Plasmid PV-GMPQ1972 Backbone

To determine the presence or absence of PV-GMPQ1972 backbone sequence, MON 87769 and conventional soybean DNA were digested with either the combination of restriction enzymes Lgu I and Bst X I or restriction enzymes Bsr G I and Psh A I and analyzed by Southern blot using probes 1- 3 and 6 (Figure V-1). These probes span the plasmid backbone sequences of PV-GMPQ1972 not contained in T-DNA I or T-DNA II.

The results are summarized in Table V-2.

Conventional soybean DNA (A3525) was used as a control to determine the endogenous background hybridization of MON 87769 on the Southern blot used to confirm the absence of plasmid backbone (Figure V-5). Conventional soybean control DNA digested

with a combination of Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and Psh A I (lanes 3 and 7) and hybridized with the backbone pr obes (probes 1-3 a nd 6) (Figure V-5) simultaneously showed no hybridization bands. This indicates that no endogenous soybean DNA sequences hybridized to the plasmid backbone probes.

To ensure that each of the backbone probes were capable of hybridizing to their intended target, both plasmid PV-GMPQ1972 DNA digested with Bam H I and probe template spikes were mixed with conventiona l soybean DNA (predigested with Lgu I and Bst X I)

and included on the Southern blot (Figure V-5). PV-GMPQ1972 DNA digested with

Bam H I, mixed with predigested conve ntional soybean DNA, and hybridized simultaneously with probes 1-3 and 6 (Figure V-5) produced the expected size bands of approximately 2.3 and 7.9 kb (lane 9). The prob e template controls, mixed with digested conventional soybean DNA, and hybridized w ith the backbone probes produced four expected bands at approximately 0.7, 0.9, 1.5, and 1.8 kb (lanes 10 and 11). Also, a nonspecific hybridization spot ex ists between lanes 5 and 6. There is no DNA present at the location of the hybridization spot since lanes 5 and 6 contai n the “short r un” analysis,

and therefore does not affect the results of the Southern blot. These results indicate that all the backbone probes labelled properly and hybridized to thei r intended targets.

Southern blot analysis of MON 87769 DNA digested with either a combination of Lgu I

and Bst X I (lanes 2 and 6) or Bsr GI and Psh A I (lanes 4 and 8) and hybridized simultaneously with probes 1-3, and 6 showed no detectable hybridization signal (Figure V-5). This indicates that MON 87769 does not contain any detectable backbone sequence from the transformation vector PV-GMPQ1972.Monsanto Company 09-SY-200U Page 59 of 395 Figure V-5. Southern Blot Analysis of MON 87769 with PV-GMPQ1972 Backbone

Probes The blot was hybridized simultaneously with four overlapping 32 P-labeled probes that span the entire backbone sequence (pro bes 1-3 and 6, Figure V-1) of plasmid PV-GMPQ1972. Each lane contains approxi mately 10 μg of digested genomic DNA

isolated from leaf tissue. La ne designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and Psh A I) 9: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 10: Conventional soybean ( Lgu I and Bst XI) spiked with probe templates [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~0.1 genomic equivalent]



## → Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 60 of 395 V.C. Confirmation of the Absence of T-DNA II Sequence

The T-DNA II insert was segreg ated away from MON 87769 by traditional breeding after

the initial transforma tion. To confirm the absence of T-DNA II region, MON 87769 and conventional soybean DNA were digested with either a combination of the restriction

enzymes Lgu I and Bst X I or Bsr G I and Psh A I and analyzed by Southern blot (Figure V-6) using probes 4 and 5 (Figure V-1). Th e results are summarized in Table V-2.

Conventional soybean DNA was used as a cont rol in order to dete rmine the endogenous background of MON 87769 on the Southern bl ot used to confirm the absence of T-DNA II sequences (Figure V-6). C onventional soybean DNA digested with a combination of Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and Psh A I (lanes 3 and 7) and hybridized simultaneously with the probes 4 and 5 produced no detectable hybridization bands. This indicates that no endogenous soybean DNA sequences hybridized to the T-DNA II probes.

To ensure that both of the overlapping T-DNA II probes were capable of hybridizing to their intended target, both plas mid PV-GMPQ1972 DNA digested with Bam H I and probe template spikes were mixed with conventional soybean DNA (predigested with Lgu I and Bst X I) and used on the Southern blot (Figure V-6). PV-GMPQ1972 DNA digested with Bam H I and analyzed using the two overlapping T-DNA II probes produced the expected size bands of approxi mately 7.9 and 2.3 kb (lane 9). The probe template controls, mixed with conventional soybean, and analyzed using the T-DNA II probes produced two expected bands at approximately 2.0 and 1.9 kb (lanes 10 and 11). The 0.1 genomic equivalent of the probe templa tes resulted in a fairly faint hybridization signal (lane 11), but is dete ctable on the actual autoradi ography film. These results indicate that the T-DNA II probes labelled properly and hybridized to their intended

targets.

To confirm the absence of T-DNA II sequ ences not associated with T-DNA I, MON 87769 DNA was analyzed by S outhern blot using probes 4 and 5 (Figure V-1) that span the T-DNA II sequence of PV-GMPQ1 972. The overlapping probes spanning T-DNA II contain the right border sequence, the E9 3 ′ nontranslated region sequence, and the left border sequence that are also co ntained on T-DNA I. Th erefore, given the common genetic elements shared between T-DNA I and T-DNA II, the T-DNA II probes are expected to hybridize to fr agments derived from T-DNA I. Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6) produced a single band of approximately 1.6 kb. This band is consis tent with the 1.6 kb band detected with the

E 9 3 ′ nontranslated region + left border sequence (lanes 2 and 6, Fi gure V-12). Although a band at 6.8 kb, that represents the right borde r sequence in the insert , was also expected in MON 87769, this band was not visible in Fi gure V-6. This band was not observed in the reported exposure because, as revealed by sequence analyses, there are only 43 base pairs of the right border sequence in the MON 87769 insert. Ther e were no additional fragments observed. Southern blot an alysis of MON 87769 DNA digested with Bsr G I

and Psh A I (lanes 4 and 8) produced a single band of approximately 4.2 kb. This band is

consistent with the 4.2 kb band detected with the E 9 3 ′ nontranslated region + left border sequence (lanes 4 and 8, Figure V-12). Alth ough a band at 2.0 kb that represents theMonsanto Company 09-SY-200U Page 61 of 395 right border sequence in the in sert was also expected, this band was not visible in the Figure V-6. This band was not observed in the reported exposure because, as revealed by sequence analyses, there are only 43 base pairs of the ri ght border sequence in the

MON 87769 insert. No additiona l fragments were observed. These results indicate that MON 87769 does not contain any additional de tectable T-DNA II elements other than

those associated with T-DNA I.Monsanto Company 09-SY-200U Page 62 of 395 Figure V-6. Southern Blot Analys is of MON 87769 with T-DNA II Probes The blot was hybridized simulta neously with two overlapping 32 P-labeled probes that span the entire T-DNA II sequence (probe s 4 and 5, Figure V-1) of plasmid PV-GMPQ1972. Each lane contains approxi mately 10 μg of digested genomic DNA

isolated from leaf tissue. La ne designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and Psh A I) 9: Conventional soybean ( Bsr G I and Psh A I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 10: Conventional soybean ( Bsr G I and Psh A I) spiked with probe templates [~1 genomic equivalent] 11: Conventional soybean ( Bsr G I and Psh A I) spiked with probe templates [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 63 of 395 V.D. Pj.D6D and Nc.Fad3 Cassette Copy Number and Integrity

The copy number of the inserted Pj.D6D and Nc.Fad3 coding sequences and their associated genetic elements was asse ssed by digesting MON 87769 DNA with a

combination of Lgu I and Bst X I or a combination of Bsr G I and Psh A I and hybridizing the Southern blots with individual ge netic elements from the inserted Pj.D6D and

Nc.Fad3 cassettes. The size of the genomic fragments and the T-DNA elements expected to be contained in each of those fragments is described below and summarized in Table

V-2.

Digestion of MON 87769 genomic DNA with Lgu I and Bst X I was expected to generate two border fragments of approximately 6.8 a nd 1.6 kb (Figure V-3). The 6.8 kb fragment contains genomic DNA flanking the 5 ′ end of the insert, right border sequence, 7S α ′

promoter sequence, Pj.D6D coding sequence , tml 3 ′ nontranslated region , 7S α promoter sequence, and a portion of the Nc.Fad3 coding sequence. The 1.6 kb fragment contains the remaining portion of the Nc.Fad3 coding sequence , E9 3 ′ nontranslated region

, left border sequence, and genomic DNA flanking the 3 ′ end of the insert.

Digestion of MON 87769 genomic DNA with Bsr G I and Psh A I was expected to generate one internal restriction fragmen t of approximately 1.9 kb and two border fragments of approximately 2.0 and 4.2 kb (Fig ure V-3). The 2.0 kb fragment contains genomic DNA flanking the 5 ′ end of the insert, right border sequence, 7S α ′

promoter sequence, and a portion of the Pj.D6D coding sequence. The 1.9 kb fragment contains the remaining portion of the Pj.D6D coding sequence and a portion of the tml 3 ′ nontranslated region. The 4.2 kb fragment c ontains the remaining portion of the tml 3 ′ nontranslated region, 7S α promoter sequence, Nc.Fad3 coding sequence, E9 3 ′ nontranslated region

, left border sequence, and genomic DNA flanking the 3 ′ end of the

insert.

Individual Southern blots were examined w ith the following probes: the right border +

7S α ′

promoter probe, Pj.D6D coding region probe, tml 3 ′ nontranslated region probe, 7S α promoter probe, Nc.Fad3 coding sequence probe, or E9 3’ nontranslated region + left border probe (probes 7-12, Figure V-1). Th e expected DNA fragments identified by probes 7-12 are summarized in Table V-2. To en sure that each of the probes was capable of hybridizing to the respectiv e target, plasmid PV-GMPQ1972 DNA digested with

Bam H I and combined with predigested conventional soybean DNA was used as a positive hybridization control.

V.D.1. Right Border/ 7S α ' Promotor

Conventional soybean DNA was used as a control to determine the endogenous background hybridization of M ON 87769 on the Southern blot used to assess the copy number of the right border and 7S α ′ promoter (Figure V-7). Conventional soybean DNA digested with Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and Ps hA I (lanes 3 and 7) and hybridized with the right border + 7S α ′ promoter probe (probe 7, Figure V-1) produced a single hybridization signal. This hybridizatio n signal results from the probe hybridizing to an endogenous soybean genomic sequence and is not specific to the inserted DNA.Monsanto Company 09-SY-200U Page 64 of 395 This signal was produced in both test and control lanes and, therefore, the band is considered to be endogenous background.

As a positive hybridization c ontrol, plasmid PV-GMPQ1972 DNA was digested with

Bam H I, mixed with conventi onal soybean DNA, and included on the Southern blot analysis that was hybridized wi th probe 7 (Figure V-7). The results of this experiment showed the expected size hybridization ba nds at approximately 1.3, 2.3 and 7.9 kb in addition to the endogenous hybridization ba nd observed in the conventional soybean control (lanes 10 and 11).

Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6) using probe 7 produced the expected si ngle unique band of a pproximately 6.8 kb in addition to the endogenous hybridization ba nd observed in the conventional soybean control (lanes 1 and 5; Figure V-7). Southe rn blot analysis of MON 87769 DNA digested

with Bsr G I and Psh A I (lanes 4 and 8) produced the single expected unique band of approximately 2.0 kb in addition to the e ndogenous hybridization ba nd observed in the conventional soybean control (lanes 3 and 7; Figure V-7). There were no unexpected bands present in the MON 87769 DNA sample s. This indicates that MON 87769

contains no additional de tectable Right Border or 7S α′ promoter elements other than those endogenous to the soybean ge nome or associated with the Pj.D6D gene cassette

.

V.D.2. Pj.D6D Coding Sequence

Conventional soybean DNA was used as a control to determine the endogenous background hybridization of MON 87769 on the Sout hern blot used to confirm the copy

number of the Pj.D6D coding sequence (Figure V-8). Conventional soybean DNA digested with the enzyme combination Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and

Psh A I (lanes 3 and 7) and hybridized with the Pj.D6D coding sequence probe (probe 8, Figure V-1) showed no detectable hybridizatio n bands. This indicates that there are no endogenous soybean DNA sequences that hybridized to the Pj.D6D coding sequence probe.

As a positive hybridization control, plas mid PV-GMPQ1972 DNA was digested with

Bam H I, mixed with conventional soybean DNA (predigested with Lgu I and Bst X I), and included on the Southern blot analysis that was hybridized with probe 8. The results of this experiment showed the expected size hybridization bands at approximately 1.3 and 1.9 kb (lanes 10 and 11).

Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6) using probe 8 produced th e expected single unique ba nd of approximately 6.8 kb. Southern blot analysis of MON 87769 DNA digested with Bsr G I and Psh A I (lanes 4 and 8) produced the expected unique bands of approximately 1.9 and 2.0 kb. Although these two bands appear as one band in the shor t run (lane 8), the long run (lane 4) clearly

resolves the 1.9 and 2.0 kb bands. There are no unexpected bands in the MON 87769

DNA. This indicates that MON 87769 contains no additional detectable Pj.D6D coding sequence elements other than those associated with the Pj.D6D cassette.Monsanto Company 09-SY-200U Page 65 of 395 V.D.3. tml 3 ′ Nontranslated Sequence

Conventional soybean DNA was used as a control to determine the endogenous background hybridization of MON 87769 on the Sout hern blot used to confirm the copy

number of the tml 3' nontranslated region (Figure V- 9). Conventional soybean control DNA digested with Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and Psh A I (lanes 3 and 7) and hybridized with the tml 3' nontranslated region probe (probe 9, Figure V-1) showed no detectable hybridization bands. This i ndicates that there are no endogenous soybean DNA sequences that hybridized to the tml 3' nontranslated region probe.

As a positive hybridization control, pl asmid PV-GMPQ1972 DNA was digested with

Bam H I, mixed with conventional soybean DNA (predigested with Lgu I and Bst X I) and

included on the tml 3' nontranslated region Southern bl ot. The results for this probe showed the expected size band at ap proximately 1.9 kb (lanes 10 and 11).

Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6) using probe 9 produced th e expected single unique ba nd of approximately 6.8 kb. Southern blot analysis of MON 87769 DNA digested with Bsr G I and Psh A I (lanes 4 and 8) produced two expected unique bands of approximate ly 1.9 and 4.2 kb. There were no additional bands detected using the tml 3 ′ nontranslated region pr obe. This indicates

that MON 87769 contains no additional, detectable tml 3 ′ elements other than those

associated with the Pj.D6D gene cassette.

V.D.4. 7S α Promoter

Conventional soybean DNA was used as a control to determine the endogenous background hybridization of MON 87769 on the Sout hern blot used to confirm the copy

number of the 7S α Promoter (Figure V-10). The c onventional soybean DNA, digested

with Lgu I and Bst X I (lanes 1 and 5) or Bs rG I and Psh A I (lanes 3 and 7) and hybridized

with the 7S α promoter probe (probe 10, Figure V- 1), produced two hybrid ization signals. These hybridization signals result from th e probe hybridizing to endogenous soybean genomic sequences and are not specific to the inserted DNA. These signals were produced in both test and control lanes and therefore the bands are considered to be endogenous background.

As a positive hybridization c ontrol, plasmid PV-GMPQ1972 DNA was digested with

Bam H I, mixed with conventional soybean DNA (predigested with Lgu I and Bst X I), and

included in the 7S α promoter Southern blot analysis. The results of this experiment showed the expected size band at approxi mately 3.1 kb in addition to the endogenous hybridization bands observed in the conventional soybean co ntrol (lanes 10 and 11).

Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I ( lanes 2 and 6) produced the expected single unique band of approximately 6.8 kb in addition to the endogenous hybridization bands ob served in the conventional soybean control (lanes 1 and 5). Southern blot analys is of MON 87769 DNA digested with Bsr G I and Psh A I (lanes 4 and 8) produced the expected si ngle unique band of approximately 4.2 kb in addition to the endogenous hybr idization bands observed in the conventional soybeanMonsanto Company 09-SY-200U Page 66 of 395 control (lanes 3 and 7). The migration of approximately 6.8 kb and 4.2 kb fragments is slightly higher than indicated by molecula r weight marker band sizes. The altered migration may be due to the difference in salt concentrations between the DNA sample and the molecular weight marker 

### ( Sambrook and Russell, 2001 

### ) . There were no unexpected bands detected in the MON 877 69 DNA samples. This indicates that

MON 87769 contains no additional detectable 7S α promoter elements other than those endogenous to the soybean genome or associated with the Nc.Fad3 gene cassette.

V.D.5. Nc.Fad3 Coding Sequence

Conventional soybean DNA was used as a control to determine the endogenous background hybridization of MON 87769 on the Sout hern blot used to confirm the copy

number of the Nc.Fad3 coding sequence (Figure V-11). Conventional soybean DNA digested with Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and Psh A I (lanes 3 and 7) and hybridized with the Nc.Fad3 coding sequence probe (probe 11, Figure V-1) produced no detectable hybridization bands. This indi cates that there are no endogenous soybean DNA sequences that hybridized to the Nc.Fad3 coding sequence probe.

As a positive hybridization control, plas mid PV-GMPQ1972 DNA was digested with

Bam H I, mixed with conventional soybean DNA (digested with Lgu I and Bst X I), and

included on the Nc.Fad3 coding sequence Southern blot analysis using probe 11. The results of this experiment showed the expe cted size hybridization band at approximately 3.1 kb (lanes 10 and 11).

Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6) using probe 11 produced the two expect ed unique bands of approximately 1.6 and 6.8 kb. The migration of the approximately 1.6 kb fragment is slightly lower than indicated by the molecular weight marker ba nd sizes. The altered migrations may be due to the difference in salt concentrations between the DNA sample and the molecular weight marker (Sambrook and Russell, 2001). Southern blot analysis of MON 87769 DNA digested with Bsr G I and Psh A I (lanes 4 and 8) produced the expected single unique band of approximately 4.2 kb. There were no unexpected bands observed on the Southern blot using MON 87769 DNA. This indicates that MON 87769 contains no

additional detectable Nc.Fad3 coding sequence elements other than those associated with

the Nc.Fad3 cassette.

V.D.6. E9 3 ′ Nontranslated Sequence/Left Border

Conventional soybean DNA was used as a control to determine the endogenous background hybridization of MON 87769 on the Sout hern blot used to confirm the copy

number of the E9 3 ′ nontranslated r egion and left border se quence (Figure V-12). Conventional soybean DNA digested with Lgu I and Bst X I (lanes 1 and 5) or Bsr G I and

Psh A I (lanes 3 and 7) and hybridized with the E9 3 ′ nontranslated region + left border probe (probe 12, Figure V-1) produced no detect able hybridization bands. This indicates that there are no endogenous soybean DNA sequences that hybridized to the E9 3 ′

nontranslated r egion and left border sequence.Monsanto Company 09-SY-200U Page 67 of 395 As a positive hybridization control, pl asmid PV-GMPQ1972 DNA was digested with

Bam H I, mixed with conventional soybean DNA (digested with Lgu I and Bst X I), and

included in the E9 3 ′ nontranslated r egion and + left border Southern blot analysis (Figure V-12). The results of this experiment showed the two expected size bands at approximately 2.3 and 7.9 kb (lanes 10 and 11).

Southern blot analysis of MON 87769 DNA digested with Lgu I and Bst X I (lanes 2 and 6) using probe 12 produced the expected si ngle unique band of approximately 1.6 kb. Southern blot analysis of MON 87769 DNA digested with Bs rG I and Psh A I (lanes 4 and 8) produced the expected single unique band of approxi mately 4.2 kb. There were no additional bands detected using the E9 3 ′ nontranslated region + left border probe. This

indicates that MON 87769 contai ns no additional detectable E9 3 ′ nontranslated region or

left border elements other than those associated with the Nc.Fad3 gene cassette.Monsanto Company 09-SY-200U Page 68 of 395 Figure V-7. Southern Blot Analysis of MON 87769 with Right Border/P-7s α ′ Probe The blot was hybridized with a 32 P-labeled probe that spanned the right border and 7s α ′ promoter sequence (probe 7, Figure V-1). E ach lane contains approximately 10 μg of digested genomic DNA isolated from leaf tis sue. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8 MON 87769 ( Bsr G I and Psh A I) 9: Empty 10: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 69 of 395 Figure V-8. Southern Blot An alysis of MON 87769 with CS- Pj.D6D Probe The blot was hybridized with a 32 P-labeled probe that spanned the Pj.D6D coding sequence (probe 8, Figure V-1). Each lane contains approximate ly 10μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and Psh A I) 9: Empty 10: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 70 of 395 Figure V-9. Southern Blot Analysis of MON 87769 with T -tml Probe The blot was hybridized with a 32 P-labeled probe that spanned the tml nontranslated region (probe 9, Figure V-1). Each lane co ntains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and PshA I) 9: Empty 10: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 71 of 395 Figure V-10. Southern Blot Analysis of MON 87769 with P- 7s α Probe The blot was hybridized with a 32 P-labeled probe that spanned the 7s α promoter sequence (probe 10, Figure V-1). Each lane contains approximately 10 μg of digested genomic

DNA isolated from leaf tissue. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and Psh A I) 9: Empty 10: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 72 of 395 Figure V-11. Southern Blot An alysis of MON 87769 with CS- Nc.Fad3 Probe The blot was hybridized with a 32 P-labeled probe that spanned the Nc.Fad3 coding sequence (probe 11, Figure V-1). Each lane contains approximatel y 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bsr G I and Psh AI) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and Psh A I) 9: Empty 10: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 73 of 395 Figure V-12. Southern Blot Analysis of MON 87769 with T- E9 /Left Border Probe The blot was hybridized with a 32 P-labeled probe that spanned the E9 nontranslated region and left border (probe 12, Figure V-1). Each lane contains approximately 10 μg of digested genomic DNA isolated from leaf tis sue. Lane designa tions are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) 2: MON 87769 ( Lgu I and Bst X I) 3: Conventional soybean ( Bsr G I and Psh A I) 4: MON 87769 ( Bs rG I and Psh A I) 5: Conventional soybean ( Lgu I and Bst X I) 6: MON 87769 ( Lgu I and Bst X I) 7: Conventional soybean ( Bsr G I and Psh A I) 8: MON 87769 ( Bsr G I and Psh A I) 9: Empty 10: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 11: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~0.1 genomic equivalent]

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on 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

1.6 2.0 40 20 15

10 8.1 7.1 6.1

5.1

4.1

3.1

2.0

1.6

1.0

0.5 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

1.6 2.0 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 09-SY-200U Page 74 of 395 V.E. Southern Blot Analyses of MON 87769 Across Multiple Generations

In order to assess the stability of the T- DNA in MON 87769 across multiple generations, Southern blot analyses were performed usi ng DNA obtained from multiple generations of MON 87769. DNA from four generations was analyzed by Southern blot using six overlapping probes that span the entir e T-DNA I region of plasmid PV-GMPQ1972 (probes 13-18, Figure V-2). For referen ce, the breeding history of MON 87769 is presented in Figure V-13. The expected Southern hybrid ization DNA banding pattern for these analyses is summarized in Table V-2.

V.E.1. Generational Stability of the Insert

Conventional soybean DNA from A3525 was us ed as a control to determine the endogenous background hybridization of MON 87769. As shown in Figure V-14, conventional soybean DNA digested with a combination of Lgu I and Bst X I (lane 4) produced several hybridization signals. This is expected because several genetic

elements within T-DNA I were derived from soybean. These hybridization signals result from the probes hybridizing to endogenous soybean gene sequences and are not specific to the inserted DNA. These signals were produced in all lane s, including those containing the conventional soybean DNA a nd, therefore, are considered to be endogenous background hybridization.

To ensure that each of the T-DNA probes was capable of hybridizing to their intended target, plasmid DNA and probe template spikes were used as controls on the Southern blots. Plasmid PV-GMPQ1972 DNA controls digested with Bam H I, mixed with conventional soybean DNA (digested with Lgu I and Bst X I), and analyzed using probes 13-15 (lane 1, Figure V-14a) produced the expe cted bands at approximately 1.3, 1.9, 2.3, 3.1, and 7.9 kb, in addition to the endogenous background hybridization produced by the conventional soybean DNA (refer to Table V-2). Conventional soybean DNA spiked with probes 13-15 (lanes 2 and 3) produced the expected bands at 1.6, 1.7, and 2.0 kb, in addition to the endogenous background hybrid ization produced by the conventional soybean DNA.

Plasmid PV-GMPQ1972 DNA digested with Bam H I, mixed with conventional soybean DNA (digested with Lgu I and Bst X I) and analyzed with probes 16-18 (lane 1, Figure V-14b) produced the expected bands at a pproximately 2.3, 3.1, and 7.9 kb, in addition to the endogenous background hybr idization produced by the conventional soybean DNA (refer to Table V-2). Conventional soybean DNA spiked with probes 16-18 (lanes 2 and 3) produced expected bands at 0.8, 1.0, and 1.3 kb, in addition to the endogenous background hybridization.

Southern blot analysis of MON 87769 DNA from four generations digested with Lgu I

and Bst X I (lanes 5-9, Figure V-14 a) and hybridized simultaneously with probes 13-15 produced the expected band of approximate ly 6.8 kb in addition to the endogenous background hybridization observed in the conven tional soybean DNA controls (lanes 4). Southern blot analysis of MON 87769 DNA from all four generations digested with Lgu I

and Bst X I (lanes 5-9, Figure V- 14b) using probes 16-18 pr oduced the two expectedMonsanto Company 09-SY-200U Page 75 of 395 bands of approximately 1.6 and 6.8 kb in addition to the endogenous background hybridization observed in the conventional soyb ean control DNA (lane 1). Also, several nonspecific hybridization spots ex ist in lanes 4, 8, and 9. These nonspecific hybridization spots are faint and do not affect the results of the Southern blot. The observed results are consistent with the results presented in Figures V-4a and V-4b, and confirm that the single copy of the insert in MON 87769 is stab ly maintained across multiple generations of its breeding history.

V.E.2. Confirmation of the Absen ce of PV-GMPQ1972 Backbone Sequence

The four generations of MON 87769 utilized to assess generational stability also were

examined for the absence of backbone se quence by Southern blot. MON 87769 and control DNA samples were digested with Lgu I and Bst X I and the blot was hybridized simultaneously with four overlapping probes, which taken together, span the backbone sequence of plasmid PV-GMPQ1972 (p robes 1-3 and 6, Figure V-1).

Conventional soybean DNA was used as a cont rol in order to dete rmine the endogenous background hybridization of MON 87769 on the Southern blot used to confirm the absence of plasmid backbone across multiple generations (Figure V-15). Conventional soybean DNA digested with a combination of Lgu I and Bst X I (lane 4) and hybridized with the backbone probes (probes 1-3 and 6, Fi gure V-1) showed no hybridization bands. This indicates that there are no endogenous soybean DNA sequences that hybridized to the plasmid backbone probes.

To ensure that each of the backbone probes was capable of hybridizing to their intended target, plasmid PV-GMPQ1972 DNA and probe template spikes were used as positive hybridization controls on the Southern blot (Figure V-15). PV-GMPQ1972 control DNA digested with Bam H I, mixed with conventional soybean, and analyzed with the backbone probes (probes 1-3 and 6; Figure V- 1) produced the expected size bands of approximately 2.3 and 7.9 kb (lane 1; refer to Table V-2). The probe template controls, mixed with digested conventional soybean DNA and analyzed with the backbone probes, produced four expected bands at approximately 0.7, 0.9, 1.5, a nd 1.8 kb (lanes 2 and 3).

These results indicate that all the backbone probes labelle d properly and hybridized to their intended targets.

To confirm the abse nce of PV-GMPQ1972 backbone sequence, MON 87769 DNA was analyzed by Southern blot using probes 1- 3 and 6 (Figure V-1) that span the entire backbone sequence of PV-GMPQ1972. Sout hern blot analysis of MON 87769 DNA digested with a combination of Lgu I and Bst X I (lanes 5-9) showed no detectable hybridization signal. These resu lts are consistent with the da ta depicted in Figure V-5.

This indicates that MON 87769 does not contai n any detectable backbone sequence from the transformation vector PV-GMPQ1972.

V.E.3. Confirmation of the Absence of T-DNA II Sequence

The four generations of MON 87769 material utilized to asse ss generational st ability also

were examined for the absence of T-DNA II sequence by Southern blot analysis.Monsanto Company 09-SY-200U Page 76 of 395 MON 87769 and control DNA samples were digested with Lgu I and Bst X I and the blot was hybridized simultaneously with two overlapping probes, which span the T-DNA II sequence of plasmid PV-GMPQ1972 (probes 4 and 5, Figure V-1).

Conventional soybean DNA was used as a cont rol in order to dete rmine the endogenous background of MON 87769 on the S outhern blot used to conf irm the absence of T-DNA II sequences (Figure V-16). Conve ntional soybean DNA digested with a combination of Lgu I and Bst X I (lanes 4) and hybridized w ith the T-DNA II probes produced no detectable hybridization bands. This indi cates that there are no endogenous soybean DNA sequences that hybridized to the T-DNA II probes.

To ensure that both of the overlapping T-DNA II probes were capable of hybridizing to their intended target, plasmid PV-GMPQ1972 DNA and probe template spikes were used as positive hybridization contro ls on the Southern blot (Figure V-16). PV-GMPQ1972 DNA digested with Bam H I, mixed with conventional soybean DNA, and analyzed using the two overlapping T-DNA II probes produced the expected size bands of approximately 7.9 and 2.3 kb (lane 1; refer to Table V-2). The probe template controls, mixed with conventional soybean DNA and analyzed using the T-DNA II probes, produced two expected bands at approximately 2.0 and 1.9 kb (lanes 2 and 3). Lanes 1 and 2 have an apparent smear throughout the lane due to background hybridizati on. These results indicate that the T-DNA II probes labelled properly and hybridized to their intended

targets.

To confirm the absence of T-DNA II se quences not associated with T-DNA I, MON 87769 DNA from four gene rations was analyzed by Southern blot using probes 4 and 5 (Figure V-1) that span the T-DNA II sequence of PV-GMPQ1972. The overlapping probes spanning T-DNA II cont ain the right border sequence, the E9 3 ′ nontranslated region sequence, and the left border sequence that al so are contained on T DNA I. Therefore, the T-DNA II probes are expected to hybridize to fragments

derived from T-DNA I. S outhern blot analysis of MON 87769 DNA from all four generations digested with Lgu I and Bst X I (lanes 5-9) produced two bands of approximately 1.6 and 6.8 kb. The 1.6 kb band is consistent wi th the 1.6 kb band

detected with the E 9 3 ′ nontranslated region + left borde r sequence (lanes 2 and 6, Figure V-12). The hybridization band at 6.8 kb that represents the right border sequence in the insert was faint in the reported exposure beca use there are only 43 base pairs of the right border sequence in the MON 87769 insert. No additional fragments were observed. The

observed results are consistent with the results presented in Figure V-6, and confirm that all four generations of MON 87769 do not contain any additional detectable T-DNA II

elements other than those associated with T-DNA I.Monsanto Company 09-SY-200U Page 77 of 395 Figure V-13. MON 87769 Breeding Diagram All generations shown were self-pollinated ( ⊗ ). The R1 generation was used for segregation analysis and th e selection of plants homoz ygous for the T-DNA I insert (Section III). R5 seed material was us ed for commercial variety development.

Generation R4 was used in the molecular char acterization analyses. Generations R3, R4,

R5 a , R6 b , and R6 c were used in the molecular generation stability analyses.Monsanto Company 09-SY-200U Page 78 of 395 Figure V-14. Generational Stability of MON 87769 with T-DNA I Probes The blot was hybridized with overlapping 32 P-labeled probes that spanned the T-DNA (probes 13-18, Figure V-2). Each blot was hybridized simulta neously with three of the overlapping probes. Figure V-14a was hybrid ized with probes 13-15 and Figure V-14b was hybridized with probes 16-18. Each lane c ontains approximately 10 μg of digested genomic DNA isolated from leaf tissue. The breeding history of MON 87769 is

illustrated in Figure V-13. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 2: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~1 genomic equivalent] 3: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~0.1 genomic equivalent] 4: Conventional soybean ( Lgu I and Bst X I) 5: R3: MON 87769 ( Lgu I and Bst X I) 6: R4: MON 87769 ( Lgu I and Bst X I)

7: R5 a : MON 87769 ( Lgu I and Bst X I)

8: R6 c : MON 87769 ( Lgu I and Bst X I)

9: R6 b : MON 87769 ( Lgu I and Bst X I)

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel. 20 15

2.0 8.1 7.1 6.1 5.1
4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9

20 15

2.0 8.1 7.1 6.1

5.1

4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9 a) b)

20 15

2.0 8.1 7.1 6.1 5.1
4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9

20 15

2.0 8.1 7.1 6.1

5.1

4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9

20 15

2.0 8.1 7.1 6.1 5.1
4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9

20 15

2.0 8.1 7.1 6.1 5.1
4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9

20 15

2.0 8.1 7.1 6.1

5.1

4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9

20 15

2.0 8.1 7.1 6.1

5.1

4.1

3.1

1.6 10 40

1.0

0.5 20 15

2.0 8.1 7.1 6.1

5.1

4.1

3.1

1.6 10 40

1.0

0.5 1 2 3 4 5 6 7 8 9 a) b)Monsanto Company 09-SY-200U Page 79 of 395 Figure V-15. Generational Stability of MON 87769 with PV-GMPQ1972 Backbone

Probes The blot was hybridized with four overlapping 32 P-labeled probes that span the entire backbone sequence (probes 1, 2, 3, and 6, Figur e V-1) of plasmid PV-GMPQ1972. Each lane contains approximately 10 μg of digested genomic DNA is olated from leaf tissue. The breeding history of MON 87769 is illustrate d in Figure IV-17. Lane designations are

as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 2: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~1 genomic equivalent] 3: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~0.1 genomic equivalent] 4: Conventional soybean ( Lgu I and Bst X I) 5: R3: MON 87769 ( Lgu I and Bs tX I) 6: R4: MON 87769 ( Lgu I and Bst X I)

7: R5 a : MON 87769 (Lgu I and Bs tX I)

8: R6 c : MON 87769 (Lgu I and Bst X I)

9: R6 b : MON 87769 (Lgu I and Bst X I )

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel. 23.1

9.4

6.6

4.4

2.3

2.0

0.6 1 2 3 4 5 6 7 8 9

23.1

9.4

6.6

4.4

2.3

2.0

0.6 23.1

9.4

6.6

4.4

2.3

2.0

0.6 1 2 3 4 5 6 7 8 9Monsanto Company 09-SY-200U Page 80 of 395 Figure V-16. Generational Stability of MON 87769 with T-DNA II Probes The blot was hybridized with two overlapping 32 P-labeled probes that span the entire TDNA II (probes 4 and 5, Figure V-1) of plas mid PV-GMPQ1972. Each lane contains approximately 10 μg of digest ed genomic DNA isolated from leaf tissue. The breeding history of MON 87769 is illustrated in Figure V-13. Lane designations are as follows:

Lane 1: Conventional soybean ( Lgu I and Bst X I ) spiked with PV-GMPQ1972 ( Bam H I) [~1 genomic equivalent] 2: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~1 genomic equivalent] 3: Conventional soybean ( Lgu I and Bst X I) spiked with probe templates [~0.1 genomic equivalent] 4: Conventional soybean ( Lgu I and Bst X I) 5: R3: MON 87769 ( Lgu I and Bst X I) 6: R4: MON 87769 ( Lgu I and Bst X I)

7: R5 a : MON 87769 ( Lgu I and Bst X I)

8: R6 c : MON 87769 ( Lgu I and Bst X I)

9: R6 b : MON 87769 ( Lgu I and Bst X I)

→ Symbol denotes size of DNA, in kilobase pairs, obtained from MW markers on ethidium bromide stained gel.Monsanto Company 09-SY-200U Page 81 of 395 V.F. Organization of the Genetic Elements in MON 87769

The organization of the genetic elements w ithin the insert of MON 87769 was confirmed by PCR and DNA sequence analyses. Overla pping DNA fragments spanning the entire length of the insert and genomic DNA directly adjacent to the insert were amplified by PCR. The amplified DNA fragments were subjected to DNA sequence analyses. The DNA sequence of the insert contains 7367 base pairs beginning at base 9387 of PV-GMPQ1972 located in the ri ght border region, and ending at base 288 in the left border region of PV-GMPQ1972. In additi on, 933 base pairs of soybean genomic DNA flanking the 5' end of the insert and 831 ba se pairs of soybean genomic DNA flanking the

3' end of the insert were also determine d. A comparison of the soybean genomic DNA flanking the insert in MON 87769 to the sequen ce of the insertion site in conventional soybean demonstrated that no major rearrangements occurred at the insertion site during

transformation. Results confirm that the DNA sequence of the insert in MON 87769 is identical to that of plasmi d PV-GMPQ1972, and the organiza tion of the insert genetic elements is as depicted in Figure V-3.

V.G. Inheritance of the Introduced Traits

The genotypic stability and segregat ion of T-DNA I in MON 87769, initially demonstrated by Southern blot analysis (Section V.E), was confirmed by performing a Chi-square analysis on zygosity data generated for the T- tml 3' genetic element over three generations. The Chi-square analysis is base d on testing the observe d segregation ratio to the expected segregation ratio according to Mendelian principles. The breeding path for these segregation data on MON 87769 is desc ribed in Figure V-17. The R0 plant was self-pollinated to produce R1 seed, wh ich is expected to segregate 1:2:1 (1 homozygote:2 hemizygote:1 null segregant) for the inse rt. A homozygous selection (R1 plant) was identified from the segreg ating population by Invade r and Southern blot analyses. The selected R1 plant was self -pollinated to produce R2 seed, which was expected to be fixed for the trait, mean ing all seed are homozygous for T-DNA I. The homozygosity of subsequent generations was confirmed using the Invader assay (data not shown).

Homozygous R4 MON 87769 plants were crossed to a conventional soybean variety to produce hemizygous F1 seed. Seed from the resulting F1 plants were harvested to produce the F2 generation. The F2 plan ts are expected to segregate 1:2:1 (1 homozygote:2 hemizygote:1 null segregant) for the insert. Individual plants were tested for the presence and zygosity of th e insert using an Invader assay for the T- tml 3' genetic element 

### ( Gupta et al., 2008 

### ) . Hemizygous positive F2 plants were selected and self-pollinated to produce F3 seed. The F3 plants were tested for the presence and zygosity of the insert using the T- tml 3' Invader assay. Hemi zygous F3 plants were selected and self-pollinated to produce F4 see d. Likewise, the F4 pl ants were tested for the presence and zygosity of the insert using the T- tml 3 ′ Invader assay. Like the F2 progeny, the F3 and F4 progeny each were pred icted to segregate 1:2:1 (1 homozygote:2 hemizygote:1 null segregant) for the insert.

The Chi-square ( χ 2) test for each generation was computed as:Monsanto Company 09-SY-200U Page 82 of 395 χ

2 = ∑ [( | o – e | )2 / e]

where o = observed frequency of the genotype and e = e xpected frequency of the genotype. The level of statistical signi ficance was predetermined to be 5% (p ≤ 0.05). Table V-3 shows the segregation data obt ained with Invader-based assay. The Chi-square value for the F2, F3, and F4 ge nerations indicated no significant difference between the observed and expected segregation ratios. These results are consistent with molecular characterization data, indicating a si ngle insertion site of the insert within

MON 87769 and show the insert follows the e xpected Mendelian patt ern of segregation.Monsanto Company 09-SY-200U Page 83 of 395 Figure V-17. Breeding Path for Genera ting Segregation Data on MON 87769Monsanto Company 09-SY-200U Page 84 of 395Table V-3. Chi-square (χ2) analysis of MON 87769 Progeny1:2:1 SegregationGeneration1TotalPlantsTested2Observed #PlantsHomozygousPositiveObserved #PlantsHemizygousPositiveObserved #PlantsHomozygousNegativeExpected #PlantsHomozygousPositiveExpected #PlantsHemizygousPositiveExpected #PlantsHomozygousNegativeχ2ProbabilityF24713231111.7523.511.750.20.9087F317445814843.58743.50.90.6278F4222601026055.511155.51.50.4821F2 progeny were from the cross of homozygous MON 87769 with a soybean variety that did not contain the MON 87769 insert. F3 and F4progeny were from self-pollinated F2 and F3plants hemizygous positive for the MON 87769 insert.2Plants were tested for the presence of thetml3′genetic element by Invader analysis.Monsanto Company 09-SY-200U Page 85 of 395 V.H. Conclusions of Molecular Characterization

Molecular analyses were performed to ch aracterize the integrated DNA insert in MON 87769. Southern blot analyses were used to determine the DNA insert number (number of integration site s within the soybean genome), copy number (the number of copies within one inse rt), intactness of the Pj.D6D and Nc.Fad3 gene expression cassettes, and absence of plasmid backbone and T-DNA II sequences in the plant. The stability of the DNA insert across multiple generations also was demonstrated by Southern blot fingerprint analysis. In addition, DNA sequenc ing analyses were performed to confirm the organization of the elements within the MON 87769 DNA insert, and to confirm the sequence identi ty between the plasmid PV-GMPQ1972 T-DNA

and the insert.

Data show that one intact copy of the Pj.D6D and Nc.Fad3 expression cassettes was integrated at a single chromosomal locus contained within a ~10 kb Lgu I restriction fragment. No additional elements from the transformation vector PV-GMPQ1972, linked

or unlinked to the intact DNA insert were detected in the genome of MON 87769. Additionally, backbone sequence from PV-GMP Q1972 was not detected and it also was

confirmed that MON 87769 does not contai n any additional detectable T-DNA II

elements other than those a ssociated with T-DNA I. Gene rational stability analysis demonstrated that the expected Southern blot fingerprint of MON 87769 has been maintained across four generations of breed ing, thereby confirming the stability of the DNA insert over multiple generations. These ge nerations were also shown not to contain any detectable T-DNA II or backbone seque nce from plasmid PV-GMPQ1972. In addition, DNA sequence analyses confirme d the sequence identities between the MON 87769 insert and the portion of the T-DNA from PV-GMPQ1972 that was integrated into the soybean genome. These re sults also confirmed the organization of the genetic elements within the Pj.D6D and Nc.Fad3 expression cassettes of MON 87769,

which was identical to those cassettes in plasmid PV-GMPQ1972. Fi nally, heritability and stability of the insert within M ON 87769 were as expected across multiple generations, which corroborates the molecular in sert stability analysis and establishes the genetic behavior of the DNA insert at a single chromosomal locus.Monsanto Company 09-SY-200U Page 86 of 395 VI. CHACTERIZATION OF THE INTRODUCED Pj Δ 6D AND Nc Δ 15D

PROTEINS

This section summarizes the safety assessment of the Pj Δ 6D and Nc Δ 15D proteins produced in MON 87769 ba sed on the following:

1. Characterization of their identity and function,

2. Isolation from immature soybean seed for use in subsequent la boratory and regulatory safety evaluations,

3. Quatitation of expression levels in key soybean tissues,

4. An assessment of the pote ntial allergenicity, and

5. An evaluation of potential toxicity and hu man and animal dietary risk assessment.

Results show that the Pj Δ 6D and Nc Δ 15D proteins share sequen ce similarity to several proteins that are ubiquitous in the human diet and are consumed directly in many common foods. These proteins are expressed at low levels in the seed and not detectable in other tissues analysed. Both proteins lack biologically-relevant amino acid sequence similarities to known protein allergens and t oxins. Additionally, both proteins are rapidly digested in simulated gastric and intestinal fl uids and do not exhibit any signs of toxicity when administered to mice via oral gavage. Ultimately, the safety assessment supports the conclusion that dietary exposure to the Pj Δ 6D and the Nc Δ 15D proteins derived from MON 87769 poses no meaningful risk to human or animal health.

These results, all of which have been subm itted to FDA as part of the pre-market consultation, support a conclusion of safe di etary consumption for humans and animals. The following sections provide further details regarding the safety assessment of the Pj Δ 6D and Nc Δ 15D proteins produced in MON 87769.

VI.A. Identity and Function of the Pj Δ 6D and Nc Δ 15D Proteins

Fatty acid desaturases are enzymes that c onvert a single bond be tween two carbon atoms (C-C) to a double bond (C=C) in a fatty acyl chain (Stump f, 1980). The resultant double

bond often is referred to as an unsaturated bond, and the reactions catalyzed by these enzymes are known as desaturation reactions. The degree of unsaturation of a fatty acid chain is a major determinant of the fluidity of biological membrane s that consist of a bilayer of phospholipids (Agu ilar and Mendoza, 2006). All organisms synthesize unsaturated fatty acids de novo using a common metabolic pathway (Somerville and Browse, 2000; Voet and Voet, 1995). Hen ce, enzymes involved in this metabolic pathway, including fatty acid desaturases, ar e conserved across all kingdoms (Los and Murata, 1998).

The Pj Δ 6D and Nc Δ 15D proteins are members of a fam ily of integral membrane fatty acid desaturases found in all eukaryotic orga nisms (plants, animals, fungi) and some prokaryotes, e.g. cyanobacteria (Hashimoto et al., 2008; Los and Murata, 1998). FattyMonsanto Company 09-SY-200U Page 87 of 395 acid desaturases are non-heme iron-contai ning enzymes that introduce a double bond

between defined carbons of fatty acyl chains with st rict regioselectivity and stereoselectivity (Shanklin and Cahoon, 1998). Solubilization of integral membrane desaturases requires detergents which are likely to disrupt the association with one or more required accessory proteins; therefore, th e functional activity of integral membrane proteins usually is studied in the context of an intact membrane system (Reed et al., 2000; Sayanova et al., 2001). Due to the tremendous difficultly in the purification and assay of the activity of these integral membrane fatty desaturase proteins (Warude et al., 2006), the tertiary structure of the integral memb rane desaturase prot eins is not known.

VI.A.1. Pj Δ 6D Protein

The Pj Δ 6D protein expressed in MON 87769 is nearly identical to the native protein produced by Primula juliae . The protein consists of 446 amino acids with a calculated molecular mass of 50,985 Da and a predicted is oelectric point of 8.8 . Analysis of the Pj Δ 6D amino acid sequence demonstrates three hi stidine motifs that are characteristic of integral membrane desaturases (Figure VI-1). The active site of integral membrane

desaturases consists of three histidine motif s with a total of eight essential histidine residues (Shanklin et al., 1994). The protein is thought to be folded in such a way that the histidine boxes align to form the di-iron binding site at the cata lytic center of the desaturases (Los and Murata, 1998). Analysis of Pj Δ 6D deduced amino acid sequence has shown that, similar to other Δ 6 desaturases (Nakamura and Nara, 2004) Pj Δ 6D contains the amino-terminal cytochrome b5 domain carrying the heme-binding motifs (Figures VI-1 and VI-2). As in other Δ 6 desaturases, the first His residue of the third His-box in Pj Δ 6D is substituted with glutamine (QXXHH inst ead of HXXHH) and, therefore, is called Q-box. The cytochrome b5 domain and glutamine residue are essential for the activity of Δ 6 desaturases because mutations abolish the enzyme activity (Sayanova et al., 2001; Sayanova et al., 2000).

The Pj Δ 6D protein also contains multiple membrane-spanning regions that are characteristic of the integral membrane desa turase proteins (Figure VI-2) based on the patterns observed from hydropathy plots.Monsanto Company 09-SY-200U Page 88 of 395 Figure VI-1. Deduced Amino Acid Sequence of Pj Δ 6D from PV-GMPQ1972 Three histidine-motifs are represented by black sh aded blocks while the heme binding motif is shaded in light gray.

Figure VI-2. Hydropathy Plot of the Deduced Amino Acid Sequence of Pj Δ 6D The deduced amino acid sequence was analyzed by the DNAStar-Protean soft ware package. The positive half of the hydropathy plot represents regions that are hydrophobic. Assuming ~20 amino acids are required to span a membrane, there would be at least six transmembrane spanning domains predicted. The heme binding motif, the two histidine rich motifs, and the Q-box are shown below the hydropathy plot. The first ~80 residues comprise the cytb5 domain.

The functional activity of the Pj Δ 6D protein was confirmed in vivo after expression from the yeast expression vector in S. cerevisiae . There are only four fatty acids present in

S. cerevisiae (palmitic acid [16:0], stearic acid [18:0], palmitoleic acid [16:1] and oleic acid [18:1]). S. cerevisiae cells containing the Pj.D6D expression vector were grown in the absence or presence of exogenous fatty acids. In the absence of the exogenous fatty acids, yeast cells were shown to produce two new fatty acids as, cis -6, cis -9- hexadecadienoic acid (16:2 c6,9) and cis -6, cis -9-octadecadienoic acid (18:2 c6,9), which are both the products of Δ 6 desaturation of the endogenous monounsaturated fatty acids palmitoleic acid (16:1) and oleic acid (18: 1). Cultures grown in the presence of exogenous oleic acid showed even higher levels of cis -6, cis -9-octadecadienoic acid (18:2 c6,9). Both GLA and SDA were produced wh en an equal mixture of LA and ALA was

included in the medium.

The minimum components required for in vitro activity of an integral membrane

desaturase would include an intact membrane system containing the desaturase and theMonsanto Company 09-SY-200U Page 89 of 395 requisite electron tr ansfer proteins. In the case of Pj Δ 6D, this would include a source of cytb5 and b5 reductase. Additionally, the membranes would need to be comprised of lipids that could serve as an appropriate subs trate. Enzymatic activity specific to the Pj Δ 6D protein was observed in the crude hom ogenate obtained from very young (stage 0-1, green immature seed, up to 4 mm) fr esh MON 87769 seed. The starting seed material was collected on the day of the assay, cotyledons removed, and a homogenate prepared by gently grinding the tissue at 4 °C with a mortar and pestle. When 14 C-ALACoA was incubated with the immatu re seed homogenate of MON 87769, 14 C-SDA was detected in the assay mixture. The formati on of SDA is a unique reaction attributed to the presence of the Pj Δ 6D protein in MON 87769.

The results obtained from the yeast expres sion system and crude SDA immature seed

extracts demonstrate the Δ 6 desaturase function of the Pj Δ 6D protein encoded by the Pj.D6D gene.

VI.A.2. Nc Δ 15D Protein

The Nc Δ 15D protein is identical to the native protein produced by Neurospora crassa

, with the exception of a single amino acid change from threonine to alanine at the first

amino acid after the start codon th at was introduced to facilitate the insertion of the gene into the plant transformation v ector. The MON 87769-expressed Nc Δ 15D protein consists of 429 amino acids, has a calcu lated molecular mass of 49,195 Da, and a predicted isoelectric point of 7.1. Analysis of the Nc Δ 15D amino acid sequence demonstrates the presence of three histidine motifs that are characteristic of integral membrane desaturases (Figure VI-3). The activ e site of integral membrane desaturases is thought to be comprised of th ree histidine-rich motifs with a total of nine essential histidine residues (Shanklin et al., 1994). Similar to Pj Δ 6D, the Nc Δ 15D proteins shows multiple membrane-spanning regions (Figure VI-4) based on the patterns observed from hydropathy plots.

Figure VI-3. Deduced Amino Acid Sequence of Nc Δ 15D from PV GMPQ1972 Three histidine-motifs are represented by shaded blocks. 1 MAVTTRSHKA AAATEPEVVS TGVDAVSAAA PSSSSSSSSQ KSAEPIEYPD IKTIRDAIPD
61 HCFRPRVWIS MAYFIRDFAM AFGLGYLAWQ YIPLIASTPL RYGAWALYGY LQGLVCTGIW

121 ILA HECGH GA FSRHTWFNNV MGWIGHSFLL VPYFSWKFS H HRHH RFTGHM EKDMAFVPAT
181 EADRNQRKLA NLYMDKETAE MFEDVPIVQL VKLIAHQLAG WQMYLLFNVS AGKGSKQWET
241 GKGGMGWLRV SHFEPSSAVF RNSEAIYIAL SDLGLMIMGY ILYQAAQVVG WQMVGLLYFQ
301 QYFWVHHWLV AITYLHHTHE EVHHFDADSW TFVKGALATV DRDFGFIGKH LFHNIIDH HV

361 VHH LFPRIPF YYAEEATNSI RPMLGPLYHR DDRSFMGQLW YNFTHCKWVV PDPQVPGALI
421 WAHTVQSTQMonsanto Company 09-SY-200U Page 90 of 395 Figure VI-4. Hydropathy Plot of the Deduced Amino Acid Sequence of Nc Δ 15D The deduced amino acid sequence was analyzed in the DNAStar-Protean soft ware package. The positive half of the hydropathy plot represents regions that are hydrophobic. Assuming ~20 amino acids are required to span a membrane, there are at least six transmembrane spanning domains predicted. The locations of the th ree histidine-rich motifs are shown below the hydropathy plot.

The functional activity and specificity of the Nc Δ 15D protein was confirmed in vivo via the expression of the yeast expression vector containing Nc.Fad3 (Nc Δ 15D-encoding gene) in S. cerevisiae as described for Pj Δ 6D protein in Section VI.A.1. S. cerevisiae was grown in the absence or presence of various 18 and 20 carbon fatt y acids. In the absence of the exogenous fatty acids, the y east cells produced ALA from oleic acid. The

Δ 15D protein is the only known enzyme capable of catalyzing this r eaction. Results also demonstrated that other 18 ( e.g., LA and GLA) or 20 carbon fatty acids (e.g., di-homo- γ

linolenic acid and arachidonic acid tested) were substrates for Nc Δ 15D and all of the exogenous fatty acid substrates were desaturated th ree carbons from the methyl terminus (omega-3 desaturation).

A demonstration of the functional activity of the Nc Δ 15D protein in the crude homogenate from immature MON 87769 soybean seed was not successful because the 18:3 fatty acid isomers (ALA and GLA) from the assay were not distinguishable. It should be noted that soybean seed contains an endogenous Δ 15 desaturase; therefore, this

Δ 15 desaturation reaction occurs in the convent ional control soybean seed. The fatty acid composition of yeast containing the Nc.Fad3 vector and MON 87769 demonstrate the omega-3 desaturation function of the Nc Δ 15D protein encoded by Nc.Fad3 gene.

VI.B. Characterization of the Pj Δ 6D and Nc Δ 15D Proteins Produced in

MON 87769

The safety assessment of crops improved through biotechnology includes characterization of the introduced protein( s), confirmation of their functional and physicochemical properties, and confirmation of the safety of the proteins produced from the inserted genes. A common approach used for the safety assessment of the proteins introduced into crop plants by biotechnology is to use heterologously expressed proteinsMonsanto Company 09-SY-200U Page 91 of 395 as surrogates for the plant-expressed protei ns. In such cases, the physicochemical and functional properties of the plant-produced a nd heterologously produced proteins must be established to justify th e use of the surrogate protein for sa fety testing. In the case of the two integral membrane desaturase proteins, Pj Δ 6D and Nc Δ 15D, safety assessment studies were conducted with proteins isolated directly from MON 87769 immature seed. Therefore, no equivale nce study was required.

Expression of desaturase protei ns is higher during seed devel opment, therefore, the level of Pj Δ 6D and Nc Δ 15D is higher in immature seeds than mature seeds (Tables VI-1 and VI-2). Purification of integral membrane desa turases is a challenging task that requires removal of membranes and replacement of the lipids surrounding the protein’s hydrophobic membrane-spanning regions with an a ppropriate detergent that will keep the protein in solution (Wiener, 2004). To purify the integral membrane Pj Δ 6D and Nc Δ 15D proteins from immature seeds as the source material for safety testing, the membranes were isolated and a panel of detergents was tested for ability to release these proteins from the seed membranes while maintaining thei r solubility. As a re sult, the zwitterionic detergent Fos-choline 12 was selected. Fos-choline 12 is a member of a relatively new class of detergents that are phospholipid analogs. These lipid-like detergents have a phosphocholine head group, but in contrast to phospholipids, possess simple hydrophobic tails. Fos-choline 12 has been rapidly a dopted for use in NMR studies of membrane proteins due to its structur e-stabilizing properties (Choow ongkomon et al., 2005; Farès et al., 2006; Oxenoid and Chou, 2005). Furthermore, Fos-choline 12 was an essential tool in allowing for the refolding of the integr al membrane protein di acylglycerol kinase (Gorzelle et al., 1999) and had less deleteriou s effects than other tested detergents on functional activities of some integral membra ne proteins (Li et al., 2001; Narayanan et al., 2007). After the Pj Δ 6D and Nc Δ 15D proteins were solubilized from the membranes, multiple chromatographic steps were applied to further purify these proteins. Standard precautions to retain the inte grity of the protein during purification were undertaken, including conducting all chromat ographic steps in a cold room, inclusion of protease inhibitors at critical steps, and the addition of reducing agents and glycerol to buffers. This approach allowed the isolation of these desaturases in soluble form that enabled the characterization of their physico-chemical properties. Appendix C describes the purification and char acterization of Pj Δ 6D and Nc Δ 15D proteins.

Although the activity of integral membrane de saturases can be demonstrated in crude extracts when the appropriate substrates are supplied, as with other desaturases (Reed et al., 2000), it has not been possible to assay Pj Δ 6D and Nc Δ 15D enzymatic activities following their solubilization from the membranes. This most likely is due to the desaturases’ requirement for the requisite elec tron transfer proteins co-localized in the

membrane.

To characterize the purified Pj Δ 6D and Nc Δ 15D proteins produc ed in MON 87769, a number of analyses were performed. The an alyses employed for the characterization of MON 87769-produced Pj Δ 6D and Nc Δ 15D proteins included:

1. N-terminal sequence analysis,Monsanto Company 09-SY-200U Page 92 of 395 2. Matrix-assisted laser desorption ionization - time of flight (MALDI-TOF) mass spectrometry to generate a tryptic peptide map,

3. Immunoblot analysis to es tablish protein identity through immunoreactivity with Pj Δ 6D and Nc Δ 15D-specific antibodies,

4. Sodium dodecyl sulfate polyacrylam ide gel electrophoresis (SDS-PAGE) to assess the apparent molecular weight of the protein, and

5. Glycosylation analysis to evaluate poten tial post-translational modification of the protein.

Results show that the identi ty of the MON 87769-produced Pj Δ 6D and Nc Δ 15D proteins was confirmed by: 1) their recognition by anti-Pj Δ 6D and anti-Nc Δ 15D antibodies, respectively; 2) the identification of the firs t 15 amino acids of the expected N-terminus sequence of each protein; 3) the identificati on of tryptic peptide masses of each protein that yielded >40% overall coverage of the e xpected protein sequence; 4) confirmation of the apparent molecular weights estimated to be 46 kDa; and, 5) confirmation that the proteins are non-glycosylated. Taken together, these data confirm the identity of the Pj Δ 6D and Nc Δ 15D proteins isolated from MON 87769. The Pj Δ 6D and Nc Δ 15D proteins purified from MON 87769 immature seed were used in the protein safety assessment, which is described in section VI E.

VI.C. Expression of Pj Δ 6D and Nc Δ 15D Proteins in MON 87769

Pj Δ 6D and Nc Δ 15D are integral membrane proteins, and their detection in plant tissue extracts is a difficult task using a standard ELISA format. When plant tissue is extracted in neutral buffers that do not contain detergen t, cell membranes tend to form vesicles of different orientation depending on the ch arge and hydrophobicity of the surface 

### ( Rosenberg, 1996 

### ) . Hydrophilic portions of the inte gral membrane proteins are often trapped inside membrane vesicles and not avai lable for interaction w ith antibody. Due to

their membrane lo calization, the Pj Δ 6D and Nc Δ 15D proteins require detergentcontaining buffers in order to be effectively solubilized from the membranes and to decrease the potential for aggregation. Because detergent in the extraction buffer interferes with the protein’ s ability to inter act with antibody, extracts containing solubilized Pj Δ 6D and Nc Δ 15D proteins are not well suited for analysis in liquid phase assays such as an ELISA, but are compatible with a solid phase assay such as western blot. For western blot (o r immunoblot), protein extract s are separated on SDS-PAGE performed in the presence of a denaturing dete rgent. The subsequent electrotransfer of proteins from the gel to a blotting membrane yields an immunoblot that can be probed in buffer conditions that facilitate specif ic protein-antibody interactions. For Pj Δ 6D and

Nc Δ 15D protein detection in MON 87769, optimi zed tissue extraction conditions were

combined with standard ized electrophoretic, bl otting, and immunodetection methodologies to utilize Pj Δ 6D and Nc Δ 15D peptide antibodies specific to a fragment (peptide) of soluble portion of the respect ive proteins. This method allowed for the reproducible and accurate estimation of the Pj Δ 6D and Nc Δ 15D protein levels in plant tissue samples. Densitometric analysis of the Pj Δ 6D- and Nc Δ 15D-specific immunoblots yielded the reported quantitative values of Pj Δ 6D and Nc Δ 15D proteins by interpolation from standard curves prepared using purified Pj Δ 6D and Nc Δ 15D protein standards,Monsanto Company 09-SY-200U Page 93 of 395 respectively. Limit of quantitation (LOQ) for Pj Δ 6D and Nc Δ 15D in each plant tissue type was determined from the corresponding immunoblot and was defined as the lowest

amount of the standard that could be reliabl y determined. Limit of detection (LOD) was defined as the lowest amount of Pj Δ 6D and Nc Δ 15D protein standards visually observed on X-ray films. The levels of the Pj Δ 6D and Nc Δ 15D proteins in various tissues of

MON 87769 that are relevant to the risk assessment were estimated by densitometric analysis of X-ray films exposed to immunoblots probed with Pj Δ 6D- and Nc Δ 15Dspecific antibodies and visualized using chem iluminescent detection reagents (refer to Appendix D).

VI.C.1. Protein Expression Analysis of Tissues from the 2006 U.S. Field Trials

Tissue samples for analysis were collected from five field locations in the U.S. during the 2006 soybean growing season. The trial locations were in the states of Iowa (two sites), Illinois, Michigan, and Ohio, which represen t major soybean-growing regions of the U.S. and provide a range of environmental condi tions that would be encountered in the commercial production of soybean. At each si te, three replicated plots of MON 87769 and conventional soybean control (A3525) we re grown using a randomized complete block field design. Over-season leaf (OSL), fo rage, root, mature seed, and immature seed were collected from each replicat ed plot from all field sites. A description of the tissues collected is provided below.

Twenty sets of the youngest expanded trifol iate leaves were randomly collected from plants of each plot at each site. The twenty leaves were combined to form the leaf sample for each plot. OSL samples were collected as follows:

Over-season leaf (OSL) Development stage Days after planting (DAP)

OSL-1 V3-V4 25-37

OSL-2 V6-V8 38-46

OSL-3 V10-V12 46-59

OSL-4 V14-V16 56-73

The above ground portion of six plants was collected from each plot at each site at the R6 stage and combined to form the forage sample . Roots remaining from four to six plants after the collection of the forage samples from each plot were combined to form the root sample. Forage and root samples were collected as follows:

Tissue Development stage Days after planting (DAP) Forage R6 81-108

Root R6 81-108

Immature seed was collected from each plot , with a sufficient number of plants being sampled to yield ~15-56 g of tissue. Mature seed was harvested from all plants of each plot yielding 3.0 to 8.1 lb of mature seed per plot. Immature and mature seed were

harvested as follows:Monsanto Company 09-SY-200U Page 94 of 395 Tissue Development stage Days after planting (DAP) Immature seed R5 to early R6 76-96

Mature seed R8 (95% mature pod color) 120-153

All tissue samples, except mature seed, were stored and shipped on dry ice for processing and analysis. Mature seed was stored and shipped at room temperature. All tissue samples were stored in a -80 oC freezer upon re ceipt. Tissue samples were extracted and analyzed using an immunoblot according to applicable standard operating procedures (SOP). Appendix D describes the materials a nd methods used for th e protein expression analysis.

The levels of Pj Δ 6D and Nc Δ 15D proteins in OSL-1 th rough OSL-4, forage, root,

immature and mature harvested seed were determined using immunoblot and the results

are summarized in Tables VI-1 and VI-2. Protein levels for al l tissue types were calculated on a microgram ( μ g) per gram (g) fresh weight (FW) basis. For tissues displaying measurable quantities of Pj Δ 6D and Nc Δ 6D proteins, moisture content also was measured and protein levels we re also expressed on a microgram ( μ g) per gram (g) dry weight of tissue (DW) basis.

The expression of the Pj Δ 6D and Nc Δ 15D proteins is driven by a 7S α ' and 7S α seed specific promoter, respectively. As expected, both Pj Δ 6D and Nc Δ 15D proteins were

undetectable across all field sites in OSL-1, OSL-2, OSL-3, OSL-4, and root tissues of

MON 87769 due to the lack of a measurable si gnal specific to each protein at or above the tissue-specific LOD of the immmunoblot (Table s VI-1 and VI-2). Both proteins were detected in immature seed, mature seed, and at low levels in forage because this tissue usually contains a small amount of immature seed. The mean Pj Δ 6D protein levels across all sites for immature seed, mature seed and forage were 100, 1.8, and 16 μ g/g DW, respectively (Table VI-1). The mean Nc Δ 15D protein levels across all sites for immature seed, mature harvested seed and forage were 200, 10, and 14 μ g/g DW, respectively (Table VI-2). The Pj Δ 6D and Nc Δ 15D proteins were not detected in the conventional soybean control, A3525.Monsanto Company 09-SY-200U Page 95 of 395 Table VI-1. Summary of Pj Δ 6D Protein Levels in Tissues Collected from
MON 87769 Produced in the 2006 U.S. Growing Season

Tissue Type Pj Δ 6D Mean ( μ g/g FW) (SD) 1 Range 2

( μ g/g FW) Pj Δ 6D Mean ( μ g/g DW) (SD) 3 Range ( μ g/g DW) LOD ( μ g/g FW)

OSL-1 < LOD n.a. n.a. n.a. 0.2

OSL- 2 < LOD n.a. n.a. n.a. 0.2

OSL -3 < LOD n.a. n.a. n.a. 0.1

OSL- 4 < LOD n.a. n.a. n.a. 0.1

Root < LOD n.a. n.a. n.a. 0.1

Forage 4.3 (2.4) 1.0-7.4 16 (9.5) 3.6-28 0.1

Immature

seed 27 (15) 5.6-45 100 (63) 19-210 0.2

Mature 4

seed 1.7 (0.86) 0.45-3.0 1.8 (0.95) 0.50-3.2 0.4

n.a – not applicable; LOD – Limit of Detection.

1 Protein quantities are expr essed as micrograms ( μ g) of protein per gram (g) of tissue on a fresh weight

(FW) basis. When applicable, an ar ithmetic mean and a standard deviatio n (SD) were calculated for each tissue type across all sites (n=15 for all tissues).

2 When applicable, minimum and maximum values were de termined for each tissue type across all sites.

3 When applicable, protein quantities are expressed as “ μ g/g” of tissue on a dry weight (DW) basis. The

dry weight values were calculated by dividing the fresh weight values by the dry weight conversion factors obtained from moisture analysis data.

4 For the Pj Δ 6D levels in mature seed, the low end of range was below the LOQ of the western blot assay,

but above the LOD of 0.4 μ g/g FW that was determined by serially diluting protein standard to the lowest amount that produced a visible band in the western blot. The reported values were based on the protein signal observed on the western blot and extrapolation of the standard curve.

Limit of Quantitation (LOQ) for the Pj Δ 6 desaturase western blot are as follows:

Limit of Quantitation ( μ g/g FW) 1

Tissue Type Pj Δ 6D Western Blot

OSL-1, OSL-2, OSL-3 and OSL-4 1.0

Root 1.0

Forage 1.0

Immature Seed 4.0

Mature Seed 2.0

1 For each tissue type, the Limit of Quantitation (LOQ) was calculated based on the lowest amount of protein standard visualized and recorded from the western blot in the study.Monsanto Company 09-SY-200U Page 96 of 395 Table VI-2. Summary of Nc Δ 15D Protein Levels in Tissues Collected from
MON 87769 Produced in the 2006 U.S. Growing Season

Tissue Type Nc Δ 15D Mean ( μ g/g FW) (SD) 1 Range 2

( μ g/g FW) Nc Δ 15D Mean ( μ g/g DW) (SD) 3 Range ( μ g/g DW) LOD ( μ g/g FW)

OSL-1 < LOD n.a. n.a. n.a. 0.5

OSL-2 < LOD n.a. n.a. n.a. 1.0

OSL-3 < LOD n.a. n.a. n.a. 0.5

OSL-4 < LOD n.a. n.a. n.a. 1.0

Root < LOD n.a. n.a. n.a. 0.5

Forage 3.7 (1.7) 1.3-7.9 14 (6.8) 4.6-30 1.0

Immature

seed 55 (21) 20-85 200 (89) 66-330 4.0

Mature

seed 9.5 (5.9) 4.3-23 10 (6.5) 4.8-25 2.0

n.a – not applicable; LOD – Limit of Detection.

1 Protein quantities are expressed as micrograms ( μ g) of protein per gram (g) of tissue on a fresh weight

(FW) basis. When applicable, an ar ithmetic mean and a standard deviatio n (SD) were calculated for each tissue type across all sites (n=15 for all tissues).

2 When applicable, minimum and maximum values were determined for each tissue type across all sites.

3 When applicable, protein quantities are expressed as “ μ g/g” of tissue on a dry weight (DW) basis. The

dry weight values were calculated by dividing the fresh weight values by the dry weight conversion factors obtained from moisture analysis data.

Limit of Quantitation (LOQ) for the Nc Δ 15 desaturase western blot are as follows:

Limit of Quantitation ( μ g/g FW) 1

Tissue Type Nc Δ 15D Western Blot

OSL-1, OSL-2, OSL-3 and OSL-4 2.0

Root 2.0

Forage 1.0

Immature Seed 10.0

Mature Seed 2.0

1 For each tissue type, the Limit of Quantitation (LOQ ) was calculated based on the lowest amount of protein standard visualized and recorded from the western blot in the study.Monsanto Company 09-SY-200U Page 97 of 395 VI.D. Assessment of the Potentia l for Allergenicity of the Pj Δ 6D and Nc Δ 15D

Proteins Produced in MON 87769

The Nc Δ 15D and Pj Δ 6D proteins have been assessed for their potential allergenicity according to the recommendations of the Codex Alimentarius Commission (Codex, 2003). Both proteins are from nonallergenic so urces, lack structural similarity to known allergens, are digested in simulated gastric and intestinal fluids, and constitute a small portion of the total protein present in MON 87769 seed.

The Pj Δ 6D protein is from Primula juliae , an organism that is not known to be allergenic. Bioinformatics analyses demonstrated that the Pj Δ 6D protein does not share structurally or immunologically relevant amino acid sequen ce similarities with kno wn allergens and, therefore, is highly unlikely to contain immunologically cross-re active allergenic epitopes. Digestive fate experiments conducted with the Pj Δ 6D protein demonstrated that the fulllength protein is rapidly dige sted in SGF, a characteristic shared among many proteins with a history of safe consumption. The tran siently stable protein fragments in the SGF assay were quickly degraded during a short expo sure to SIF. Rapid digestion of the fulllength Pj Δ 6D protein in SGF and SIF, together with rapid degradation of the transiently stable fragment from the SGF assay by SIF, in dicates that it is highly unlikely that the Pj Δ 6D protein and its fragment will reach abso rptive cells of the intestinal mucosa. Finally, the Pj Δ 6D protein represents no more than 0.00043% of the total protein in

MON 87769 mature seed.

The Nc Δ 15D protein is derived from Neurospora crassa , a fungus that is ubiquitous in the environment, is not allergenic, and found in the digestive tracts of vertebrate species, including humans. Bioinformatics an alyses demonstrated that the Nc Δ 15D protein does not share structurally or immunologically rele vant amino acid sequence similarities with known allergens and, therefore, is highly unl ikely to contain immunologically crossreactive allergenic epitopes. Digestive fate experiments conducted with the Nc Δ 15D protein demonstrated that th e full-length protein is ra pidly digested in SGF, a characteristic shared among ma ny proteins with a history of safe consumption. The transiently stable protein frag ments from the SGF assay of Nc Δ 15D were very quickly degraded during short exposure to SIF. Rapid digestion of the full-length Nc Δ 15D protein in SGF and SIF, together with rapid de gradation of the transiently stable fragment from the SGF assay by SIF, indicates th at it is highly unlikely that the Nc Δ 15D protein and its fragment will reach absorptive cells of the intestinal mucosa. Finally, the

Nc Δ 15D protein represents no more than 0.00239% of the total protein in MON 87769

mature seed.

Taken together, these data lead to the conclusion that the Pj Δ 6D and Nc Δ 15D proteins do not pose a significant allergenic risk.

VI.E. Assessment of the Pote ntial for Toxicity of the Pj Δ 6D and Nc Δ 15D Proteins

The Pj Δ 6D and Nc Δ 15D proteins have been assessed for their potential toxicity. Homologues of Nc Δ 15D and Pj Δ 6D are present in all kin gdoms, from prokaryotes toMonsanto Company 09-SY-200U Page 98 of 395 humans. Many organisms that either are c onsumed as food or used to produce food constituents contain homologous desaturases, supporting the general familiarity of these proteins in human and animal diets. The pr oteins lack structural similarity to known toxins or biologically active proteins known to have adverse effects to mammals. The Pj Δ 6D and Nc Δ 15D proteins are present at a very low level in the harvested seed of MON 87769 and, therefore, constitute a negligib le portion of the tota l protein present in

food and feed derived from MON 87769. The Pj Δ 6D and Nc Δ 15D proteins were readily digestible in simulated gastri c and intestinal fluids.

Acute toxicology studies were conducted with Pj Δ 6D and Nc Δ 15D purified from MON 87769 immature seed. The Pj Δ 6D and Nc Δ 15D did not cause any adverse effects on mice at a NOAEL of 4.7 mg/kg and 37.3 mg/ kg BW, respectively. Human margin of safety was calculated by comparing the NOAEL with acute dietary consumption of soybean. The margin of safety was estimated to be 29,000 for Pj Δ 6D and 47,000 for

Nc Δ 15D. Estimated dietary intake of the Pj Δ 6D and Nc Δ 15D proteins by animals was at extremely low levels compared to the consumption of total proteins present in animal feed. These data indicate that the Pj Δ 6D and Nc Δ 15D proteins are safe for human and animal consumption.Monsanto Company 09-SY-200U Page 99 of 395 VII. COMPOSITIONAL AND NUTR ITIONAL ASSESSMENT OF MON 87769

Compositional analyses were conducted to a ssess whether the nutrients and antinutrient levels in harvested seed and forage derive d from MON 87769 are comparable to those in the conventional soybean control, A3525. In addition, 10 commercial conventional soybean varieties were included in the analys is as references to establish a range of natural variability for each analyte, where th e range of variability is defined by a 99% tolerance interval for that particular analyte. Compositional analysis included the significant nutrients, antinutri ents, and key secondary metabolites, consistent with OECD guidelines (OECD, 2001). As expected, MON 87769 contains SDA and GLA. Due to the known spontaneous isomerization of polyuns aturated fatty acids, small amounts of

the trans-isomers of SDA and ALA that are fo rmed during seed extraction and processing are also expected in MON 87769 analysis. Therefore, MON 87769 harvested seed was also analyzed for SDA, GLA, trans-SDA a nd trans-ALA. Results of the comparisons indicate that except for the intended fatty acid change, MON 87769 is compositionally and nutritionally equivalent to conventional soybean that is currently in commerce and has a history of safe consumption by humans and animals.

Seed and forage of MON 87769 and the conve ntional soybean control were harvested from soybean grown in three re plicated plots at each of fi ve sites across the U.S., during the 2006 growing season. Samples we re collected from MON 87769 and the conventional soybean control from all three plots; samples from the three different commercial reference varieties grown at each site were collected from a single plot. Forage was collected at the R6 plant growth stage and soybean seed was collected at physiological maturity. The seed and forage collected from MON 87769, the conventional control, and the reference va rieties were analyzed for compositional components. Appendix E describes the materi als and methods used for the compositional analysis.

In all, 75 different analytical components were measured, 68 in seed and seven in forage. Of the measured components, 26 in seed had more than 50% of the observations below the assay limit of quantitation (LOQ) and were excluded from the statistical analysis. Thus, statistical analyses were conducted fo r 49 components (42 in seed and seven in forage). The overall data set was examined for evidence of biologi cally relevant changes using a mixed model of variance. Six sets of statistical analyses were conducted, five based on the data from each of the replicated field sites (individual-site) and the sixth analysis based on data from a combination of all five field sites (combined-site). The compositional data set was examined for evidence of statistically significant differences between MON 87769 and the conventional soybean control. Statistically significant

differences were determined at the 5% leve l of significance (p< 0.05) using established

statistical methods.

A 99% tolerance interval was calculated for each compositional component, except for the fatty acid components that are intentionall y changed. This interval is expected to contain, with 95% confidence, 99% of th e values obtained fr om the population of commercial reference varieties. It is important to establish the 99% tolerance interval from representative conventional soybean vari eties for each of the analytes, because suchMonsanto Company 09-SY-200U Page 100 of 395 data illustrate the compositional variability th at occurs naturally in commercially grown varieties. By comparison to the 99% to lerance interval, any significant difference (p<0.05) between MON 87769 and the conven tional control can be assessed for biological relevance in the cont ext of the natural variability in soybean. This comparative evaluation also considers natural ranges in soybean component levels published in the

literature or in the International Life Sc iences Institute Crop Composition Database (ILSI-CCD, 2008). 2

VII.A. Intended Changes to Fatty Ac id Levels in MON 87769 Soybean Seed

MON 87769 was developed to contain SDA at le vels of approximately 20 to 30% of total fatty acids. As expected, composition analysis showed that the levels of SDA in MON 87769 harvested seed ranged from 16.83 to 33.92% of total fatty acids, with a mean of 26.13% (Table VII-1). Associated with the expected levels of SDA in MON 87769 is the production of GLA from the Δ 6 desaturation of linoleic acid (LA) by Pj Δ 6D (Figure VII-1). The GLA levels in MON 87769 ranged from 6.07 to 8.03% of total fatty acids, with a mean of 7.09%. Lo wer levels of two othe r fatty acids, trans-SDA (mean = 0.18%, range = 0.058 - 0.26% of tota l fatty acids) and trans-ALA (mean = 0.44%, range = 0.38 - 0.48% of total fatty acids) also were observed (Table VII-1). The

formation of trans-ALA and trans-SDA is due to the known spontaneous transisomerization of unsaturated fatty acids during seed extr action and processing. Trans isomerization of unsaturated fatty acids are kno wn to increase with increasing degree of

unsaturation 

### ( Chardigny et al., 1996 

### ) . As SDA and ALA represent a significant proportion of total fatty acids in MON 87769 (approximately 35 - 40% in total), transALA and trans-SDA are expected to be seen in the fatty acid anal ysis of MON 87769.

Since SDA, GLA, and the two trans-isomer analytes were not detected in the conventional soybean control seed, statis tical comparisons between MON 87769 and the conventional soybean control were not possibl e for these analytes. The mean values, standard errors, and the range of values for the introduced fatty acids observed in MON 87769 are presented as % total fatty acids and % dry weight in Table VII-1.

2 ILSI-CCD. 2008. International Life Sciences Institute Crop Composition Database. Version 3.0 http://www.cropcomposition.org. Search criteria soybean seed, all locations, all years, all proximates, amino acids, fatty acids, bio-actives, fiber, dry weight other than moisture [Accessed June 3, 2008].Monsanto Company 09-SY-200U Page 101 of 395 Table VII-1. Combined-Site Summary of SDA, GLA, trans-SDA and trans-ALA

Levels in MON 87769 Seed

Analytical Component (Units)1 MON 87769 Mean (S.E.) 2 MON 87769 (Range)

Combined-Site Seed Fatty Acid (% Total FA)

18:4 Stearidonic (SDA) 26.13 (1.64) [16.83 - 33.92]

18:3 γ -Linolenic (GLA) 7.09 (0.19) [6.07 – 8.03]

18:4 6c,9c,12c,15t (trans-SDA) 0.18 (0.019) [0.058 - 0.26]

18:3 9c,12c,15t (trans-ALA) 0.44 (0.0091) [0.38 – 0.48]

Combined-Site Seed Fatty Acid (% DW)

18:4 Stearidonic (SDA)

18:3 γ -Linolenic (GLA)

18:4 6c,9c,12c,15t (trans-SDA)

18:3 9c,12c,15t (trans-ALA) 3.94 (0.15)

1.09 (0.023)

0.027 (0.0023)

0.068 (0.0018) [2.77 - 4.91]

[0.93 - 1.22]

[0.011 - 0.036]

[0.055 - 0.081]

1DW = dry weight; 2 S.E. = standard error.

VII.B. Compositional Comparisons of Soybean Seed and Forage from

MON 87769 and the Conventional Control

Of the 42 comparisons made in the combined-s ite analysis between harvested seed from

MON 87769 and the conventional soybean control, a total of 28 statistically significant differences, representing six fa tty acids and 22 non-fatty acid analytes, were observed. In forage, no significant differences were observed for any analyte for the seven comparisons made between MON 87769 and th e conventional soybean control in the combined-site analysis. A summary of the significant differences (p<0.05) from the

combined-site statistical ev aluation of MON 87769 versus conventional soybean control

can be found in Table VII-2. Of the 28 detect ed differences in seed, 27 (except LA) were small in magnitude and/or their mean co mponent values were all within the 99% tolerance interval for the population of convent ional references grown at the same time

and location as MON 87769 and also within th e range of values found in the published literature and ILSI-CCD values. Therefore, th ese differences were not considered to be biologically meaningful from a food/feed safety and/or nutritional perspective. The LA

levels in seed were lower for MON 87769 in the combined-site anal ysis and outside ofMonsanto Company 09-SY-200U Page 102 of 395 the 99% tolerance interval fo r the population of conventional references. These results were expected, because LA is the startin g material from which SDA and GLA are produced and, therefore, its levels were e xpected to be significantly different in MON 87769 seed compared to conventional soybean seed.

Further assessment of the statistically si gnificant differences observed between

MON 87769 and the conventional s oybean control are discussed in the sections below. The analytes that are significantly diffe rent between MON 87769 and the conventional soybean control in the combined-site anal ysis are presented in Table VII-2. The

statistical summaries of the combined-site anal ysis data, individual-si te analysis data, and reported literature and ILSI-CCD ranges for the analytical components present in the harvested seed are included in Appendix E.

VII.B.1. Fatty Acid Levels in Soybean Seed

Statistical comparisons of fatty acids be tween MON 87769 and the conventional soybean control seed could be made only for the eight fatty acids present in both MON 87769 and the conventional soybean control. Out of the eight fatty acids compared, significant differences (p<0.05) were observed for six fatty acids in the combined-site and in more than one individual-site analysis (Table V II-2). In MON 87769 seed, oleic acid, LA, and ALA were found to be significan tly different from the conv entional soybean control in

the combined-site and in the five individual-si te analyses. Since oleic acid, LA, and ALA are directly involved in the pathway to SD A (Figure VII-1), thei r concentrations are interdependent with that of other fatty acids a nd, therefore, were expected to be different

in MON 87769 seed. Arachidic acid was fou nd to be significantly different from the control in the combined-site analysis and in four of the five indi vidual-site analyses. Palmitic acid and behenic acid values were significantly different in the combined-site analysis and in two individual-site analyses . Given the intended shift in the fatty acid metabolism toward an increase in SDA content in MON 87769, differences in fatty acid levels were expected. Except for LA, the diffe rences in fatty acid levels were relatively small in absolute magnitude (<4 wt% of total fatty acids) and/or their mean values and ranges in MON 87769 seed were within the 99 % tolerance interval for the population of

the conventional reference variet ies. Therefore the differences in fatty acid levels with the exception of the intended change are not c onsidered to be biol ogically meaningful from a food/feed safety and/or nutritional perspective. The levels of LA in MON 87769 were significantly lower than the control in the combined-site as well as in the five individual-site analyses. Since LA is the starting material from which SDA and GLA are produced (Figure VII-1), LA leve ls were expected to be significantly lower across sites.Monsanto Company 09-SY-200U Page 103 of 395 Pathways where soybean endogenous desaturases work

Pathways where exogenous desaturases work

Figure VII-1. Fatty acid biosynthesis in plants and the introduced changes to produce MON 87769

VII.B.2. Nutrient Levels Other than Fatty Acids in Soybean Seed

Soybean seed also was analyzed for the following 26 nutrients: proximate (5), ADF, NDF, amino acids (18), and vitamin E. Ninet een nutrient analytes in the seed were statistically different (p<0.05) between M ON 87769 and the conventio nal control in the combined-site analysis. For five of these 19 nutrient analytes (proline, arginine, cystine, glycine, and phenylalanine), st atistically significant differe nces were observed in the combined-site analysis and in more than one individual-site analys es (Table VII-2).

Proline values in MON 87769 were found to be significantly different from the conventional soybean control in the combined-site analysis and in three of the five individual-site analyses. The magnitude of the differences in proline between

MON 87769 and the conventional control from the combined-site and individual-site analyses were very small, ranging from 2.94 to 5.77%. Statistically significant differences for arginine, cystine, glycine, and phenylalanine were observed in the

combined-site and in two individual-site an alyses. The magnitude of the differences between MON 87769 and conventional soybean c ontrol for arginine, cystine, glycine, and phenylalanine also were very small ( ≤ 10%). The mean and range values for proline, arginine, cystine, glycine, and phenylalani ne in MON 87769 seed were within the 99% tolerance interval for the population of the conventional reference varieties.

Eight of the 19 nutrient analytes that were significantly differe nt between MON 87769 and conventional control (aspartic acid, glutam ic acid, histidine, isoleucine, leucine, lysine, valine and protein) were observed in the combined-site analysis and in one individual-site analysis. The magnitude of these differences were very small (<10%).

18:0

stearic acid 18:1

oleic

acid 18:2

linoleic

acid 18:3

α -linolenic acid Δ 9 Δ 12

Δ 6

18:3

γ -linolenic

acid 18:4

stearidonic

acid Δ 15 Δ 6 Δ 15Monsanto Company 09-SY-200U Page 104 of 395 For the remaining six of 19 nutrient analytes, significant differences only were found in the combined-site analysis (alanine, meth ionine, serine, thre onine, tyrosine and carbohydrates). The magnitude of these differe nces also were very small (<10%). The mean and range of values for all 19 nutrient analytes that were significantly different

between MON 87769 and conventional control in the combined-site analysis and in the individual-site analyses were within the 99% tolerance interval for the population of conventional references and within the range of values found in th e published literature and/or the ILSI-CCD (Tables VII-2 and E-14) . Therefore, these differences were not considered to be biologically meaningful from a food/feed safety and/or nutritional perspective.

VII.B.3. Naturally Occurring Antinut rient Levels in Soybean Seed

Soybean seed contains several well-describe d antinutritional factors according to OECD 

### ( 2001 

### ) , which include: trypsin inhibitors, lectin s, isoflavones (genis tein, daidzein and glycitein), stachyose, raffinose, and phytic ac id. The analytes th at are significantly

different between MON 87769 and the conventi onal soybean control are presented in

Table VII-2.

Trypsin inhibitors are heat-labil e antinutrients that interfere w ith the digestion of proteins and result in decreased animal growth (Liene r, 1994). Lectins are also heat labile, and lectins can inhibit growth and cause death in animals if raw soybean is consumed (Liener, 1994). Both trypsin inhibitors and lectins are inactivated during processing of soybean protein products or soybean meal, and if pro cessed appropriately, the final edible soybean

fractions contain minimal levels of these anti nutrients. No statistical differences were observed for all comparisons of trypsin inhi bitors and lectin levels between MON 87769

and conventional control.

There are three basic categories of isoflavones in soybean seed; namely, daidzein, genistein, and glycitein. Although they ha ve been reported to possess biochemical activities, including estr ogenic, anti-estrogenic, and hypochol esterolemic effects, it is not universally accepted that the isoflavones are anti nutrients as they have also been reported

to have beneficial anti-carci nogenic effects (OECD, 2001). Ge nistein and daidzein levels in MON 87769 were found to be significantly different (p>0.05) from the conventional soybean control in the combined-site analysis and in four of the five individual-site analyses. The mean and range of values for these two isoflavones for MON 87769 in the combined-site analysis and in each of four individual-site an alyses were lower than the values observed in the conventional soybean co ntrol (Table VII-2). Glycitein levels in MON 87669 were found to be significantly lowe r from the conventional soybean control

in the combined-site and in one individual-site analysis. It is well documented that the soybean isoflavone levels are greatly in fluenced by many factors, ranging from environmental conditions, variety, and agronomic practices 

### ( Messina, 2001; Nelson et al.,
2001; OECD, 2001 

### ) . Furthermore, the significant differences are not biologically meaningful since the mean levels of genistein and daidzein in MON 87769 are well

within the 99% tolerance intervals establishe d from conventional soybean varieties, and

within the literature and ILS I-CCD (Tables VII-2 and E-14). Therefore, these differencesMonsanto Company 09-SY-200U Page 105 of 395 do not raise any nutritional, antinutritional or other biological or toxicological concerns and are not considered biologically relevant.

Stachyose and raffinose are low molecular weig ht carbohydrates presen t in soybean seed

that are considered to be antinutrients due to the gas production and resulting flatulence caused by consumption. There were no signi ficant differences (p>0.05) observed for stachyose and raffinose betw een MON 87769 and the conve ntional soybean control (Table VII-2).

Phytic acid is present in soybean seed, wh ere it chelates mineral nutrients, including calcium, magnesium, potassium, iron, and zinc, rendering them biologically unavailable to monogastric animals consuming the seed (Liener, 2000). Unlike trypsin inhibitors, phytic acid is not heat labile, and remains stable through most soybean processing steps. No significant differences (p>0.05) in phytic acid levels were observed when MON 87769 was compared to the conventional soybean control (Table VII-2).

Based on the data and information presented above, it is reasonable to conclude that MON 87769 is compositionally equivalent to conventional soybean with regard to the antinutrients in soybean seed.

VII.B.4. Proximate and Fiber Levels in Soybean Forage

Combined-site analysis of forage showed no significant differences (p>0.05) between MON 87769 and the conventional soybean control. In one of the five individual-site analyses, total fat and ADF were signifi cantly different (p<0.05) between MON 87769 and the conventional control forage. All mean and range of values obtained from MON 87769 in the five individual-site analyses were within the cal culated 99% tolerance interval for the population of conventional references gr own at the same time and locations, therefore, th e two differences found in one indi vidual-site analysis were not considered to be biologically meaningful from a food/feed safety and/or nutritional perspective.

VII.B.5. Compositional Equivalence of MON 87769 to Conventional Soybean

The compositional data presented in Secti on VII.B establishes that MON 87769 seed is compositionally equivalent to conventional soybean seed, with the exception of the intended change in fatty acid composition. As expected, MON 87769 contains SDA and GLA. Due to the known spontaneous isomeri zation of polyunsaturated fatty acids during the seed extraction and processing, small amou nts of the trans-isomers of SDA and ALA are also seen in MON 87769 compostion anal ysis. In addition, the forage from MON 87769 is compositionally equivalent to conventional soybean forage.Monsanto Company 09-SY-200U Page 106 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in theCompositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial Reference VarietiesMean Difference(Test minus Control)Component (Units)1MON 87769MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval2Statistical Differences Observed in Combined-Site AnalysisSeed Amino Acid (% DW)Alanine 1.78 1.74 2.51 0.001 [1.76 - 1.84] [1.45, 2.02]Arginine 3.23 2.95 9.35 <0.001 [3.00 - 3.61] [2.13, 3.62]Aspartic Acid 4.54 4.36 4.04 0.007 [4.41 - 4.73] [3.45, 5.29]Cystine 0.62 0.6 3.23 <0.001 [0.56 - 0.65] [0.49, 0.68]Glutamic Acid 7.63 7.29 4.7 <0.001 [7.42 - 7.90] [5.51, 9.04]Glycine 1.79 1.73 3.6 0.003 [1.76 - 1.87] [1.39, 2.05]Histidine 1.09 1.05 3.42 <0.001 [1.06 - 1.14] [0.86, 1.27]Isoleucine 1.87 1.78 4.95 <0.001 [1.75 - 1.97] [1.34, 2.28]Leucine 3.19 3.09 3.28 <0.001 [3.13 - 3.32] [2.45, 3.76]Lysine 2.67 2.6 2.69 <0.001 [2.61 - 2.75] [2.13, 3.06]Methionine 0.6 0.58 2.99 0.038 [0.54 - 0.62] [0.48, 0.66]Phenylalanine 2.14 2.06 3.63 0.002 [2.08 - 2.24] [1.61, 2.55]Proline 2.09 1.99 5.13 <0.001 [2.03 - 2.19] [1.53, 2.45]Serine 2.2 2.14 2.55 0.043 [2.08 - 2.25] [1.75, 2.51]Monsanto Company 09-SY-200U Page 107 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial ReferenceVarieties (Continued)Mean Difference(Test minus Control)Component (Units)1MON 87769 MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval2Seed Amino Acid (% DW)Threonine 1.6 1.57 1.9 0.035 [1.54 - 1.65] [1.30, 1.82]Tyrosine 1.4 1.34 4.43 0.013 [1.27 - 1.50] [1.03, 1.67]Valine 1.98 1.88 5.08 <0.001 [1.84 - 2.08] [1.42, 2.41]Seed Fatty Acid (% Total FA)16:0 Palmitic 12.06 11.77 2.5 <0.001 [11.53 - 12.54] [7.28, 14.20]18:1 Oleic 15.18 19.19 -20.92 0.001 [12.66 - 18.80] [12.56, 27.98]18:2 Linoleic 22.78 54.93 -58.53 <0.001 [16.46 - 30.81] [50.46, 59.96]18:3 Linolenic 11.18 9.2 21.51 0.016 [10.20 - 11.80] [3.72, 13.46]20:0 Arachidic 0.34 0.31 9.88 <0.001 [0.31 - 0.37] [0.20, 0.45]22:0 Behenic 0.29 0.32 -8.3 0.023 [0.26 - 0.31] [0.22, 0.49]Seed Proximate (%DW)Carbohydrates 36.45 38.68 -5.78 <0.001 [33.23 - 39.93] [26.76, 45.99]Protein 41.92 39.75 5.47 <0.001 [40.92 - 43.36] [33.37, 46.00]Seed Isoflavone (μg/g DW)Daidzein 1187.81 1807.36 -34.28 0.006 [957.23 - 1838.91] [0, 2594.50]Genistein 733.64 1136.52 -35.45 0.007 [576.70 - 1118.40] [254.31, 1976.30]Monsanto Company 09-SY-200U Page 108 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial ReferenceVarieties (Continued)Mean Difference(Test minus Control)Component (Units)1MON 87769 MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval2Glycitein (μg/g DW) 82.73 102.18 -19.04 0.004 [65.37 - 106.72] [0, 243.40]Statistical Differences Observed inMore than One Individual SiteSeed Amino Acid (% DW)Arginine Site IA-2 3.42 3.11 10.03 0.003 [3.36 - 3.50] [2.13, 3.62]Arginine Site OH 3.11 2.86 8.67 0.012 [3.07 - 3.15]Cystine Site IA-2 0.62 0.6 4.05 0.005 [0.62 - 0.63] [0.49, 0.68]Cystine Site IL 0.63 0.61 3.21 0.01 [0.62 - 0.64]Glycine Site IA-1 1.8 1.72 4.38 0.046 [1.79 - 1.80] [1.39, 2.05]Glycine Site OH 1.76 1.71 2.87 0.024 [1.76 - 1.77]Phenylalanine Site IA-1 2.13 2.03 5.18 0.024 [2.08 - 2.16] [1.61, 2.55]Phenylalanine Site OH 2.12 2.05 3.69 0.015 [2.12 - 2.13]Proline Site IA-1 2.12 2.0 5.77 0.039 [2.11 - 2.13] [1.53, 2.45]Proline Site IA-2 2.12 2.06 2.94 0.014 [2.09 - 2.15]Proline Site OH 2.05 1.95 4.98 0.011 [2.03 - 2.06]Seed Fatty Acid (% Total FA)16:0 Palmitic Site IL 12.31 12.0 2.51 0.024 [12.24 – 12.39] [7.28 – 14.20]16:0 Palmitic Site MI 12.11 11.79 2.66 0.019 [12.03 - 12.19]18:1 Oleic Site IA-1 13.42 18.45 -27.29 0.001 [13.14 - 13.80] [12.56, 27.98]Monsanto Company 09-SY-200U Page 109 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial ReferenceVarieties (Continued)Mean Difference(Test minus Control)Component (Units)1MON 87769 MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval218:1 Oleic Site IA-2 13.56 18.53 -26.84 0.003 [12.93 - 14.36]18:1 Oleic Site IL 17.89 20.89 -14.35 0.01 [17.52 - 18.18]18:1 Oleic Site MI 12.92 17.44 -25.95 <0.001 [12.66 - 13.16]18:1 Oleic Site OH 18.1 20.65 -12.31 0.046 [16.73 - 18.80]18:2 Linoleic Site IA-1 18.46 54.9 -66.39 <0.001 [18.24 - 18.68] [50.46, 59.96]18:2 Linoleic Site IA-2 21.19 55.33 -61.7 <0.001 [20.36 - 22.78]18:2 Linoleic Site IL 30.48 54.33 -43.9 <0.001 [30.26 - 30.81]18:2 Linoleic Site MI 18.4 55.6 -66.92 <0.001 [16.46 - 19.58]18:2 Linoleic Site OH 25.37 54.5 -53.45 <0.001 [25.06 - 25.75]18:3 Linolenic Site IA-1 11.11 9.85 12.85 <0.001 [11.08 - 11.13] [3.72, 13.46]18:3 Linolenic Site IA-2 11.14 9.96 11.84 0.018 [11.10 - 11.18]18:3 Linolenic Site IL 10.27 7.59 35.25 0.002 [10.20 - 10.38]18:3 Linolenic Site MI 11.76 10.59 10.99 0.001 [11.72 - 11.80]18:3 Linolenic Site OH 11.63 8.02 45.05 0.001 [11.41 - 11.75]20:0 Arachidic Site IA-1 0.33 0.3 10.66 0.005 [0.32 - 0.34] [0.20, 0.45]20:0 Arachidic Site IL 0.36 0.34 8.18 0.025 [0.36 - 0.37]Monsanto Company 09-SY-200U Page 110 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial ReferenceVarieties (Continued)Mean Difference(Test minus Control)Component (Units)1MON 87769 MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval220:0 Arachidic Site MI 0.32 0.29 10.39 0.029 [0.31 - 0.32]20:0 Arachidic Site OH 0.36 0.33 8.57 0.011 [0.35 - 0.36]22:0 Behenic Site IA-1 0.29 0.31 -8.9 0.018 [0.26 - 0.31] [0.22, 0.49]22:0 Behenic Site IL 0.31 0.33 -6.71 0.046 [0.30 - 0.31]Seed Isoflavone (μg/g DW)Daidzein Site IA-1 995.39 1550.96 -35.82 0.001 [978.37 - 1026.28] [0, 2594.50]Daidzein Site IA-2 1076.04 1583 -32.03 0.002 [999.02 - 1130.31]Daidzein Site MI 1662.22 2750.13 -39.56 0.016 [1389.19 - 1838.91]Daidzein Site OH 1125.54 1668.07 -32.52 0.002 [1094.38 - 1183.11]Genistein Site IA-1 594.53 973.04 -38.9 <0.001 [584.75 - 612.91] [254.31, 1976.30]Genistein Site IA-2 656.8 1044.68 -37.13 0.001 [612.27 - 687.55]Genistein Site MI 1000.9 1683.74 -40.55 0.01 [841.05 - 1118.40]Genistein Site OH 760.07 1143.19 -33.51 0.003 [750.00 - 773.91]Statistical Differences Observed in One SiteSeed Amino Acid (% DW)Aspartic Acid Site OH 4.43 4.27 3.72 0.021 [4.41 - 4.47] [3.45, 5.29]Glutamic Acid Site OH 7.51 7.17 4.67 0.02 [7.42 - 7.59] [5.51, 9.04]Histidine Site OH 1.07 1.03 3.2 0.003 [1.06 - 1.07] [0.86, 1.27]Monsanto Company 09-SY-200U Page 111 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial ReferenceVarieties (Continued)Mean Difference(Test minus Control)Component (Units)1MON 87769 MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval2Isoleucine Site OH 1.87 1.75 6.53 0.025 [1.84 - 1.90] [1.34, 2.28]Leucine Site OH 3.16 3.05 3.42 0.016 [3.14 - 3.18] [2.45, 3.76]Lysine Site OH 2.62 2.56 2.46 0.02 [2.61 - 2.63] [2.13, 3.06]Valine Site OH 1.96 1.84 6.62 0.027 [1.94 - 2.01] [1.42, 2.41]Seed Fiber (% DW)Acid Detergent Fiber Site IL 16.16 17.76 -9 0.005 [15.91 - 16.61] [10.36, 22.77]Neutral Detergent Fiber Site IL 17.06 17.87 -4.52 0.043 [16.72 - 17.25] [10.91, 22.59]Seed Proximate (% DW)Ash Site OH 5.83 5.71 2.12 0.022 [5.78 - 5.93] [5.16, 6.64]Protein Site OH 41.37 39.54 4.63 0.008 [40.92 - 41.70] [33.37, 46.00]Seed Antinutrient (% DW, unless noted)Lectin (H.U./mg DW) Site MI 1.49 3.44 -56.79 0.019 [0.55 - 2.14] [0, 16.00]Phytic Acid Site IA-1 1.28 1.17 9.49 0.041 [1.22 - 1.34] [0.51, 1.59]Seed Isoflavone (μg/g DW)Glycitein Site IL 84.86 120.49 -29.57 0.044 [83.23 - 87.74] [0, 243.40]Monsanto Company 09-SY-200U Page 112 of 395Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositional Analysis ofMON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (Continued)Mean Difference(Test minus Control)Component (Units)1MON 87769MeanA3525MeanMean Difference(% of A3525)Significance(p-Value)TestRangeCommercialTolerance Interval2Forage - Statistical Differences Observed in One SiteForage Fiber (% DW)Acid Detergent Fiber Site OH 32.29 28.31 14.09 0.009 [30.74 - 33.95] [19.24, 38.36]Forage Proximate (% DW)Total Fat Site IA-2 4.87 6.27 -22.39 0.037 [4.40 - 5.28] [1.46, 9.88]1DW = dry weight; FA = fatty acid.2With 95% confidence, interval contains 99% of the values expressed in the populationof commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 113 of 395 VII.C. Compositional Comparisons of Processe d Fractions from Soybean Seed of

MON 87769 and the Conventional Control

For preparing soybean processed fractions, seed samples were collected from a field trial conducted with MON 87769, the conventional s oybean control, and eight conventional varieties at two field sites (Monmouth, IL and Carlyle, IL) in the U.S. during the 2006 growing season. The seed samples were proces sed into defatted toasted soybean meal (DT soybean meal); refined, bleached, and deodorized soybean oil (RBD oil); protein isolate; and crude lecithin fractions. The processed fractio ns were analyzed according to the principles

outlined in the OECD consensus document fo r soybean composition (OECD, 2001). The DT soybean meal was analyzed for proximates (m oisture, protein, fat, and ash), ADF, NDF, amino acids, trypsin inhibitors and phytic acid. The RBD oil was analyzed for fatty acids and vitamin E ( α -tocopherol). The protei n isolate fraction was analyzed for amino acids and moisture. The crude lecithin fraction was analyzed for phosphatides ( α -phosphatidic acid,

α -phosphatidyl-choline, α -phosphatidyl-ethanolamine, and α -phosphatidylinositiol). The methods used for composition analysis of th e processed fractions are summarized in Appendix E, Section E.6. The statisti cal analysis compared MON 87769 and the

conventional control across the two sites (combined-site). Statistical summary of the composition of each processed fraction and summary of the significant differences observed between the processed fractions prepared fr om the seed of MON 87769 and the conventional

control are included in A ppendix E, Section E.6.

As expected, apart from the intended fatty acid changes, the composition of the soybean processed fractions from MON 87769 is equi valent to the composition of conventional soybean control (OECD, 2001). Thus, th e processed fractions from MON 87769 are

concluded to be as safe and nutritious as the processed fractions prepared from conventional soybean from a food/feed perspective.

Collectively, these compositional data from pr ocessed fractions support the conclusion that MON 87769, with the exception of the expected changes in fatty acid composition, does not have biologically meaningful differences from conventional soybean from a food/feed safety and/or nutritional perspective.

VII.D. Safety and Nutritional Assessm ent of the Intended Changes in MON 87769

Beside the components typically present in soybean, MON 87769 is expected to contain SDA, GLA, and small amounts of trans-ALA and trans-SDA resulting from the intended change. As expected, the intended change al so decreased the levels of LA in MON 87769. The safety of SDA soybean oil is supporte d by the results of a published 90-day/onegeneration reproductive rat toxi city study in which a no observ able adverse effect level (NOAEL) of 1 g SDA/kg body weight/day (4 g SDA soybean oil/kg body weight/day) was

determined 

### ( Hammond et al., 2008 

### ) . In addition to this, many published studies on SDA and

GLA from other sources corroborat e the safety of SDA soybean oil. A discussion of the safety and nutritional impact resulting from the intended fatty acid change in MON 87769 is included in Appendix F.Monsanto Company 09-SY-200U Page 114 of 395 VII.E. Safety and Nutrition Assessment Conclusion

In conclusion, having demonstrated: (a) the compositional equivalence of MON 87769 seed (except for the intended fatty acid change) and forage to seed and forage from conventional soybean already on the market, (b) the safety of the expected fatty acids resulting from the intended change, (c) the history of safe use of the introduced proteins, and (d) familiarity of the host organism from which the genes are de rived, MON 87769 is as safe and nutritious as conventional soybean for food and feed use with the added nutritional improvement of SDA, an omega-3 fatty acid.Monsanto Company 09-SY-200U Page 115 of 395 VIII. PHENOTYPIC, AGRONO MIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides an evaluation of the phenotypic and agronomic characteristics, and

the environmental interactions of MON 87769 compared to the control, A3525. The A3525 control is a conventional soybean variet y that has a genetic background similar to

MON 87769 but does not contain the Pj.D6D or Nc.Fad3 genes.

These data support a determination that M ON 87769 is no more likely to pose a plant pest risk or to have a significant envir onmental impact compared to conventional soybean. The conclusions are based on the results of the multiple evaluations reported

herein.

Phenotypic and agronomic characteristic s of MON 87769 were evaluated in a comparative manner to assess plant pest pot ential (OECD, 1993). These assessments included 14 plant growth and development characteristics, five seed germination parameters, two pollen characteristics, six pl ant-symbiont interaction characteristics, and observations for plant-insect and plant-disease interactions and plant responses to abiotic stressors. Results from the phenotypic and agronomic assessments indicate that MON 87769 neither possesses characteristics that would increase plant pest potential, nor would it have a significant e nvironmental impact compared to conventional soybean.

Data on environmental interactions also indicate that MON 87769 does not confer any increased susceptibility or tolerance to specif ic diseases, insects, or abiotic stressors.

VIII.A. Characteristics Measured for Assessment

In the phenotypic, agronomic, and envi ronmental interactions assessment of MON 87769, data were collected to evaluate specific aspects of altered plant pest potential based on requirements of USDA -APHIS set forth at 7 CFR § 340.6. The MON 87769 plant characterization encompas ses six general data categories: 1) germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive growth (including pollen characteristics); 4) seed retention on the plant and lodging; 5) plantsymbiont associations; and 6) pl ant interactions with insect, di sease, and abiotic stressors.

An overview of the characteristics a ssessed is presented in Table VIII-1.

The phenotypic, agronomic, and environmental interactions data were evaluated on the 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 87769 was compared to an appropriate conventiona l control, A3525, which had a genetic background similar to MON 87769 but di d not possess the genes introduced in MON 87769 ( Pj.D6D and Nc.Fad3) , the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change. In addition, multiple commercial soybean varieties (see Appendix G and Tables G-1 and G-2) were included to provide a range of values that are representative of co mmercial soybean varieties for each measured phenotypic, agronomic, and environmental interaction characteristic.Monsanto Company 09-SY-200U Page 116 of 395 Data collected from the commercial referenc e varieties reflect a range of selection and breeding for desirable characteristics and th erefore can provide context for interpreting experimental results.

Table VIII-1. Phenotypic, Agronomic and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials

Characteristics Characteristics

measured Evaluation timing 1 Evaluation description (measurement endpoints)
Dormancy, germination 5, 8, and 13 days after planting Percent normally germinated, abnormally germinated, viable hard (dormant), dead,

and viable firm-swollen seed

Plant phenotypic

and agronomic

characteristics Seedling vigor V2–V4 Rated on a 1-9 scale, where 1-3 = excellent, 4-6 = average, and 7-9 = poor vigor Early stand count V2–V4 Number of emerged plants per plot,

standardized to 20 ft rows Growth stage

assessment 1 Every 2-3 weeks;

V2–R8 Average soybean plant growth stage per plot Days to 50% flowering Flowering; R1–R2 Calendar day number when approx. 50% of the plants in each plot were flowering Pollen viability Flowering; R1–R2 Viable and nonviable pollen based on pollen grain staining characteristics Pollen morphology Flowering; R1–R2 Diameter of viable pollen grains Flower color Flowering; R2 Color of flowers: purple, white, or mixed

Plant pubescence Maturity; R8 Pube scence on plants in each plot categorized as hairy, hairless, or mixed Plant height Maturity; R8 Distance from the soil surface to the uppermost node on the main stem of five representative plants per plot Lodging Maturity; R8 Rated on 0- 9 scale, where 0 = completely erect and 9 = completely flat or lodged Pod shattering Maturity; R8 Rated on 0-9 scale, where 0 = no shattering and 9 = completely shattered Final stand count Maturity; R8 Number of plants per plot, standardized to

20 ft rows

Seed moisture Harvest Percent moisture content of harvested seed 100 seed weight (g) Harvest Weight of 100 harvested seed Test weight (lb/bu) Harvest Weight of a bushel of harvested seed Yield (bu/ac) Harvest Bushels of harvested seed produced per acre, adjusted to 13% moisture

Plant

environmental

interactions Plant response to abiotic stressors, disease damage, and arthropod damage Variable, from planting to harvest Each plot rated using a continuous 0-9 or 0-5 rating scale of increasing symptomology

Arthropod abundance R1 – R2

R3 – R5

R6 – R8 Number of specific pest and beneficial arthropods collected per plot using beat sheet sampling method Plant × symbiont relationship 4 and/or 6 weeks after emergence in greenhouse Nodule number and dry weight, root and shoot dry weight, shoot total nitrogen

1 Soybean plant growth stages were determined usin g descriptions and guidelines outlined in Soybean Growth and Development (Iowa State University, 2004).Monsanto Company 09-SY-200U Page 117 of 395 VIII.B. Interpretation of Phenotypic and Environmental Interaction Data

Plant pest risk assessments for biotec hnology-derived crops are, by OECD (1993) standards, comparative assessments. Familiarity provides a basis from which the potential environmental impact of a biotec hnology-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 conventio nal plant variety. Familiarity considers the biology of the crop, the intr oduced trait, the receiving en vironment and the interaction of these factors, and provides a basis for comparative environmental risk assessment between a biotechnology-derived plant and its conventiona l counterpart.

Expert knowledge and experience with conven tionally bred soybean were the basis for selecting appropriate endpoints and estimatin g the range of responses that would be considered typical for soybean. Thus , assessment of phenotypic and agronomic

characteristics and environmental intera ctions was essential to compare the biotechnology-derived plant to the conven tional counterpart. An overview of the characteristics assessed is presented in Table V III-1. A subset of the data relating to well

understood weediness criteria (e.g., dormancy, pre-harvest seed loss characteristics, and lodging) was used to assess whether there is an increased weediness potential, an element of APHIS’s plant pest determin ation. Based on all of the da ta collected, an assessment

was made whether the biotechno logy-derived plant is likely to pose an increased plant pest risk compared to the conventional counterpart.

During the processes of data collection, summarization, and analysis, experienced scientists familiar with each experimental desi gn and evaluation criteria were involved in all steps. This level of oversight ensure d that the evaluation system was functioning appropriately, measurements were taken pr operly, and data were consistent with expectations based on experience with the cr op. In addition, the overall dataset was

evaluated for evidence of biol ogically relevant changes, and for possible evidence of an unexpected plant response. Data then were submitted to statistical analysis.

VIII.C. Interpretation of De tected Differences Criteria

Comparative plant characteriz ation data between a biot echnology-derived crop and the control are interpreted in the context of contri butions to increased pl ant pest potential as assessed by APHIS. Under the framework of familiarity, characteristics for which no differences are detected suppor t a conclusion of no increase d plant pest potential of the biotechnology-derived crop compared to th e conventional crop. Characteristics for

which differences are detected are considered in the step-wise method (Figure VIII-1 or a similar method). All detected differences fo r a characteristic ar e considered in the

context of whether the difference would in crease the plant pest potential of the biotechnology-derived crop. Ultimately, a wei ght of evidence approach considering all

characteristics and studies was used for the overall risk assessment of differences and their significance. In deta il, Figure VIII-1 illustrates th e stepwise assessment process employed:Monsanto Company 09-SY-200U Page 118 of 395 Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or ecological change for the crop in te rms of plant potential and subsequent steps are not considered. If the answer is “yes” or uncertain the subsequent step is considered.

Figure VIII-1. Schematic Diagram of Data Interpretation Methods

▪ Steps 1 & 2. Evaluate Detected Statistical Differences. Combined-site and

individual-site statistical analyses are conducted and ev aluated on each measured

characteristic. Differences detected in the individual-site analysis must be observed in the combined-site analysis to be consider ed further for plant pest potential. Any difference detected in the combined-site analysis is further assessed.

▪ Step 3. Evaluate Differences Relative to Reference Range. If a difference is

detected in the combined-site analysis ac ross multiple environments, then the test

substance mean value is assessed relative to the reference substances.

▪ Step 4. Evaluate Differences in the Context of the Crop. If the test substance mean

is outside the variation of the reference s ubstances (e.g., reference range), the test

substance mean is considered in the cont ext of known values common for the crop.

▪ Step 5. Plant Pest Potential. If the test substance mean is outside the range of values

common for the crop, the detected difference is then assessed for wh ether or not it is

adverse in terms of plant pest potential.

▪ Step 6. Conduct Risk Assessme nt on Identified Hazard. If an adverse effect

(hazard) is identif ied, risk assessment on the difference is conducted. The risk

assessment considers contributi ons to enhanced plant pest potential of the crop itself, the impact of differences detected in othe r measured characteristics, and potential for Hazard identification & risk

assessment on difference Outside variation for crop? No

Yes

No

Yes Plant pest potential? Yes Outside variation of study No Not adverse; the direction or magnitude of the

detected difference in the

measured characteristic

does not contribute to a biological or ecological change for the crop in

terms of plant pest potential Yes Statistical differences detected in combined-site analysis? No Differences detected in the combined-site and individual-site analyses are evaluated* Step 1

Step 2

Step 3

Step 4 The measured

characteristic does not contribute to a biological or ecological change for the crop in terms of plant pest potential

Step 5

Step 6

*See text for interpretation of differences detected in the individual-site analysisMonsanto Company 09-SY-200U Page 119 of 395 and effects of trait transfer to feral popula tions of the crop or a sexually compatible species.

VIII.D. Phenotypic, Agronomic and Envi ronmental Interactions Characteristics

As a significant part of the evaluation of MON 87769, plant phenotypic and agronomic characteristics including seed dormancy and germination, phenotypic, agronomic and environmental interactions, pollen characteri stics, and symbiont interactions were evaluated. The phenotypic, agronomic, and environmental interaction evaluations are based on replicated laboratory, greenhouse, and/ or multi-site field trials and experiments. In evaluating the phenotypic and agronomic characteristics of MON 87769, data were collected that address specific plant pest risks, as defined by APHIS.

VIII.D.1. Seed Dormancy and Germination Characteristics

APHIS considers the potential for weediness to constitute a plant pest factor (7 CFR § 340.6). Seed germination and dormancy mechanisms vary with species, and their genetic basis tends to be complex. Seed dormancy (e.g., hard seed) is a survival mechanism that prevents germination when conditions for survival are poor and is an important

characteristic that is often associated with plants that are considered as weeds (Anderson, 1996; Lingenfelter and Hartwig, 2003). Thus, information on germination and dormancy

is relevant to a weediness assessment. Howeve r, it is important to note that it is not

uncommon to observe low levels of hard seed in soybean (Mullin and Xu, 2001; Debeaujon et al., 2007). Standardized germ ination assays are av ailable and routinely used to measure the germination characteristics of soybean seed. The Association of Official Seed Analysts, an internationa lly recognized seed te sting organization, recommends a temperature range of 20/30 ° C as optimal for germination of soybean (AOSA, 2007) (Table VIII-2).

Comparative assessments of seed dormanc y and germination characteristics were conducted on MON 87769 and A3525, where A3525 se rved as a comparable control as it has a genetic background similar to MON 87769 but does not contain the Pj.D6D or Nc.Fad3 gene. In addition, nine commercial soybean varieties were included as references to provide a range of values for each assessed characteristics that is representative of commerci al soybean. The seed lots for MON 87769, the conventional soybean control and reference varieties were produced during 2006 in Iowa (IA), Illinois (IL), and Indiana (IN), which represent enviro nmentally relevant conditions for soybean production for this product. In addition to the AOSA recommended temperature range of 20/30 °C, seed were tested at five othe r temperature regimes of 10, 20, 30, 10/20, and

10/30 ° C to assess seed germination characterist ics. For the alternating temperature regimes of 10/20, 10/30, or 20/30 ° C, the lower temperature was maintained for 16 hours and the higher temperature for eight hours. The temperature re gimes and types of

observation are listed in Tabl e VIII-2. The details of the materials, experimental methods, and data from all individual produc tion sites are presented in Appendix H.

A total of 25 comparisons were made betw een MON 87769 and the conventional control for seed germination characteristics across the three seed production sites (Table VIII-3). No statistically or biological ly significant differences we re detected between MON 87769Monsanto Company 09-SY-200U Page 120 of 395 and the conventional control for percent norm al germinated, abnormal germinated, viable hard, dead, or viable firm-swollen seed in the AOSA temperature regime (20/30 °C) or for percent germinated, viable hard, dead, or viable firm-swollen seed in the additional temperature regimes (10, 20, 30, 10/20, 10/30 °C). In some cases, it was not possible to conduct an analysis of variance because there was no variability presen t in the data. For these comparisons, the values for MON 87769 and the control were all the same, indicating no biol ogical differences.

One viable hard seed was observed at ea ch of the 10 °C and 10/20 °C temperature regimes for MON 87769 produced from the IL site. In soybean, it is not uncommon to observe low levels of hard seed (Mullin and Xu, 2001; Potts et al., 1978). Thus, the

observance of a total of two hard seeds out of 7,188 seeds of MON 87769 evaluated in this study was not unexpected.

In the AOSA temperature regime of 20/ 30 oC, the germination means of both

MON 87769 and the conventional control were outside the range of the nine reference varieties included (Table VIII-3). However, no statistically significant differences in percent normal germinated or abnormal ge rminated seeds were detected between

MON 87769 and the conventional control. Furthermore, the germination of both MON 87769 (89.1%) and the conventional cont rol (92.1%) at 20/30 oC exceeded the accepted standard of 80% minimum germination for certified soybean seed recommended by the official seed certifying agencies of North America (AOSCA, 2003). Thus, the germination values of MON 87769 were not different from the A3525 conventional control, an established commercial soybean variety, and were within the range of accepted germination values for certified soybean seed.

The biological characteristics evaluated we re used to characterize MON 87769 in the context of a plant pest risk assessment. The results de monstrate that the assessed dormancy and germination characteristics of MON 87769 were not di fferent from those

of the conventional control. The observe d dormancy and germination values of MON 87769 were within the range of accepted values for soybean. The results, in particular the lack of change in hard seed, are indica tive of no increased plant pest or weediness potential of MON 87769 relative to conventional soybean.Monsanto Company 09-SY-200U Page 121 of 395Table VIII-2. Seed Dormancy and Germination Characteristics EvaluatedEvaluationTiming1Temperature Regimes and SeedCharacteristics EvaluatedAOSA2Additional Temperatures20/30oC 10oC, 20oC, 30oC, 10/20oC and 10/30oCDay 5Normal germinatedGerminatedDeadDeadDay 8Normal germinatedGerminatedAbnormal germinatedDeadDeadHard viable and non-viable3Firm swollen viable and non-viable3Day 13No data collectedGerminatedDeadHard viable and non-viable3Firm swollen viable and non-viable31Seed in the 20/30oC temperature regime were evaluated in Days 5 and 8 (accordingto AOSA guidelines), while seed in the additional temperatureregimes were evaluated on Days 5, 8, and 13.2Association of Official Seed Analysts (AOSA, 2007).3Hard and firm swollen were confirmed to be viable or non-viable by Tetrazolium (Tz) test (AOSA, 2000).Monsanto Company 09-SY-200U Page 122 of 395 Table VIII-3. Germination Charac teristics of MON 87769 and A3525

Temperature Regime Germination

Characteristic 1 Mean % ( S.E. ) 2 Reference Ran g e 3

MON 87769 Control Min. Max.

10 °C Germinated 99.8 (0.1) 100.0 (0.0) 99.8 100.0

Viable Hard 0.1 (0.1) 0.0 (0.0) 0.0 0.0

Dead 0.1 (0.1) 0.0 (0.0) 0.0 0.3

Viable Firm-swollen 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

20 °C Germinated 100.0 (0.0) † 100.0 (0.0) 99.8 100.0

Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

Dead 0.0 (0.0) † 0.0 (0.0) 0.0 0.3

Viable Firm-swollen 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

30 °C Germinated 99.7 (0.1) 99.6 (0.2) 99.3 100.0

Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

Dead 0.3 (0.1) 0.4 (0.2) 0.0 0.8

Viable Firm-swollen 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

10/20 °C Germinated 99.8 (0.2) 99.8 (0.1) 99.5 100.0

Viable Hard 0.1 (0.1) 0.0 (0.0) 0.0 0.0

Dead 0.2 (0.2) 0.2 (0.1) 0.0 0.5

Viable Firm-swollen 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

10/30 °C Germinated 100.0 (0.0) † 100.0 (0.0) 99.5 100.0

Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

Dead 0.0 (0.0) † 0.0 (0.0) 0.0 0.5

Viable Firm-swollen 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

20/30 °C Normal Germinated 89.1 (2.4) 92.1 (2.5) 93.8 98.5

(AOSA) Abnormal Germinated 10.7 (2.4) 7.7 (2.4) 1.4 6.0

Viable Hard 0.0 (0.0) † 0.0 (0.0) 0.0 0.0

Dead 0.3 (0.2) 0.2 (0.1) 0.0 0.5

Viable Firm-swollen 0.0 (0.0) 0.1 (0.1) 0.0 0.0

Note: The data in this table are the combined-site resu lts for the seeds from the three production sites. The experimental design was a split-plot where the whole-plot treatment was seed production site arranged in a randomized complete block and the sub-plot treatment was seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No statistically significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525). The germination characteristic means for seeds from each indivi dual production site are

included in Appendix H.

† No statistical comparisons were made due to lack of variability in the data.

1 Germinated seed in the AOSA temperature regime (20/30 °C) were categor ized as either normal

germinated or abnorma l germinated seed.

2 Means based on four replicates (n = 4) of 100 seeds. The total percentage of all germination

characteristics of MON 87769 or the control in some temperature regimes is greater than 100% due to numerical rounding of the means. SE = Standard Error.

3 Minimum and maximum mean values from among nine commercial reference soybean varieties.Monsanto Company 09-SY-200U Page 123 of 395 VIII.D.2. Field Phenotypic, Agronomic Characteristics and Environmental

Interactions

Plant growth, development, and yield character istics were assessed und er field conditions as part of the plant characterization assessment of MON 87769. These data were developed to provide APHIS with a detaile d description of any unintended phenotypic

differences in MON 87769 relative to the conventional soybean control, A3525, and commercial soybean. Environmental interacti ons also were assessed as an indirect indicator of phenotypic changes to MON 87769 relative to the same comparators described above. The purpose of these fiel d evaluations was to assess whether the introduced genes ( Pj.D6D and Nc.Fad3 ), the proteins produ ced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change altered the phenotypic and agronomic characteristics or the plant- arthropod, plant-disease, or plant-abiotic

stressor interactions of MON 87769 compared to the c ontrol. Certain growth, reproduction, and pre-harvest seed loss characteristics (such as lodging and pod shattering) can be used in the assessment of whether MON 87769 has enhanced plant pest potential.

Data were collected at 21 field locations over two consecutive years (17 locations in 2006 and four in 2007) to thoroughly evaluate phenotypic, agronomic, and environmental

interaction characteristics. These 21 locatio ns provided a diverse range of environmental and agronomic conditions representative of commercial soybean production areas in the U.S. (Table VIII-4). The experiment at each field site was established in a randomized complete block design with three replications. Fourt een phenotypic and agronomic characteristics were evaluated: early stand c ount, seedling vigor, plant growth stage, days to 50% flowering, flower color, plant pub escence, plant height, lodging, pod shattering, final stand count, seed moisture, 100 seed we ight, test weight, and yield. In addition,

observational data were collected on plant response to abio tic stressors, di sease damage, and arthropod damage. These data were co llected four times during the growing season at all 21 sites over the two years to assess the susceptibility a nd tolerance of MON 87769 to specific abiotic stressors, diseases, or arthropod pests compar ed to that of the conventional soybean control. Additionally, data were collected on the abundance of specific pest and beneficial arthropods in the plots to assess the interactions between MON 87769 and these arthropods. Th e categories and timing of phenotypic

characteristic and environmental interaction evaluations are included in Table VIII-1.

The methods and detailed results of these indi vidual site data comparisons are presented and discussed in Appendix G, while the combined-site analyses are summarized below.

The results of this assessment showed the in troduced trait did not unexpectedly alter the phenotypic or agronomic char acteristics of MON 87769 co mpared to conventional soybean and are indicative of no increased weediness of MON 87769. The lack of biologically meaningful differences in plant response to abiotic stressors, disease damage, arthropod damage, and the abundance of various pest and beneficial arthropods further indicate that the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change are unlikely to increase plan t pest potential.Monsanto Company 09-SY-200U Page 124 of 395 VIII.D.2.1. Field Phenotypic and Agronomic Characteristics

A total of 14 phenotypic and agronomic characteris tics were evaluated. In the combinedsite analysis of the data from the 2006 a nd 2007 field trials, no statistically significant

differences were detected between MON 87769 and the control for any of the assessed phenotypic characteristics (Tables VIII-5 and VIII-6). All plants of MON 87769 and the control had purple flowers and hairy pubescence at each site as expected (Appendix G, Tables G-3 and G-5). Furthe rmore, no differences in plan t growth stage were observed between MON 87769 and the control for any of the 126 and 27 observations conducted among the sites in 2006 and 2007, respectively (Appendix G).

The phenotypic and agronomic characteristics evaluated in this study were used to provide a detailed description of any diffe rences between MON 87769 compared to the nontransformed control (A3525). A subset of these characterist ics were useful to assess the weediness poten tial of MON 87769 compared to the conventional soyb ean control.

The results from the 2006 and 2007 field trials demonstrate that the assessed phenotypic and agronomic characteristic s of MON 87769 were not a ltered compared to the

conventional control as a result of the introduction of the genes (Pj. D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change. The observed values of phenotypic and agronomic characteristics of MON 87769 were within the range expected fo r soybean. These results are indicative of no increased plant pest or weediness potenti al of MON 87769 compared to conventional soybean.Monsanto Company 09-SY-200U Page 125 of 395 Table VIII-4. Field Phenotypic Eval uation Sites for MON 87769 during 2006 and

2007

Site Code County, State USDA-APHIS

Notification Number

2006 Field Sites IA1 Jefferson, IA 06-045-22n
IA2 Benton, IA 06-045-22n IA3 Jefferson, IA 06-074-05n IA4 Guthrie, IA 06-074-05n IL1 Stark, IL 06-045-22n
IL2 Warren, IL 06-045-22n IL3 Clinton, IL 06-074-05n IN1 Parke, IN 06-045-22n
IN2 Boone, IN 06-045-22n
KS Pawnee, KS 06-045-22n
MI Ottawa, MI 06-074-05n
MO1 Macon, MO 06-045-22n MO2 Lincoln, MO 06-045-22n NE York, NE 06-045-22n NE2 York, NE 06-074-05n OH Fayette, OH 06-074-05n WI Walworth, WI 06-074-05n

2007 Field Sites IL Stark, IL 07-052-104n
IN Boone, IN 07-052-104n
MI Ottawa, MI 07-052-104n WI Walworth, WI 07-052-104nMonsanto Company 09-SY-200U Page 126 of 395Table VIII-5. Plant Growth and Development Data across 17 Locations during 2006MON 87769 Control Reference Range1Phenotypic Characteristic(units)Mean (S.E.) Mean (S.E.) Minimum MaximumEarly stand count (#/plot)217.0 (8.1) 219.7 (9.1) 150.5 298.6Seedling vigor (1-9 scale)3.2 (0.2) 3.1 (0.2) 2.3 5.0Days to 50% flowering2195.4 (1.1) 195.6 (1.1) 184.7 206.5Flower color3Purple Purple Purple PurplePlant pubescence3Hairy Hairy Hairy HairyPlant height (in)36.2 (1.0) 36.1 (0.9) 30.4 43.9Lodging (0-9 scale)1.1 (0.2) 1.3 (0.2) 0.0 4.3Pod shattering (0-9 scale)0.3 (0.1) 0.3 (0.1) 0.0 1.5Final stand count (#/plot)199.6 (8.0) 203.4 (8.2) 154.3 271.3Seed moisture (%)12.2 (0.3) 12.2 (0.3) 11.0 16.0100 seed weight (g)15.1 (0.2) 15.4 (0.2) 14.0 18.0Test weight (lb/bu)456.5 (0.5)†56.1 (0.4)†49.7 57.7Yield (bu/ac)452.7 (1.7)†55.7 (1.9)†28.8 66.9Note: The experimental design was a randomized complete block. S.E. = Standard Error. Means based on n = 51, except where denoted by†inwhich means are based on n = 48.No statistically significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525).1Reference range = Minimum and maximum mean values among 18 commercial reference soybean varieties.2Calendar day number when approximately 50% ofthe plants in each plot were flowering.3Flower color and plant pubescence data were categoricaland not statistically analyzed (see Appendix G, Table G-3).4Test weight and yield data from the IA4 site were excluded from the statistical analysis because an inaccurate measuring device was used tocollect the data.Monsanto Company 09-SY-200U Page 127 of 395Table VIII-6. Plant Growth and Development Data across Four Locations during 2007MON 87769 Control Reference Range1Phenotypic Characteristic(units)Mean (S.E.) Mean (S.E.) Minimum MaximumEarly stand count (#/plot)357.8 (28.1) 354.2 (31.6) 180.0 492.7Seedling vigor (1-9 scale)2.8 (0.4) 2.5 (0.3) 1.0 6.0Days to 50% flowering2205.4 (1.5) 205.3 (1.5) 201.5 211.7Flower color3Purple Purple Purple PurplePlant pubescence3Hairy Hairy Hairy HairyPlant height (in)38.2 (2.4) 39.3 (1.6) 30.6 49.4Lodging (0-9 scale)1.7 (0.6) 1.8 (0.6) 0.0 3.7Pod shattering (0-9 scale)0.2 (0.1) 0.0 (0.0) 0.0 0.3Final stand count (#/plot)331.6 (21.8) 339.6 (21.3) 158.7 455.7Seed moisture (%)11.8 (0.3) 11.7 (0.3) 10.6 12.2100 seed weight (g)417.3 (0.9)†17.0 (0.8)†13.9 20.0Test weight (lb/bu)54.2 (0.9) 54.5 (0.7) 51.2 57.0Yield (bu/ac)46.4 (1.6) 50.4 (2.0) 39.1 53.5Note: The experimental design was a randomized complete block. S.E. = Standard Error. Means based on n = 12, except where denoted by†inwhich means are based on n = 9.No statistically significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525).1Reference range = Minimum and maximum mean values among 12 commercial reference soybean varieties.2Calendar day number when approximately 50% ofthe plants in each plot were flowering.3Flower color and plant pubescence data were categoricaland not statistically analyzed (see Appendix G, Table G-5).4100 seed weight data from the WI site were excluded from the statistical analysis because the balance used to weigh the samples did not havesufficient precision.Monsanto Company 09-SY-200U Page 128 of 395 VIII.D.2.2. Environmental Interaction Analyses

Evaluations of environmental interactions were conducted as part of the plant

characterization for MON 87769. In the 2006 and 2007 U.S. field trials conducted for evaluation of phenotypic and agronomic ch aracteristics of M ON 87769, data were collected on plant response to abiotic st ressors (e.g., drought, nutrient deficiency, wind, etc.), disease damage, and arthropod damage . These data were used to assess the susceptibility and tolerance of MON 87769 to specific abiotic stressors, diseases, or arthropod pests compared to that of the conve ntional soybean control. Additionally, data were collected on the abundance of specific pest and beneficial arthropods in the plots to

assess the interactions between MON 87769 and these arthropods (Appendix I; Tables I-1 through I-10). These data are used as part of the environmenta l risk assessment to evaluate plant pest poten tial and impact on NTOs for MON 87769 compared to the conventional soybean control. In addition, multiple commercial soybean varieties were included in the analysis to establish a range of natural variability for each characteristic. The lack of biologically meaningful differe nces in plant response to abiotic stressors, disease damage, arthropod damage, or the ab undance of various pest and beneficial arthropod indicate that the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change are unlikely to be bi ologically meaningful in te rms of increased plant pest potential.

Plant response to abiotic stressors, disease damage, and arthropod damage were assessed qualitatively four times during the growing season at 17 sites in 2006 and four sites in 2007. The observed abiotic stressors, diseases, a nd arthropod pests were “natural” (i.e.,

no artificial infestation or in terference was used) and, theref ore, often varied between

observations at a site and between sites. For each abiotic stress response, disease damage, and arthropod damage observation at a site, the reported values represent the range of ratings observed across the three re plicatons at each site. MON 87769 and the control were considered qualitatively different in response to a st ressor on a particular observation date if the range of ratings acr oss all three replications of MON 87769 and the conventional soybean control did not overlap (e.g., “none” rating vs. “slightmoderate” rating). The ratings observed among the commercial reference soybean varieties provide qualitative assessment data common to the crop for each stressor assessed. Arthropod abundance data were collect ed quantitatively from plots three times during the growing season at f our sites each in 2006 and 2007 to assess the interactions between MON 87769 and these arthropods.

A total of 703 comparative observations of pl ant response to abiotic stressors, disease damage, and arthropod damage and 260 comp arative observations of the abundance of various pest and beneficial ar thropods were conducted in fiel d trials over tw o consecutive years to assess environmental interactions of MON 87769 compared to the conventional control. No biologically mean ingful differences were obser ved across sites over two U.S. soybean growing seasons between MON 87769 and the conventional control in their susceptibility or tolerance to specific abiotic stressors, diseas es, or arthropod pests or in

their interactions wi th specific pest and beneficial arthropods, as indicated by theMonsanto Company 09-SY-200U Page 129 of 395 arthropod abundance data. Thus , the results from the field trials demonstrate that the

assessed interactions between MON 87769 and ab iotic stressors, dise ases, and arthropods

were not altered as a result of the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change, compared to the conven tional control and were within the range expected for soybean. These results are indica tive of no increased plan t pest potential or adverse ecological interac tions of MON 87769 relative to conventional soybean.

VIII.D.3. Pollen Characteristics

In determining the potential for a biotechno logy-derived plant to increase weedy or

invasive characteristics in ot her plants, APHIS considers the potential for gene flow and introgression of the biotechnology- derived trait into other plant varieties or w ild relatives. Information on pollen morphology and viability are pertinent to this assessment, and therefore, were assessed for MON 87769. In addition, morphological characterization of pollen produced by MON 87769 and the conventi onal soybean control are relevant to APHIS’s plant pest risk assessment because they add to the detailed description of the phenotype of MON 87769 compared to conventional soybean.

The purpose of this evaluation was to as sess the morphology and viability of pollen collected from MON 87769 compared to a conve ntional soybean control. Pollen was collected from MON 87769, the control (A3525) , and four commercial reference soybean varieties grown under similar agronomic conditions in a field trial in Missouri. The trial was arranged in a randomized complete bl ock design with three replications. A

minimum of 20 flowers were collected from each plot. Pollen was extracted, combined among flowers collected from the same pl ot, and stained with Alexander’s stain (Alexander, 1980). Pollen viability was eval uated for each sample and pollen grain diameter was measured for ten representative viable pollen grains per replication. General morphology of the pollen was observed for each of the three replications of MON 87769, the control, and the reference soybean varieties (s ee Appendix J).

No statistically significant di fferences were detected (p >0.05) between MON 87769 and the control for percent viable pollen or pollen grain diameter (Table VIII-7). Furthermore, no visual differences in gene ral pollen morphology were observed between

MON 87769 and the control. These results de monstrate that the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced fr om the introduced genes (Pj Δ 6D and

Nc Δ 15D), and the intended fatty acid change did not alter the overall morphology or viability of MON 87769 pollen compared to the conventional soybean control. The absence of statistically significant diffe rences between the pollen collected from MON 87769 compared to the conventiona l soybean control for the assessed

characteristics demonstrate that the obser ved values of MON 87769 were within the range of values expected for soybean. Thus, these data further support no change in plant pest potential for MON 87769 compared to the nontransformed control and other soybean

varieties.Monsanto Company 09-SY-200U Page 130 of 395 Table VIII-7. Pollen Grain Diameter and Viability Analyses

Pollen

Characteristic Mean (S.E.) Reference Range 1

MON 87769 Control Minimum Maximum

Viability (%) 98.2 (0.91) 98.7 (0.33) 94.3 98.7

Diameter (μm) 23.4 (0.20) 24.3 (0.25) 23.4 23.8

S.E. = Standard Error. Means based on n = 3. Note: No statistically significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525).

1 Reference range is the minimum and ma ximum mean value observed among the four

commercial reference soybean varieties.

VIII.D.4. Symbiont Interactions

Members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae form a highly complex and specific symbiotic relationship with leguminous plants, including soybean (Gage, 2004). The Rhizobium -legume symbiosis results in th e formation of root nodules, providing an environment in which differentia ted bacteria called b acteroids are capable of reducing or “fixing” atmospheric nitr ogen. The product of nitrogen fixation, ammonia, can then be utilized by the plant. In soybean, atmospheric nitrogen is fixed into organic nitrogen through a symbio tic association w ith the bacterium Bradyrhizobium
japonicum . As a result of this re lationship, no nitrogen inputs are needed for agricultural production of soybean.

As part of the plant pest risk assess ment, APHIS considers the impact of the biotechnology-derived crop to agricultural or cultivation practices (7 CFR § 340.6). Changes in the symbiotic relationship with Rhizobiaceae and Bradyrhizobiaceae could directly impact cultivation practices (e.g., th e need to add additional nitrogen to soybean production). Thus, the purpose of this evaluation was to assess whether the B. japonicum- soybean symbiosis of MON 87769 had be en altered as a result of the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change, compared to the conventional soybean control.

The relative effectiveness of the symbiotic association be tween a leguminous plant and its rhizobial symbiont can be assessed by various methods. Assessment of nodule number and mass along with plant growth an d nitrogen status ar e commonly used to assess differences in the symbiotic association between a legume and its associated rhizobia (Israel et al., 1986). It should be noted, however, that nodule number relative to nodule dry weight may be variable in soybean experiments because some nodules may be larger in diameter and less numerous while others are not as developed (smaller) but more abundant (Israel et al., 1986; Appunu et al., 2006).Monsanto Company 09-SY-200U Page 131 of 395 MON 87769, a conventional soybean control (A3525), and reference soybean varieties were produced in a greenhouse from seeds pl anted in pots containing nitrogen-deficient potting medium. Seeds were inoculated with a solution of B. japonicum . The pots were arranged in a randomized strip-plot or ra ndomized complete block design with eight replicates. At four and/or six weeks after emergence, plan ts were excised at the surface of the potting medium, then shoot and root plus nodule material were removed from the pots. Nodules were separated from roots prior to enumerat ion and determination of dry weight. Detailed information on materials a nd methods used for symbiont evaluation is presented in Appendix K.

An initial study was conducted to assess the symbiotic interactions between B. japonicum and MON 87769; however, result s of the study were vari able and did not provide

conclusive evidence to assess the objectives of the study. Th erefore, a subsequent study was conducted using a refine d methodology in which a nega tive isoline control was

included to determine whether differen ces detected between MON 87769 and the conventional control in the initial study were associated with the trait or the plant transformation process. Additionally, refe rence soybean varietie s representing more diverse soybean germplasm were used to determine whether the measured endpoint values of MON 87769 are within the range of re sponses familiar to what is expected for soybean. The results from both studies were co nsidered in assessing the impact from the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and intended fatty acid change on the symbiotic relationship between B. japonicum and MON 87769, plant pest potential, and cultivation practices relative to nitrogen inputs. The re sults from both studies are summarized below and in Appendix K.

In the initial study, four out of the ten measurement endpoi nts assessed among the four and six week sampling times were signifi cantly different betw een MON 87769 and the control, although the detected differences we re not consistent among the two sampling times (Table VIII-8). Soybean variet ies are known to be heterogeneous for characteristics associated with symbiotic nitrogen fixation (Sinclair et al., 1991; Pazdernick et al., 1996; Graham et al., 2004). Since MON 87769 was derived from a single seed of the conventi onal control variety A3525, it wa s not possible to conclude

whether the detected differences between MON 87769 and the control were associated with the introduction of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change or were due to inherent variability with in the A3525 variety. In addition, the mean values for both MON 87769 and the control were greater than th e range of the reference varieties for five of the ten measurement endpoints among the two sampling times. This likely was due to varietal differences, since both MON 87769 and the conventional control have the same genetic background, and indicates that the refere nce varieties used in the experiment may not have sufficiently represented the inherent variability in soybean for characteristics associated with symbiotic nitrogen fixation. Therefore, an additional assessment was conducted using refined methodology to further investigate the symbiotic interactions

between B. japonicum and MON 87769 and to determine wh ether the measured endpoint values of MON 87769 are within the range of re sponses familiar to what is expected for soybean.Monsanto Company 09-SY-200U Page 132 of 395 In the subsequent study, the negative isoline of MON 87769, derived from progeny of the regenerated R0 plant, was incl uded as a control, in additi on to the conventional soybean control (variety A3525), to determine if differences between MON 87769 and the

conventional control were due to the introduction of the tra it or the plant transformation process. Additionally, six conventional refe rence soybean varietie s representing more diverse soybean germplasm from maturity group s II to V were included in the evaluation to determine whether the measured endpoint values of MON 87769 are within the range of responses familiar to what is expected for soybean. The evaluation was conducted using the same methods and measurement endpoints as in the first study with the exception of the following refinements. The data were collected at a single time point of six-weeks after emergence when the soybean pl ants were at approximately the R4 (full pod) growth stage and not at four weeks af ter emergence. Since nodule number and size

and N-fixation increase over time until the R5 -R6 stage (Iowa State University, 2004; Lersten and Carlson, 2004), it was determined that the six-week time point was more appropriate for assessing symbiotic interactions between soybean and B

. japonicum. Consequently, pots were arranged in the greenhouse in a randomized complete block design with eight replications instead of the randomized st rip-plot design used in the initial study. In addition, smaller pots and a single B. japonicum inoculation, instead of two inoculations (as was perf ormed in the initial study), were used to further reduce variability within the test system. The c oncentration of inoculum was reduced from approximately 1 × 10 8 cells to approximately 1 × 10 7 cells of B. japonicum due to the use of smaller pots and to reduce th e potential for pseudo-nodulation. MON 87769 was compared to the conventional control as well as to the negative is oline for nodule number, nodule dry weight, shoot dry weig ht, root dry weight, and s hoot nitrogen. A summary of the results is provided below, while the deta ils of the materials and methods are provided in Appendix K.

No statistically significant di fferences were detected be tween MON 87769 and either the conventional or negative isoline control fo r any measured parameter, including nodule number, shoot nitrogen (percent and total nitr ogen), and biomass (dry weight) of nodules, shoot material, or root material (Table VIII -9). Apart from shoot total nitrogen (% dry weight), each measurement endpoint evalua ted for MON 87769 was within the range of

the reference varieties. The mean shoot total nitrogen of MON 87769 (3.77%) was only slightly outside the reference range (2.85 – 3.76%). The percent differences between MON 87769 and the negative isoline were le ss than or equivalent to the percent

differences between MON 87769 and the conve ntional control for five of the six measurement endpoints (Table VIII-10).

The results from the refined assessment s upport the hypothesis that differences between

MON 87769 and the conventional control observed in the firs t study were not attributed

to the introducti on of the genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and intended fatty acid change or plant transformation process. The results also de monstrate that the measured endpoint values of MON 87769 are within the range expected for soybean. Considering all of the data, the evidence supports a conclusion that the introduction of the genes ( Pj.D6D and

Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and intended fatty acid change does not alte r the symbiotic relationship between B. japonicumMonsanto Company 09-SY-200U Page 133 of 395 and MON 87769 compared to that of conventio nal soybean. Thus, th ere is no expected impact to cultivation practices relative to nitrogen inputs and no increased plant pest potential of MON 87769 compar ed to conventional soybean.Monsanto Company 09-SY-200U Page 134 of 395Table VIII-8. Symbiont Interaction Assessment of MON87769 and the Conventional Control at 4- and 6-Weeksafter Emergence in anInitial AssessmentMeasurementEndpointMean (S.E.) Reference Range1SamplingTimeMON 87769 Control Minimum MaximumNodule Number(per plant)4 week77.63 (2.91) 68.29 (4.26)44.25 75.886 week 180.13 (11.39)* 140.50 (14.56)60.00 128.25Nodule Dry Wt(g/plant)4 week0.13 (0.01)* 0.10 (0.01)0.07 0.106 week 0.30 (0.03) 0.25 (0.02)0.13 0.22Root Dry Wt(g/plant)4 week0.89 (0.05) 0.82 (0.05)0.81 0.886 week 1.94 (0.07) 1.71 (0.13)1.35 1.67Shoot Dry Wt(g/plant)4 week 1.81 (0.14)1.49 (0.08)1.33 1.536 week 4.57 (0.25)* 3.50 (0.30)2.64 3.71Shoot TotalNitrogen(% dry wt)4 week 2.83 (0.10)2.74 (0.10)2.34 2.466 week 2.54 (0.05)* 2.80 (0.09)2.11 2.21Note: Pots were arranged in eight replicated blocks (n = 8) in a greenhouse for 4-and 6-week sampling periods using a randomizedstrip-plot design. SE = Standard Error.* Indicates a statistically significant difference (p<0.05) between MON 87769 and the conventional control (A3525).1Reference range is the minimum and maximum mean value observed among the three commercial reference soybean varieties.Monsanto Company 09-SY-200U Page 135 of 395Table VIII-9. Symbiont Interaction Assessment of MON 87769,the Conventional Control, and the Negative Isoline at6-Weeks after Emergence in a Refined AssessmentMean (S.E.)Reference Range1MeasurementEndpointMON87769A3525 Negative Isoline Minimum MaximumNodule Number(per plant)208 (24) 185 (23) 206 (16)83 265Nodule Dry Wt(g/plant)0.56 (0.04) 0.49 (0.06) 0.53 (0.04)0.40 0.80Root Dry Wt(g/plant)1.53 (0.12) 1.63 (0.08) 1.57 (0.17)1.08 2.58Shoot Dry Wt(g/plant)6.21 (0.54) 6.29 (0.32) 6.36 (0.52)5.05 11.25Shoot TotalNitrogen(% dry wt)3.77 (0.06) 3.66 (0.07) 3.74 (0.13)2.85 3.76Shoot TotalNitrogen (g)0.23 (0.02) 0.23 (0.01) 0.23 (0.01)0.19 0.33Note: Pots were arranged in a greenhouse ina randomized complete block design with eight replications (n = 8). S.E. = Standard Error.No significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525) or the negative isoline for anymeasured endpoint.1Reference range is the minimum and maximum mean value observed among the six conventional reference soybean varieties.Monsanto Company 09-SY-200U Page 136 of 395 Table VIII-10. Percent Difference between MON 87769 and the Conventional
Control or Negative Isoline Control

MON 87769

Measurement Endpoint Difference from

Conventional Control (%) 1 Difference from Negative Isoline (%)

Nodule Number (per plant) 11.3 1.0

Nodule Dry Wt (g/plant) 12.5 5.4

Root Dry Wt (g/plant) -6.5 -2.6

Shoot Dry Wt (g/plant) -1.3 -2.4

Shoot Total Nitrogen (% dry wt) 2.9 0.8

Shoot Total Nitrogen (g/plant) 0 0

1 Conventional control = variety A3525.

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

Interactions Evaluation

An extensive and robust set of information and data were used to assess whether the introduction of the genes ( Pj.D6D and Nc.Fad3 ) genes, the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change altered the plant pest potential of M ON 87769 compared to the conve ntional soybean control, A3525. Phenotypic and agronomic characte ristics of MON 87769 were evaluated and compared to those of the conventional soyb ean control. These assessments included 14 plant growth and development characterist ics; five seed dormancy and germination parameters under six different temperature regi mes; two pollen charact eristics; more than 960 observations for plant interactions with arthropods, diseases, and abiotic stressors under field conditions; six plant-symbiont inte raction characteristics; and compositional evaluation (Section VII) of 75 different compon ents (seven in forage, and 68 in soybean seed).

Results from the phenotypic and agronomic assessments demonstrate that MON 87769 does not possess characteristics that would increase plant pe st potential compared to conventional soybean. Data on environmenta l interactions indicate that MON 87769 does not confer any biologically meaningful increased suscep tibility or tolerance toMonsanto Company 09-SY-200U Page 137 of 395 specific diseases, arthopods, or abiotic stre ssors, or changes in agronomic and phenotypic characteristics. Taken together, these data support the conclusion that MON 87769 is not likely to pose increased plant pest risk compared to conventional soybean.Monsanto Company 09-SY-200U Page 138 of 395 IX. U.S. AGRONOMIC PRACTICES

IX.A. Introduction

This section provides a summary of the ke y agronomic practices in the U.S. for producing soybean and is provided to serve as a baseline for comparative purposes in an analysis of the impacts to current agronomic practices due to cultivation of MON 87769. Discussions include soybean production, growth and development, general management practices, management of weeds, insects a nd diseases, soybean ro tational crops, and volunteer soybean management. Compared to traits such as herbicide tolerance or resistance to insects that directly benef it the growers, the pr oduction of SDA through

cultivation of MON 87769 is ne utral with respect to agrono mic benefits. Information presented in the previous section demonstrated that MON 87769 is no more susceptible to diseases or pests and is phenotypically comparable to conventional soybean. Thus, there are no impacts to the inputs needed to obtain a crop from MON 87769, and as such, there are no specific impacts to most of th e agronomic practices employed for production of soybean. MON 87769 will be produced under an identity preservation system (IDP) requiring specific management to preserve the value of the SDA soybean oil. Therefore, emphasis is placed on the anticipated impact s on agronomic practices used for production of specialty type soybean upon deregulation of MON 87769.

Soybean is planted in over 30 states, demons trating its wide adaptation to soils and climate. The soil, moisture, and temper ature requirements for producing soybean are generally similar to those for corn and thus the two crops share a similar cultivation area. Proper seedbed preparation, appr opriate variety sele ction, proper plantin g dates and plant population, and good integrated pest management practices are important to optimizing the yield potential and economi c returns of soybean.

MON 87769 is expected to bring added value for growers, soybean handlers, crushers and food processors. An identity preserva tion system will be used for production, postharvest handling and processing to preserve the enhanced value of MON 87769. As such, production practices, post-harvest handling of soybean, and processing will fall under a separate production system and distribution channel termed fo r this specialty soybean. MON 87769 is expected to utilize existing ag ricultural practices employed for production and identity preservation of specialty soybean.

Annual and perennial weeds are perceived to be the greatest pest problem in soybean production. Economic thresholds for controllin g weeds in soybean require some form of weed management practice on all soybean acre age. Approximately 98% of the soybean acreage receives an herbicide application. Soybean insects and diseases are generally considered less problematic, although infe stations do occasionally reach economic thresholds requiring treatment.

Volunteer soybean is not considered a signifi cant concern in rotational crops, primarily because of climatic conditions and adequate co ntrol from tillage practices. Additionally,Monsanto Company 09-SY-200U Page 139 of 395 mechanical and chemical control methods are available to manage the occasional volunteer soybean plant. Due to the lack of weediness poten tial, introduction of MON 87769 in the soybean production system would have a negligible impact on managing soybean volunteer plants in rotation al crops such as co rn, cotton, and rice

because control measures are available for vol unteer soybean when they arise. Pre-plant tillage is the first management tool for control of emerging volunteer soybean in the spring. If volunteer soybean emerges after pl anting, shallow cultivat ion will control most of the plants and effectively reduce comp etition with the crop. Several postemergence

herbicides also are available to control volunteer soybean (conventional or glyphosatetolerant soybean) in each of the major rotational crops.

As shown in Sections VII and VIII, with the exception of the intended fatty acid change (presence of SDA and GLA in MON 87769 seed), no phenotypic, compositional, or

environmental differences between MON 87769 and conventional soybean have been observed. Therefore, it is not anticipated that commerci alization of MON 87769 in the U.S. would have a notable impact on current soybean cultivation practices, including the management of weeds, diseases, and insects.

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

IX.B.1. Soybean Production

Soybean first entered North America in the 18 th century (Hoeft et al ., 2000). Sometime during the 1930s, soybean started to be proce ssed industrially in the U.S. for edible oil and protein meal. Currently, the U.S. produ ces approximately 32% of the global soybean supply (ASA, 2008). In 2007, the U.S. exporte d 1 billion bushels (27.9 million metric tons) of soybean, which accounted for 37 perc ent of the world's soybean exports (ASA, 2008). In total, the U.S. exported $12.9 billion USD worth of soybean and soybean products globally in 2007 (ASA, 2008). China is the largest export market for U.S. soybean with purchases totalling $4.1 billion. Japan is the second largest export market with sales of $1.1 billion in the same year . Other significant markets include the European Union and Mexico.

The production of soybean is highly dependent upon soil and climatic conditions. In the U.S., the soil and climatic requirements for gr owing soybean are very similar to corn. The soils and climate in the eastern half of the U.S. provide sufficient water supplies under normal climatic conditions to produce a soybean crop. The general water requirement for a high-yielding soybean crop is approximately 20 inches of water during the growing season (Hoeft et al., 2000). Soil texture and structur e are key components determining water availability in soils, where medium-textured soils hold more available water, allowing soybean roots to penetrate deep er in medium-textured soils than in clay soils. Irrigation is used on approximately 9% of the acreage to supplement the water supply during dry periods in the western a nd southern soybean growing regions (ASA, 2008).

Most of the soybean acreage is grown as a full-season crop. Approximately 8% of the soybean acres are planted in a double-crop sy stem following winter wheat south of 35oMonsanto Company 09-SY-200U Page 140 of 395 North latitude (Boerma and Specht, 2004). However, this percentage can vary significantly from year to year. The decisi on to plant double-crop soybean is influenced by both agronomic and economic factors. Agro nomic factors include harvest date of the wheat crop, which determines the double-crop soybean planting date , and available soil

moisture. Economic factors include expected soybean price and anticipated economic return (Boerma and Specht, 2004).

The vast majority of soybean grown in the U. S. is grown for animal feed and is usually fed as soybean meal. However, approxima tely 12% of soybean grown is specialty soybean produced for a specific market or use. 3 Examples of specialty soybean include high protein, tofu, high oil, high oleic, non -biotechnology-derived (also referred to as non-genetically modified), and organic soybean. 4 The uses of these soybean varieties include human consumption, food processing or specialty products. The soybean varieties used in the specialty market are typically specified by buyers and endusers of soybean for production, and a premium relative to commodity soybean is paid for delivering a product that meets purity and quality standards (Pritchett et al., 2002; Elberhi, 2007; Sundstrom et al, 2002; Lee and Herbe k, 2004; Muth et al 2003; and Smyth and Phillips 2002) for the soybean variety. Produc t differentiation and market segmentation in the specialty soybean industry includes m echanisms to keep track of the soybean (traceability), methods for identity preser vation (IDP), including closed-loop systems, and quality assurance proce sses (e.g., ISO9001-2000 certificatio n), as well as contracts between growers and buyers that specify delivery agreements. MON 87769 is an alternate source of omega-3 fatty acid and w ill be considered a specialty soybean product

and marketed in a manner similar to other high-value specialty crops.

The U.S. soybean acreage in the past ten years has varied from approximately 64.7 to 75.7 million acres, with the lowest acreage reco rded in 2007 and the highest in 2008 (Table IX-1). Average soybean yields have varied from 33.9 to 43.3 bushels per acre over this same time period. Soybean production ranged from 2.45 to 3.19 billion bushels over the past ten years, with 2006 being the largest production year on record. According to data from USDA-NASS (2009), soybean was planted on approximately 75.7 million acres in the U.S. in 2008, producing 2.96 billion bushels of soybean (Table IX-1). Soybean acreage and production in 2007 was do wn significantly from 2006, mainly due to a large increase in corn acreage. The va lue of soybean reached $27.4 billion in the U.S. in 2008 (USDA-NASS, 2009). In compar ison, corn and wheat values in 2008 were $47.37 and $16.57 billion, respectively (USDA-NASS, 2009).

For purposes of this agronomic practices di scussion, soybean production is divided into three major soybean growing regions accounting for 99.1% of the 2008 U.S. soybean acreage: Midwest/Great Plains region (IL, IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, SD, and WI), Southeast regi on (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern Coastal region (DE, MD, NJ, NY, PA, and VA) (Table IX-2). The vast majority of soybean was grown in the Midwes t region, representing 82.1% of the total U.S. acreage.

3 http://usb.adayana.com:8080/usb/jsp/login.jsp

4 http://usb.adayana.com:8080/usb/jsp/login.jspMonsanto Company 09-SY-200U Page 141 of 395 The Southeast and Eastern Coastal regions represented 14.3% and 2.7% of the acreage, respectively. Among the three regions, the Midwest region produced the highest average yield at 38.6 bushels per acre in 2008, and aver age state yields in this region ranged from 28.0 to 47.0 bushels per acre. The average yield in th e Southeast region was 34.4 bushels per acre, with states within this region av eraging from 30.0 to 40.0 bushels per acre. The average yield in the Eastern Coastal region was 34.1 bushels per acre, with individual state averages ranging from 27.5 to 46 bus hels per acre. It is anticipated that MON 87769 will be grown mainly in the Midwest/Great Plains region, where MON 87769 is anticipated to be grown on le ss than 3.5 million acres (~5% of the total U.S. soybean acreage). MON 87769, as a specia lity soybean product is not expected to impact overall soybean production acreage in the U.S., and the impact on total soybean acreage in the Midwest/Great Plains region is an ticipated to be negligible as well because MON 87769 plantings are expected to replace existing soybean acreage. Additionally, the total U.S. soybean acreage has fluctuated over the past ten years (64.7 to 75.7 million acres) and the MON 87769 acreage is anticipated to be well within this historical

fluctuation.

Managing input costs is a major component to the economics of producing a soybean crop. Key decisions on input costs include choosing what soybean varieties to plant, amounts of fertilizer to apply, and what herbicide program to use. The average operating cost for producing soybean in the U.S. in 2006 was $93.41 per acre, according to statistics compiled by the USDA-Economic Re search Service (USDA-ERS, 2006). The value of the produc tion less operating cost was repo rted to be $161.43 per acre. A summary of potential production costs and returns are presented in Table IX-3.

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

Year Acres

Planted (×1000) Acres

Harvested

(×1000) Average

Yield (bushels/acre) Total

Production (×1000 bushels) Value (billions $)

2008

2007 75,718 64,741 74,641 64,146 39.6

41.7 2,959,174 2,677,117 27.40

26.97 2006 75,522 74,602 42.7 3,188,247 20.42 2005 72,142 71,361 43.3 3,086,432 16.93 2004 75,208 73,958 42.2 3,123,686 17.89 2003 73,404 72,476 33.9 2,453,665 18.01 2002 73,963 72,497 38.0 2,756,147 15.25 2001 74,075 72,975 39.6 2,890,682 12.61 2000 74,266 72,408 38.1 2,757,810 12.47 1999 73,730 72,446 36.6 2,653,758 12.21
Source: USDA-NASS, 2009.Monsanto Company 09-SY-200U Page 142 of 395 Table IX-2. U.S. Soybean Production by Region and State in 2008

Region/State Acres

Planted 1
(thousands) Acres

Harvested 1
(thousands) Average Yield 1
(bushels/acre) Total

Production 1
(×1000 bushels) Value 1
(billions $)
Midwest Region

Illinois 9,200 9,100 47.0 427,700 4.00

Indiana 5,450 5,430 45.0 244,350 2.27

Iowa 9,750 9,670 46.0 444,820 4.29

Kansas 3,300 3,250 37.0 120,250 1.03
Kentucky 1,390 1,380 34.0 46,920 0.42
Michigan 1,900 1,890 37.0 69,930 0.64

Minnesota 7,050 6,950 38.0 264,100 2.54

Missouri 5,200 5,030 38.0 191,140 1.72

Nebraska 4,900 4,860 46.5 225,990 2.12

North Dakota 3,800 3,760 28.0 105,280 0.96

Ohio 4,500 4,480 36.0 161,280 1.55

South Dakota 4,100 4,060 34.0 138,040 1.25

Wisconsin 1,610 1,590 35.0 55,650 0.51

Region Totals 62,150 61,450 38.6 2,495,450 23.30
Southeast Region

Alabama 360 350 35.0 12,250 0.12

Arkansas 3,300 3,250 38.0 123,500 1.09

Georgia 430 415 30.0 12,450 0.11

Louisiana 1,050 950 33.0 31,350 0.29

Mississippi 2,000 1,960 40.0 78,400 0.69

North Carolina 1,690 1,670 33.0 55,110 0.47

South Carolina 540 530 32.0 16,960 0.15

Tennessee 1,490 1,460 34.0 49,640 0.43

Region Totals 10,860 10,585 34.4 379,660 3.35
Eastern Coastal Region

Delaware 195 193 27.5 5,308 0.05

Maryland 495 485 30.0 14,550 0.13

New Jersey 92 90 29.0 2,610 0.02

New York 230 226 46.0 10,396 0.09
Pennsylvania 435 430 40.0 17,200 0.15
Virginia 580 570 32.0 18,240 0.16

Region Totals 2027 1994 34.1 68,304 0.60

1 Source: USDA-NASS, 2009.Monsanto Company 09-SY-200U Page 143 of 395 Table IX-3. U.S. Soybean Production Costs and Returns in 2006

Production Cost or Return Cate g or y Itemized Costs Return per Planted Acre ($ USD)

Total Gross Value of Production 254.84

O p eratin g Costs: Seed 32.30

Fertilize r 13.05

Chemicals 14.46

Custom o p erations 6.01
Fuel, lube and electricit y 13.51

Re p airs 11.80 Purchased irrigation

wate r 0.11

Interest on operating

ca

p ital 2.17

Total, o p eratin g costs 93.41

Hired labo r 1.78

Allocated overhead: Opportunity cost of

un p aid g rower’s labo r 15.20

Capital recovery of
machiner y and e q ui p men t 60.38

Opportunity cost of land ( rental rate ) 86.17

Taxes and insurance 7.93

General farm overhead 13.22

Total, allocated overhead 184.68

Total cost listed 278.09

Value of p roduction less total cost listed (23.25)

Value of p roduction less o p eratin g costs 161.43

Supporting Information: Yield = 46 bushels/acre, Price = $5.54/bushel, Enterprise size = 268 planted acres, Irrigated = 9%, Dry land = 91%.

Source: USDA-ERS. 2006. Soybean production costs and returns per planted crop acre, by region, excluding government payments for 2006. United States Department of Agriculture-Economic Research Service. ( http://www.ers.usda.gov/data/c ostsandreturns/Soy_all.xls ) [Accessed June 26, 2009].Monsanto Company 09-SY-200U Page 144 of 395 IX.B.2 Specialty Soybean

Commodity and specialty soybean are th e two primary production and distribution systems for soybean produced in the U.S. The majority of soybean commonly is grown and marketed through commodity markets for the oil and protein content. Commodity soybean is not consumed directly, but is crushed for meal that is predominantly used for animal feed and as a minor protein source fo r food. The oil produced during the crushing of the soybean is used for c ooking or food ingredients. Th e goal of the commodity chain is to supply a homogenous product to the e nduser. The grower producing soybean for this chain has a choice from many different varieties for production; however, harvested soybean is viewed to be the same for all co mmodity soybean varieties. At harvest, the grower either delivers soybean to a handler or stores them on farm for later delivery. The

first handler is not interested in differentia ting the commodity soybean for later use. Commodity soybean handlers typi cally have large volume stor age capacity. Similarly, commodity soybean processors crush large volumes of soybean to produce homogeneous oil and meal products. The commodity system is designed to maximize efficiency at a low profit margin resulting in commingling of different sources of soybean that do not affect the price received for the final product. This production system has been in place in the U.S. since the production of soybean be gan in earnest in the 1960s (Sonka et al., 2004).

In recent years, there has been an increased demand by c onsumers and food processers for soybean that has specific physical or ch emical characteristics that are required by certain customers to meet specif ic food or feed needs. As a result a separate specialty soybean channel has developed. This produ ction system and distribution channel is

focused on value-added traits that involve much smaller volumes than commodity soybean (Sonka et al., 2004). Specialty soyb ean varieties are produced on approximately 14% of the U.S. soybean acreage, 5 and according to the Midwest Shippers Association (MSA, 2009) this acreage could grow to over 25% of the crop acreag e in certain states within the next decade. This supply chain fo r specialty soybean typically consists of a specialty firm that contracts production of a specific variety and sets standards for quality of the harvested soybean (Lee and Herbek, 2004). In return, growers receive a premium over the price paid for commodity soybean. Growers may store harvested soybean on farm or deliver the product directly to a processor or to special containers for international shipment. The goal of this identity preserved system is to minimize handling so that value is maintained. The co st incurred from an identity preservation system is offset by the higher value received for the final product.

According to the American Soybean Asso ciation (2009), specialty soybean can be grouped into ten broad categor ies: Non-biotechnology-derive d, certified seed, organic food-grade, low saturated fat, clear hilum, tofu, natto, high sucrose, high oleic, low

5 http://usb.adayana.com: 8080/usb/jsp/login.jsp : Percent U.S. soybean acreage estimate based on

U.S. Domestic Consumption by Segment – 200 8/09. D. Ludwig, personal communication, 2009.Monsanto Company 09-SY-200U Page 145 of 395 linolenic, and high protein. The categories refe r to soybean with characteristics such as altered seed composition (e.g., low saturated fa t, high sucrose, high oleic, low linolenic, and high protein), varieties of soybean with unique physical characteristics suited to their specific uses (e.g., clear hilum for direct hum an consumption), or refer to a production process (e.g., organic, certified seed). The categories ar e not meant to be exclusive; for instance, soybean used to produc e natto or tofu may employ organic production processes, and soybean from all of the categories is of ten derived from varieties produced according to certified seed production practices. Tofu and soymilk produced from the tofu soybean category represent a large segment of the specialty soybean market and are produced from unique soybean varieties that have clea r hilum and large seed size (Lee and Herbek, 2004). Tofu varieties also must be high in protein (40% or higher) and low in oil concentration compared to commodity soybean. Clear hilum and other characteristic are required for soybean used in the production of other soybean food products consumed directly by humans, such as natto, soybean sprouts, edamame (vegetable soybean), and soy nuts (Lee and Herbek, 2004; UK Cooperative Extension Service, 2009). Organic food-grade and non-biotechnology-derived soyb ean are identity protected and produced not to contain biotechnology-de rived traits. Organic soyb ean have additional production restrictions requiring the soybean to be produced using no synthetic fertilizers or pesticides. In recent years, public and private soybean breeders have developed soybean varieties with improved nutritio nal characteristics (e.g., high sucrose, high oleic, low linolenic, and high protein). The characteristics or compos itional modifications in this group include varieties with fatty acid profile s that reduce the need for hydrogenation of the resulting soybean oil, increased sugar c oncentration, increased protein concentration,

decreased concentration of saturated a nd polyunsaturated fats, and lipoxygenase free soybean, which reduces the production of some of the flavors that are objectionable to some consumers (Lee and Herbek, 2004).

The majority of specialty soybean varietie s are offered in Group II and early Group III varieties which are adapted to the upper Mi dwest/Great Plains region (Lee and Herbek, 2004). Maturity Groups II and III soybean varieties are gr own on approximately 42 to 45 million acres, occupying the largest percentage of soybean acreage (see Section IX.C). The varieties were developed for this area primarily due to proximity to processing facilities as well as international routes of shipment and to take advantage of efficiencies in soybean breeding programs. With a fe w exceptions, the agronomic or management practices for growing specialty soybean fr om planting to harvest are similar to commodity soybean (Lee and Herbek, 2004). Since certain varieties are utilized in the production of each specialty soybean, the variet y selection is dictated by the specialty soybean buyer or processor. Nonbiot echnology-derived and organic soybean by definition must be produced utilizing only conventional soybean varieties. As with commodity soybean, weed control is extrem ely important for specialty soybean to maintain a high-yield potential and because certain weeds, such as nightshade ( Solanum
nigrum ), can stain harvested soybean, which is particularly undesirable in food-grade soybean (TCM, 2009). Since organic soybe an must be grown without synthetic fertilizers or pesticides for three or more years prior to the current crop of soybean, fertilization and pest management is mo re difficult (Lee and Herbek, 2004). Weed control in organic soybean relies on a co mbination of crop rotation, tillage, in-crop cultivation, and hand-weeding. Insect and di sease control is managed primarily throughMonsanto Company 09-SY-200U Page 146 of 395 crop rotation. Certain organic pesticides are permitted in the production of certified organic soybean.

All equipment and storage faci lities for specialty soybean mu st be clean of seed from other soybean varieties or pl ants, dirt, pathogens, and ot her foreign material. Some soybean contracts may require a special inspec tion of the handling and storage facilities. The specialty soybean for soybean foods may require special harvesting equipment since some of this soybean is harvested before full maturity (e.g., edemame or vegetable soybean).

IX.B.3. Identity Preservation

Identity Preservation (IDP) refers to a sy stem of production, handling, and marketing practices that maintains the integrity and purity of agricultural commodities (Sundstrom et al., 2002). Commodity grains, on the ot her hand, are marketed in mass according to USDA grading standards. Specialty crops requ ire some form of segregation or full-scale identity preservation to keep these grains separate from conventi onal commodity grains (Elberhi, 2007). This market segmentation w ithin the grain channeling system is driven by the need to preserve market value or ensu re a specified purity of the product. With certain specialty crops, IDP is required to prevent accidental or unintended commingling (e.g., nonbiotechnology, organic) or to segreg ate products that are approved only for certain uses (indus trial use only).

IDP grain production has been in existence fo r a long time. Agricultural producers have over many decades developed practices that allowed for differentiation between food vs. feed grain, or grain vs. seed production, or organic vs. nonorganic (Massey, 2002). Seed certification programs such as those used by th e Association of Official Seed Certifying Agencies often are cited as the model of IDP systems. These programs date back to the 1920s and 1930s when the certification process was implemented to verify the genetic purity of seed made availa ble to growers (see Section IX.B.4). Standards were established to ensure production of seed from known pedigree with high purity and quality. Similarly, commodity grain trad ers, marketing organizations, and food processors have established purity and quality tolerances for specific end-product uses. The need for segregation and IDP produc tion systems has in creased with the development of specialty crops or crops with special output traits, su ch as high oil corn, high oleic sunflower, and low-linolenic soybean (Sundstrom et al., 2002).

The production of IDP grains requires special processes in order for growers to meet buyers’ criteria for variety id entification, composition, an d quality. Buyers of IDP soybean typically contract for the production of seed-variety speci fic soybean and work directly with seed suppliers, growers, inde pendent certification agencies, intermediate processors, and freight companies to deliv er the preferred produc t within specified

tolerances. 6 Contract specifications are written to ensure the delivery of the desired

6 United Soybean Board, International Buyer’s Guide at http://www.ussoyexports.org/ussoy/buyersguide/Chap6.pdfMonsanto Company 09-SY-200U Page 147 of 395 attribute-specific product and that predetermi ned management practices are used (Massey, 2002). Lack of compliance with a product sp ecification can lead either to a price discount or rejection of a shipment by the buyer. Depending on the end use, contracts can be extensive, defining many of the produc tion, harvest and storag e activities, or less stringent dealing more with pricing, quality specifications and only the most critical production practices. Premiums are paid to growers fo r the additional risk and management only when the grain meets the c ontract specifications . Premium prices provide the impetus to maintain the specialty grain’s purity and identity separate from commodity grains (Massey, 2002). Premiums ar e affected by various factors, including the proximity of suppliers to buyers and the cost and av ailability of substitutes (Sundstrom et al., 2002). For many trait-specif ic crops, price premiums rise or fall, depending on the supply conditions for the generic commodity.

Growers of IDP grains must have good ma nagerial ability and implement certain management practices to produce and deliver grain possessing the desired physical and chemical characteristics. The production and marketing of trait-spec ific grains involves additional financial risks (Elberhi, 2007). Th e growers’ managerial ability can affect both yield performance and proficiency in mee ting the contract specifications. From a soybean buyer’s perspective, contracts he lp the buyer meet the demand for specific product qualities, improve cost efficiencies of product processing, a nd reduce transaction

costs.

While specific IDP production practices vary depending on the characteristics of the product to be delivered, general elements are implemented to ensure that th e enduser or processor receives the grain or end-product with the intended identity and desired quality. As mentioned above, many IDP systems were de veloped using the principles similar to those used in seed certification. IDP produc tion begins with a system of standards, records, and auditing that are put in place throughout the entire crop production, harvesting, handling, and marketing proces s (Sundstrom et al., 2002). Some key

considerations in the establishment of an IDP system include: 1) plan ted seed identity and tolerances, 2) appropriate fi eld isolation, 3) inspection an d clean-out of equipment and facilities, 4) end-product sampling and testing, and 5) record maintenance and identity labeling (Sundstrom et al., 2002). Each of these components are described in greater

detail below:

Planted Seed Identity and Tolerances

The purity and identity of starti ng seed should be tested and confirmed. The purity of the seed stock should equal or exceed the purity standards of the desired final product. Single or multiple quality tolerances may be established in specialized IDP programs

based on market-driven standards.

Field Isolation

Crops must be isolated either spatially or temporally fr om pollen sources that could impact the quality or purity of the harveste d seed. The amount of isolation depends on flower characteristic, sexual compatibility with neighboring crops, pollen quantity andMonsanto Company 09-SY-200U Page 148 of 395 viability, and mode of pollen dissemination. A self-pollinating crop, such as soybean, requires relatively small isolation distances to effectively preclude cross-pollination.

Equipment and Facilities

Equipment used in production should be cleaned and inspected before and after use in an IDP crop. Storage facilities and transporting ve hicles are cleaned and inspected to assure that segregation is maintained a nd no physical contamination occurs.

Sampling and Testing

In some cases, the IDP grain is sampled and tested at various stages to confirm the product identification, purity, and quality. Sp ecial consideration should be given to sampling and testing techniques th at ensure reliable results.

Record Maintenance and Identity Labeling

Records typically are maintained on field designations, harvest am ounts, storage bin locations, and product transfers. IDP products must be identified, segregated, and labeled

in the market chain.

As with current IDP systems used for specialty soybean to preserve market value or ensure purity of the product, MON 87769 will employ an IDP system based on established practices. The IDP practices will be implemented to ensure that the enduser or processor receives the soybean with the id entity, fatty acid composition of the oil, and desired quality for this value-added specialty soybean. This system will also serve to direct this speciality soybean to its intended use and away from the commodity processing stream.

IX.B.4. Soybean Seed Production

Standardized seed production practices are re sponsible for maintaining high quality seed stocks, an essential basis for U.S. agriculture. By the early 20 th century, agronomists learned how to develop specific plant varieties with desirable traits. In the U.S., state agricultural experiment stations developed many varieties which were distributed to growers for use. Growers saved seed and late r sold to neighbors; however, the desirable

traits of the varieties often were lost through random genetic changes and contamination with other crop and weed seed (Sundstrom et al., 2002). The value of seed quality (including genetic purity, vigor, absence of w eed seed, and seed-borne diseases and inert materials such as dirt) was quickly identified as a major factor in crop yields. States developed seed laws and certification agencies to ensure that purchasers who received

certified seed could be assured that the s eed met established seed quality standards (Bradford, 2006). The federal government pa ssed the U.S. Federal Seed Act of 1939 to recognize seed certification and official certif ying agencies. Regulations first adopted in 1969 under the Federal Seed Act recognize land history, field isola tion, and varietal purity standards for foundation, registered, and certified seed. Under international agreements such as the Organization for Economic Co-Operation and Development scheme, the U.S. and other countries mutually recognize minimum seed quality standardsMonsanto Company 09-SY-200U Page 149 of 395 (Bradford, 2006). The Associati on of Official Seed Certifyi ng Agencies represents state and private seed certification in the U.S., and includes intern ational member countries in North and South America, as well as in Australia a nd New Zealand.

Soybean seed is separated into four seed cl asses: 1) breeder, 2) foundation, 3) registered, and 4) certified (AOSCA, 2009). Breeder seed is seed di rectly controlled by the originating or sponsoring plan t breeding organization or firm . Foundation seed is first generation seed increased from breeder seed an d is handled to maintain specific varietal purity and identity. Registered seed is the pr ogeny of foundation seed that is handled to maintain satisfactory variety purity and identity. Certified seed is the progeny of breeder, foundation or registered seed and is two ge nerations from foundation seed. All soybean seed sold may not be officially certified; however, commercial soybean seed sold and planted for normal soybean production predomin ately is produced to meet or exceed

certified seed standards. This section of th e petition will provide a broad overview of the practices utilized in prod ucing certified seed.

Soybean seed breeders and producers have put in place practical measures to assure the quality and genetic purity of soybean seed va rieties for commercial planting. The need for such systems arose from the recogniti on that the quality of improved soybean varieties quickly deteriorated in the absence of monitoring fo r quality and genetic purity (CAST, 2007). Seed certification programs we re initiated in the early 1900s in the U.S. to preserve the genetic identity and variet y purity of seed. Th ere are special land requirements, seed stock eligibility requirements, field inspections and seed labeling

standards for seed certification. Seed certific ation services are available through various state agencies affiliated with the Association of Official Seed Certifying Agencies. Large seed producers implement their own seed quali ty assurance programs. However, large seed producers often will utilize the services of state certifying agenci es as a third-party source to perform certain fiel d inspections and audits.

The U.S soybean production for all purposes according to USDA-NASS statistics has varied from approximately 64.1 to 74.6 million acre s in the past ten year s, with the lowest acreage recorded in 2007 and the highest in 2008 (USDA-NASS, 2009; Table IX-1). This range of soybean acreage would require 105 to 125 million units (50 lbs/unit) of soybean seed. This seed volume includes allo wances for seed losses due to weather, poor yields, and quality issues. Additional allo wances are included for distribution excess, seed returns, replants and potential increases in soybean acreage. Assuming an average soybean yield of 45 bushels, or 54 units ( 50 lbs/unit) per acre, 1.9 to 2.3 million acres would be required to produce this volume of commercial certified soybean seed each

year.

Soybean seed is produced throughout most of the U.S. soybean growing regions. Soybean varieties are developed and adapte d to certain geographical zones and are separated into ten maturity groups – Group 00 to Group VIII (see Section IX.C). Seed production for these maturity groups is grow n in the respective geographical zone for each maturity group. However, the production areas generally are on the northern edge of the respective zone to minimi ze incidences of disease.Monsanto Company 09-SY-200U Page 150 of 395 Soybean seed is produced by companies which produce and sell seed, such as Monsanto Company, Pioneer Hi-Bred International, Syngenta Seeds, Kruger Seed Co., Becks Hybrids, and tollers, which are companies that produce but do not sell certified seed, such as Remington Seeds LLC and Precision Soya. Seed companies and tollers in turn contract acreage with grow ers to produce the required amount of soybean seed. Production or processing plants at these seed companies identify top soybean growers to produce the seed and also mon itor and inspect seed fields throughout the growing season. The production plants also clean, condition, and bag the harvested soybean seed as well as monitor and inspect all the processes at the plant. Production plants typically produce between 100,000 units to 2,000,000 units of soybean seed. Production plants will produce the various soybean varieties in differe nt climates or environments to spread production risks.

The entire seed production process at the majority of private seed companies and tollers is International Organization for Standardization (ISO) certified and, therefore, includes

internal and external audi ts (ISO, 2009). The ISO standards ensure desirable

characteristics of seeds and services such as quality, safety, reliab ility, and efficiency. ISO standards represent an international consensus on good manageme nt practices with the aim of ensuring that the organization can consistently deliver excellent product or services. The standards not only must meet the customer’s requirements and applicable seed regulatory requirements, but must aim to enhance customer satisfaction and achieve continual improvement of its performa nce in pursuit of these objectives.

The field operations and management practices for producing soybean seed are similar to normal soybean production. However, special a ttention is needed in certain areas to produce seed with high quality, high germina tion rates, and high genetic purity (Helsel and Minor, 1993). General guide lines specific for seed prod uction are discussed below. The seed production field should not have been planted with soybean the previous crop in order to avoid volunteer soybean plants (eve n though the risk of s oybean volunteer plants is negligible) and to ensure genetic purity.

Very early planting should be avoided becau se the seed produced from early planting often results in poorer quality seed (Helsel a nd Minor, 1993). Every effort must be made to eliminate weeds in a seed field through the use of herbicides and cultivation to prevent weed seed in the harvested soybean seed. Fields are scouted frequently for insect pests and insecticides are applied when insect pest infestations reach economic threshold levels. Foliar-applied fungicides should be considered when disease infestations are predicted in the area. Harves t should occur as soon as the mature soybean seed reaches 13% moisture content. Harvesting soybean s eed with less than 13% moisture can cause damage to the seed coat and result in split soybean seed that can affect germination and viability. Harvesting equipment must be adjusted to minimize or avoid seed damage. Harvesting equipment must be cleaned before entering the seed fields to minimize genetic contamination. Certain handling equipm ent such as auger elevators, should be avoided because they can increase seed damage.

Field inspections are vital to ensure th e soybean seed meets seed certification requirements, ISO certification standards, regulatory standards, and trait licensingMonsanto Company 09-SY-200U Page 151 of 395 agreement standards. Field inspections are conducted on seed production fields throughout the soybean growing season to evalua te variety purity, ensure soybean plants are developing properly, and fiel ds are maintained free of weed s, insects, and diseases. The fields also are mapped to ensure the seed field has the minimum federal isolation requirement of five feet (AOS CA, 2009). Some states and se ed producers have a stricter isolation requirement of 10 feet.

Production plant personnel make every effort to avoid mechanical damage to the harvested seed during the scr eening, cleaning, and bagging pro cesses. Specific methods are used to assure the genetic purity and identity of the seed is maintained throughout the handling and storage operation. Bin inspections and sample collections are conducted at storage locations at the plant to examine the physical characteristics of the soybean seed and ensure proper bin cleanout. Seed is inspected for appear ance, disease, discoloration, seed coat, mechanical damage, inert matter, and weed seed. Warm and cold germination tests are conducted on all seed lots to verify acceptable germination rates. Many seed companies will also conduct tetrazolium stai ning tests to assess seed viability.

Commercially certified soybean seed must m eet 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 crop seeds (maximum), 0.5% other varieties (maximum, includes off-colored beans and off-type seeds), 0.10% other crop seeds (maximum, not to exceed three per lb.), and 80% germination and hard seed (minimum) (AOSCA, 2009). State seed certification standa rds vary slightly from state to state and

can be more restrictive than th e seed standard s of AOSCA.

MON 87769 seed will be produced in the same manner as comm ercially certified soybean seed, such that it will meet all state and federal seed standards and labeling requirements.

IX.C. Production Management Considerations

Pre-Season

Crop rotation, tillage system, row spacing, pl anting equipment, seed or variety selection(s), and soil fertility are areas that require production deci sions well in advance of planting the soybean crop. Many of the d ecisions in this area are made immediately after harvest of the previous crop or sooner. There are many benefits to crop rotation, with the majority of the soybean acreage plan ted in a two-year corn-soybean rotation (see Section IX.G). Crop rotation is generally a long-term decision, but the rotation sequence can be modified to take advantage of a pa rticular economic or market opportunity. The decision to plant soybean in a conservation tillage or no-till system may require special equipment and will be made long before planting. In addition, this decision usually will be a long-term commitment, provided the system is successful. A decision to change row spacing is a similar long-term commitment that generally requires new equipment.Monsanto Company 09-SY-200U Page 152 of 395 The benefits of conservation tillage or no-till systems are well documented and include reduced soil erosion, reduced fu el and labor costs, and conser ving soil moisture. In 2004, approximately 29.3 million acre s (38.6%) of soybean were planted in a no-till system (CTIC, 2004). Slow soybean emergence and grow th plus lower yields have been some of the concerns associated with adoption of conservation tillage systems in soybean, especially no-till. Research in Wisconsin and Minnesota shows that soil temperatures can be four to five degrees colder in no-till than conventio nal tillage systems, which can slow emergence but have little effect on soybean yield (Pedersen, 2008a). Improved planters for establishment of good soybean populations and planting Roundup Ready soybean to effectively control weeds in no -till fields have made no-till a viable production system for soybean. Researchers still recommend some spring tillage on finetextured and poorly drained soils for prope r seedbed preparation (Pedersen, 2008a).

Most field crops, including soybean, respond very well to fertilizer when planted in soils with low fertility levels. Soybean requi res 16 essential elements for growth and development. Deficiencies in any of these elements can reduce yields (Hoeft et al., 2000). The primary or major essential nutrien ts are nitrogen, phosphor us 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 throug h the symbiotic relationship with the nitrogen-fixing Bradyr hizobia bacteria (Bradyrhizobium japonicum ) that live in the nodules on its roots. Bradyrhi zobia are unicellar, microscopic bacteria that invade the soybean plant through its root hairs (Hoeft et al., 2000). The plan t responds to this invasion by forming nodules which contain colonies of bacteria . After being established on the soybean root, bacteria in the nodule ta ke gaseous nitrogen from the atmosphere and fix it in forms easily used by the soybean plant. Since the bacteria are not native to U.S. soils, inoculation of the soybean seed is recommended when soybean has not been grown in a field for three to five years. Nitrogen fertili zer applications at planting generally do not improve yield and decreas es nodulation while increasing the plant’s dependency on the soil for nitrogen (Pedersen, 2008a). Therefore, nitrogen fertilizer seldom is applied prior to planting a soybean crop.

Soil tests are the only reliable way to determine the pH , phosphorus, and potassium levels in the soil. Liming and fertilizer requiremen ts are subsequently determined based on soil

test results. Ideal soil test results for corn are also ideal for soybean (Scott and Aldrich, 1970). In corn-soybean rotations in the Mi dwest, phosphorus and potassium fertilizers generally are applied prior to a corn crop in accordance with soil test recommendations but seldom are applied prior to a soybean crop. However, for soybean plants which require large amounts of phosphorus and potassium, fertilizer often is needed in some of the southern growing areas due to differe nces in crop rotations and soil types.

Although not common, deficiencies can oc cur in secondary nut rients (calcium, magnesium, and sulfur) or micronutrients (boron, chloride, copper, iron, manganese, molybdenum, and zinc). The availa bility of soil nutri ents is dependent on soil acidity or pH level. Soybean is adversely affected wh en the pH is below approximately 5.8 (Hoeft et al., 2000). Since soybean is grown in rotation with corn and other crops, soil pH

should be maintained at about 6.0 to 6.5 on acidic soils through the addition of limestone.Monsanto Company 09-SY-200U Page 153 of 395 Soybean varieties are developed and adapte d to certain geographical zones and are separated into ten maturity groups – Gr oup 00 to Group VIII (Hammond et al., 1991; Zhang et al., 2004). Groups 00 and 0 are the earliest maturity groups and are adapted

best to the area north of lat itude 46o north. Succeeding gr oups are adapted further south with Groups I and II within latitudes 41o a nd 46o north, and Group III within latitudes 38o and 41o North. Group 00 through Group IV soybean varieties are planted in the Midwest and Eastern Coastal regions. Groups II, III and IV account for approximately 75%, 24%, 36%, and 16%, respectively of the soybean planted in the U.S., with Group III having the largest acreage (T. Schlueter, personal co mmunication, 2008). Gr oups IV through VIII are planted in the southern states with Gr oups V, VI and VII representing 7%, 2%, and 2% of the planted soybean, respectively (T. Sc hlueter, personal communication, 2008).

Soybean variety selection is cr ucial for high yield and quality, and is the foundation of an effective management plan (Pedersen, 2008a). Soybean characteristics to consider in selecting a variety include ma turity, yield potential, diseas e and pest resistance, iron deficiency tolerance (chlorosis), lodging scor e, height, and specific soybean 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 ha ve resistance or tolerance to these pests and diseases can

be an effective and economical method of control.

Row spacing is important to maximize soybean yield. Research in the Midwest over the past 20 years consistently shows that row spac ing of less than 20 inch es is preferred for soybean regardless of tillage system, rotation sequence or planting date (Pedersen, 2008a). In the Southern stat es, the advantage from narrow rows is less consistent and less beneficial. In 2000, approximately 40% of soybean was planted in row spacing of 10 inches or less, 27% in 10.1 to 28.5 inches, and 33% in rows wider than 28.5 (Hoeft et al., 2000).

Planting and Early Season

An understanding of the growth stages of soybe an is also important for the proper timing of certain management practices, such as he rbicide and insecticide applications. In addition, the impact of certain weather conditions, insect pe sts, and diseases on soybean yield is dependent on growth stage. The syst em of soybean growth stages divides plant development into vegetative (V) and repr oductive (R) stages (Pedersen, 2008a). The vegetative stages begin with VE, which designa tes emergence. V stages continue and are numbered according to how many fully develope d trifoliate leaves are present (i.e., V1, V2, etc.). The reproductive (R) stages begin at flowering (R1) and include pod development and plant maturation. Full maturity is designated as R8.

Adequate soil moisture and warm temperatures facilitate rapid seed germination and emergence. The ideal soil temperature for s oybean germination and emergence is 77 oF (Pedersen, 2008a). However, waiting for soils to reach this soil temperature will delay planting beyond the optimum planting date th at will maximize yield. Soybean can germinate at a soil temperature of 50 oF when planted at a de pth of two inches. However, emergence is slow and can take up to thr ee weeks in Northern climates. Because of fluctuations in soil temperature in early spring, soil temperature should not be the only criteria for optimum planting time. Planti ng into a good seedbed is the most importantMonsanto Company 09-SY-200U Page 154 of 395 consideration. Planting into soil that is too wet will reduce emergence and plant population, and can lead to reduced yield.

Planting date has the greatest impact on yi eld according to research conducted in the Northern states (Hoeft et al ., 2000). Highest yields genera lly are obtained when planting is in early to mid May. Yields begin to dr op off quite rapidly when planting is delayed until late May. For example, the optimum plan ting dates for soybean in Iowa are the last week of April in the southern two-thirds of the state and the first week of May in the

northern one-third of the stat e (Pedersen, 2008a). In the So uthern U.S., planting adapted varieties before late April re sults in shorter plants and, in many cases, lower yields than when the same varieties are planted in May or early June. Planting after early June generally decreases plant height and yield due to water shorta ges in July and August.

Variations in plant spacing through row spaci ng and plant populati on have a significant effect on canopy development and soybean yiel d. Soybean has the ability to produce good yield over a wide range of plant populati ons. Most soybean varieties have the ability to branch and adjust the number of pods on branches to compensate for large differences in seeding rate. Maximum yields generally require planti ng rates that result in about 2.5 to five plants per square foot (H oeft et al., 2000). Therefore, a full stand of soybean is approximately eight to 10 plants per foot of row at ha rvest for 40-inch rows, six to eight plants per foot of row in 30-inch rows, four to six plants in 20-inch rows, and two to three plants in 10-inch rows. This translates to 109,000 to 218,000 pl ants per acre at harvest. Higher populations are recomm ended in narrow rows for maximum yields because plants are more uniformly spaced in na rrow rows. Seeding rates are generally 10 to 25% higher than the desired harvest popula tion, especially in no-till, to account for the losses in germination, emergence, and seedli ng diseases. The accuracy of the planting equipment also can impact the decision on seed ing rate. Soybean se ed traditionally has been sold by weight. Therefore, the farm er must know the number of seeds per pound for the particular soybean varieties being planted for accurate seeding rates.

Treating soybean seed with a fungicide (e.g., pyraclostrob in, metalaxyl, mefenoxam) to prevent damping-off diseases ma y be beneficial when planti ng in cold, wet soils, using reduced till and no-till planting systems, and when planting seed with a low germination rate (<80%) or low seed vigor.

Annual and perennial weeds are considered to be the greates t pest problem in soybean production (Aref and Pike, 1998). In order to maximize yields , weeds must be controlled during the early growth stages of soybean because weeds compete with soybean for water, nutrients, and light. A combination of tillage and herbicides are utilized to control weeds throughout the growing season.

Mid to Late Season

Ideal daytime temperatures for soybean growth are between 75 oF and 85 oF (Hoeft et al., 2000). Warmer temperatures result in larger plants and earlier flowering. Sustained temperatures below 75 oF will delay the beginning of flowering significantly. Seed set also is affected by temperature. Seed set is generally good when pollination followsMonsanto Company 09-SY-200U Page 155 of 395 night temperatures around 70 oF. Soybean vari eties differ in their response and tolerance

to temperatures.

Soybean is photoperiod sensitive, which mean s that it transitions from vegetative to 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. Flowering of southern varieties is initiated by a shorter day than varieties adapted to the north. The extent of vegetative growth occurring afte r the initiation of flow ering depends not only on environmental factors but also on the growth habit. Soybean varie ties are described as

either indeterminate or determinate in th eir growth habit (Scott and Aldrich, 1970). Indeterminate varieties increase their height by two to four times after flowering begins. These are grown in the Northern and Central U.S. Determinate varieties increase their height very little after flowering and gene rally are grown in the Southern U.S. Indeterminate and determinate varieties also differ in flowering characteristics. Indeterminate plants generall y bloom first at the fourth or fifth node and progress upward. Flowering on determinate plants begi ns at the eight or te nth node and progresses both downward and upward.

The first appearance of flowers signals th e beginning of the reproductive stage, namely the R1 stage (Hoeft et al., 2000). The reprodu ctive period consists of flowering, pod set,

and seed formation. Climatic conditions such as temperature and moisture supply during the flowering period will affect the number of flowers. Th e soybean plant does not form a pod for each flower. It is common for the s oybean plant to have 75% of the flowers fail to develop a pod (Scott and Aldrich, 1970). This characteristic makes soybean less susceptible than corn to s hort 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-f illing stages to prevent pod a bortion and to ensure high yields (Hoeft et al., 2000). Another critical period is dur ing the seed-filling stages to assure high rates of photosynthesis. Hi gh humidity and temperatures during seed development and maturity can result in poor seed quality since these conditions promote the development of reproductive-stage diseases.

Harvest Season

When dry matter accumulation ends, the plant is considered to be physiologically mature. The seed moisture content is approximately 55 to 60% at this stage (Hoeft et al., 2000). At this stage, namely R7, at least one normal pod on the plant reaches the mature pod color. Under warm and dry w eather conditions, seed moisture content will drop to 13 to 14% in 10 to 14 days from physiological maturity (Hoeft et al., 2000). Soybean can be harvested when the moisture content drops be low 15%. However, s oybean should be at

13% moisture to be stored without artific ial drying (Scott and Aldrich, 1970). Moisture content below 12% may increase seed cracking and seed coat damage.

Pre-harvest losses are influenced by variety, we ather, and timeliness of harvest (Scott and Aldrich, 1970). Timely harvest when the mo isture content is 13 to 14% also will minimize losses. Proper operation and adjust ment of the combine is essential to minimizing harvest losses in the field.Monsanto Company 09-SY-200U Page 156 of 395 IX.D. Weed Management

Annual weeds are typically the greatest pest problem in soybean production, followed by perennial weeds (Aref and Pike, 1998). Soybean insects and diseases are generally less problematic but may reach economic thresholds requiring treatment. Weed control in soybean is essential to optimizing yields . Weeds compete with soybean for light, nutrients, and soil moisture. Weeds can ha rbor insects and diseases, and can also interfere with harvest, caus ing extra wear on harvest e quipment (Pedersen, 2008a). The primary factors affecting soybean yield loss fr om weed competition are the weed species, weed density, and the duration of the competitio n. When weeds are left to compete with soybean for the entire growing season, yield losses can excee d 75% (Dalley et al., 2001). Generally, the competition increases with increasing weed density. The time period that weeds compete with the soybean crop influences the level of yield loss. In general, the later the weeds emerge, the less impact the weeds will have on yield. Soybean plants withstand early-season weed co mpetition longer than corn, and the canopy closes earlier in soybean than corn. In addition, canopy clos ure is much sooner when soybean is drilled or planted in narrow rows.

Crop rotations and environment have a significant impact on the adaptation and occurrence of weeds in soybean. Foxt ail spp. (foxtail species group), pigweed, velvetleaf, lambs quarters, and cocklebur are common weeds in Midwest corn and soybean fields. However, growers consider giant ragweed ( Ambrosia artemisiifolia ), lambsquarters ( Chenopodium album ), Canada thistle ( Cirsium arvense) , cocklebur ( Xanthium strumarium ), and velvetleaf ( Abutilon theophrasti ) to be the top five most problematic weeds in corn and soybean because of the difficulty to control these weeds (Nice and Johnson, 2005). The most frequently reported common weeds in the Southeast region are morning glory ( Ipomoea spp. ), prickly sida ( Sida spinosa ), johnsongrass ( Sorghum halepense ), sicklepod ( Cassia obtusifolia ), and broadleaf signalgrass ( Brachiaria platyphylla ) (Webster et al., 2005).

Cultural and mechanical weed control practices are important components of an effective weed management program (Baumann et al ., 2008). Crop rotation, narrow row spacing, and planting date are a few of the crop management practices that are implemented to provide the crop with a competitive edge ove r weeds. Although the primary purpose of tillage is for seedbed preparation, tillage stil l is used to supplement weed control with selective herbicides in soyb ean production. Approximately 98 percent of the soybean acreage received an herbicide application in 2006, indicating the importance of excellent weed control in maximizing soybean yiel d (USDA-NASS, 2007b). Herbicide-tolerant soybean was introduced to provide growers with additional options to improve crop safety and/or improve weed control. Th e Roundup Ready soybean system, i.e., planting Roundup Ready soybean and applying glyphosate in crop has become the standard weed control program in U.S. soybean produc tion. Currently, Roundup Ready soybean is planted on 91 percent of th e soybean acreage (USDA-NASS, 2007a). Consequently, glyphosate is the most widely used herbicid e in soybean, being applied on 96 percent of the soybean acreage in 2 006 (USDA-NASS, 2007b).Monsanto Company 09-SY-200U Page 157 of 395 IX.E. Management of Insects

Although insects are typically as less problema tic than weeds in U.S. soybean production, management of insect pests during the growth and development of soybean is important for protecting the yield of soybean (Aref a nd Pike, 1998). Understanding the impact of insects on soybean growth is essential for proper management (Way, 1994). It is important to understand the way that insects injure soybean as well as how the soybean plant responds to insect injury. Insect inju ry can impact yield, plant maturity, and seed quality. Injury is defined as a stimul us producing an abnormal change in plant physiological processes. Injury may produce stress whic h is a departure from optimal physiological conditions (Way, 1994). The ultimate impact of injury is damage, a measurable reduction in plant growth development or reproduction. Insect injury in soybean seldom reaches levels to cause an economic loss as indicated by the low percentage (16%) of soybean acreage that receives an insecticide treatment.

Characterizing soybean responses to insect in jury is essential in establishing economic injury levels (Way, 1994). Mo st often, soybean insects are ca tegorized or defined by the plant parts they injure, namely root-feedi ng, stem-feeding, leaf-feeding, or pod-feeding insects. The root- and stem-feeding insect groups are often the hardest to scout and typically are not detected until after they have caused their damage. The leaf-feeding insects comprise the biggest group of inse cts, but not necessar ily the most damaging

insects. Recent research on defo liation has determined that a ma jor effect of injury is to reduce light interception by the soybean canopy which in turn can have a significant effect on yield (Way, 1994). Soybean has an extraordinary capacity to withstand considerable defoliation early in the season without signif icant yield loss, whereas, defoliation during the flowering and pod filling stages poses a greater threat to yield because the soybean plant has less time to compensate for injury compared to other growth stages. Research indicates that the s oybean plant can sustain a 35% leaf loss prior to the pre-bloom period without lowering yi eld (NDSU, 2002). However, from pod-set to maturity, the plant can tolerate only a 20% defoliation level before yield is impacted.

IX.F. Management of Di seases and Other Pests

More than 100 pathogens are known to affect soybean, of which 35 are considered to be of economic importance (Bowers and Russi n, 1999; and Funderburk et al., 1999). The estimated yield losses to soybean diseases in the U.S. were 10.9, 11.9, and 14.0 million metric tons in 1996, 1997, and 1998, respectively (Wrather et al., 2000). Pathogens can affect all parts of the soybean plant, resulting in reduced quality and yi eld. The extent of losses depends upon the pathogen , the state of plant deve lopment and health when infection occurs, the severity of the disease on individual plants, and the number of plants affected (Bowers and Russin, 1999; and Funderburk et al., 1999).

One or more diseases generally can be f ound in fields wherever soybean is grown (Bowers and Russin, 1999; and Funderburk et al., 1999). Ho wever, a pathogen may be very destructive one season and difficult or impossible to find the next season. The extent and severity of soybean diseases de pend on the degree of compatibility between the host and the pathogen and the influence of the environment.Monsanto Company 09-SY-200U Page 158 of 395 According to field surveys conducted in 15 soybean producing states during 1996 to 1998, soybean cyst nematode ( Heterodera gylcines ) caused the greatest soybean yield losses (Wrather et al., 2000). Ph ytophthora root and stem rot ( Phytophthora sojae ), brown stem rot ( Phialophora gregata ), sclerotinia stem rot ( Sclerotinia sclerotiorum ), and seedling diseases followed in economical importance. As expected, yield losses varied by region. Sclerotinia stem rot caused yi eld losses in several northern states, but

not in other states. Rhizoctonia foliar blight losses were grea test in Arkansas, Louisiana, and Texas where humidity and temperatur e conditions are suitable for disease development.

Selecting resistant varieties is the primary t ool growers have for disease control (Bowers and Russin, 1999; and Funderburk et al., 1999). Resistant varieties may have morphological or physiological characterist ics that provide immunity, resistance, tolerance or avoidance to certain pathogens. Cultural practices play an important role in disease management by reducing initial in oculums or reducing the rate of disease development (Bowers and Russin, 1999; and F underburk et al., 1999). Preplant tillage can bury crop residue, which encourages the d ecomposition of fungal-resting structures. Crop rotation is routinely recommended as a disease management strategy. Rotating crops interrupts the disease cycle and allows time for the decomposition of inoculums. One exception is Rhizontonia , a soil-inhabitant pa thogen that grows on a wide variety of crops and can survive sufficiently in the so il to make crop rotation as a means of controlling this pest impractical. Row sp acing, plant population, and planting date can also be changed to manage soybean diseases.

Soybean cyst nematode (SCN) is one of the most damaging pathogens of soybean throughout the soybean growing regions of the U.S. (Pedersen, 2008b). Losses have

been estimated to be at a bout $1.5 billion in the U.S. (Ped ersen, 2008a). SCN can cause yield losses up to 50%. This pest in 2004 caused an estimated loss of 50 million bushels in Iowa (Pedersen, 2008b). Soybean cyst ne matodes feed on the roots causing severely stunted and yellow plants. The simplest, l east expensive method to reduce populations of this pest is to rotate soybean with a non-host crop, su ch as corn, small gr ains, 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 soyb ean varieties

offer sources of resistance to certain races of nematode. Alternating varieties with

different sources of resist ance may be beneficial.

High-quality seed is essential for control ling seedling diseases. The most important seedling diseases in soybean are Phytophthora, Pythium, Rhizoctonia, and Fusarium (Pedersen, 2008a). Many soybean varietie s have race-specific resistance to Phytophthora. Treating soybean seed with a fungi cide (e.g., pyraclostrobin, metalaxyl, mefenoxam) is effective against damping-o ff 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 areas such as Brazil (Dorrance et al., 2007). Soybean rust is caused byMonsanto Company 09-SY-200U Page 159 of 395 the fungus Phakopsora pachyrhizi . This disease in the U.S. was first detected in Louisiana in 2004. Foliar appl ication of fungicide s is the standard disease management practice to limit yield losses due to soybean rust at this time.

Foliar fungicide applications can effectively reduce the incidence of many diseases (Bowers and Russin, 1999; and Funderburk et al., 1999). However, the economic return from a fungicide application may be limited to select production programs, e.g., primarily to high-yield environments or when pr oducing soybean seed. According to USDANASS statistics, fungicides were applied on ap proximately 4% of the soybean acreage in 2006 (2007b).

IX.G. Crop Rotation Practices in Soybean

The well-established farming practice of crop ro tation is still a key management tool for growers. The purpose of growing soybean in rotation with other crops is to improve yield and profitability of one or both crops over time, decrease the need for nitrogen fertilizer on the crop following soybean, increas e residue cover, mitigate or break disease, insect, and weed cycles, reduce soil erosi on, increase soil organic matter, improve soil tilth, and reduce runoff of nutrients, herbicides , and insecticides (Al- Kaisi et al., 2003; Boerma and Specht, 2004). According to USDA Economic Res earch Service crop residue management studies, 95% of the soyb ean-planted acreage has been in some form of a crop rotation system since 1991 (USDA- ERS, 2006a). Corn- and wheat-planted acreage has been rotated at a slightly lower level of 75% and 70%, respectively. Although the benefits of crop rotation can be substantial, the farmer must make cropping decisions by evaluating both the agronomic and economic returns on various cropping systems. Crop rotations also afford grower s the opportunity to diversify farm production

in order to minimize market risks.

Continuous soybean production is not a co mmon practice in the Midwest and is discouraged by most extension soybean specialists to reduce the risk of diseases and nematodes (Al-Kaisi et al., 2003; Hoeft et al., 2000). Corn and soybean occupy more than 80% of the farmland in many of the Mi dwestern states, and the two-year cropping sequence of a soybean-cor n rotation is used most extensiv ely in this region. However, a soybean crop sometimes is grown after soybean and then rotated to corn in a three-year rotation sequence (soybean-soybean -corn) in the Midwest. Compared to corn, soybean shows a greater response to being grown afte r a number of years without soybean. The yields of both corn and soybean are approxi mately 10% higher when grown in rotation than when either crop is grown c ontinuously (Hoeft et al., 2000).

A combination of conservation tillage practi ces and crop rotation has been shown to be very effective in improving soil physical prope rties. Long-term studies in the Midwest indicate that a corn-soybean ro tation improves yield potential of no-till systems compared to continuous corn producti on (Al-Kaisi, 2001). The redu ction in yield of continuous corn production in no-till system s is attributed to low soil temperature during seed germination, which is especia lly evident on poorly drained soils under no-till practices.

Unique to the southern portion of the Midw est region and the mid- south states, soybean is grown in a double-cropping sy stem. Double-cropping refers to the practice of growingMonsanto Company 09-SY-200U Page 160 of 395 two crops in one year. This practice can improve income and reduce soil and water losses by having the soil covered with a plan t canopy most of the year (Hoeft et al., 2000). In the Midwest, winter wheat is harveste d in late June or July, and then soybean is planted into the wheat stubble in a no-till system to conser ve moisture. Due to the uncertainty of double-cropping yields, farmers so metimes do not plant if soils are too dry at the time of wheat harvest. Soybean is typically grown in a corn-wheat-soybean rotation sequence when grown in a double-cr opping system. In the northern soybean growing areas, wheat often will follow soybean in the rotation.

IX.H. Soybean Volunteer Management

Volunteer soybean is defined as a plant that has germinated and emerged unintentionally in a subsequent crop. Soybean seeds can rema in in a field after soybean harvest as a result of pods splitting before or during harves t. Soybean seeds also can remain in a field when pod placement on the plants is too clos e to the ground for the combine head to collect all the pods or the combine is impr operly adjusted for efficient harvesting. Volunteer soybean in rotational crops is t ypically 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 grow ing 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 is normally not a concern in rotational crops such as corn, cotton, rice, and wheat that are the significant rotationa l crops following soybean due to control measures that are available for volunteer soyb ean when they arise (Carpenter et al., 2002; OECD, 2000). Preplant tillage is the firs t management tool for control of emerging volunteer soybean in the spri ng. If volunteer soybean sh ould emerge after planting,

shallow cultivation will control most of the plants and effectively reduce competition with the crop. Several postemergence herbicides also are available to control volunteer soybean (conventional or glyphosate-tolerant soybean) in each of the major rotational crops. Table IX-4 provides c ontrol ratings on volunteer gl yphosate tolerant soybean for

several herbicides used in the major rotational crops.

To provide control of volunteer soybean in corn, postemergence applications of AAtrex (atrazine), Clarity (dicamba), Distinct (diflufenzopyr + dica mba), Hornet (flumetsulam + clopyralid) and Widematch (clopyralid + fluroxypyr) provide excellent control (Zollinger, 2005). In wheat, Bronate Advanc ed (bromoxynil), Clarity (dicamba) and Widematch postemergence provide excellent control of volunteer soybean (Zollinger, 2005).

Volunteer soybean in cotton is normally not a concern. However, hurricanes or other extreme weather conditions can damage a soybean crop preceding cotton production in the Mid-south states, where the unharvested soybean seed can produce volunteer plants. Preplant applications of paraquat or herb icide mixtures containing paraquat will effectively control volunteer glyphosate-tolerant soyb ean (Montgomery et al., 2002; Murdock et al., 2002). Recent research in North Carolina indicates Envoke (trifloxysulfuron) will provide excellent postemergence control of soybean with traits forMonsanto Company 09-SY-200U Page 161 of 395 glyphosate and sulfonylurea herbicide tolerance in Roundup Ready cotton (York et al., 2005).

Volunteer soybean in rice is rarely a concern due to the combination of preplant tillage, flooding practices, and herbicides utilized in producing rice. If volunteer plants should emerge in rice, the postemergence applic ations of Grasp (penoxsulam), Permit (halosulfuron) and Regiment (bispyribac) typi cally used for weed control in rice will effectively alleviate competition from vol unteer soybean (Dillon et al., 2006).Monsanto Company 09-SY-200U Page 162 of 395 Table IX-4. Ratings for Control of Volunteer Glyphosate-Tolerant Soybean in
Labeled Rotational Crops 1

Product Rate (Product/Acre) Soybean

V2 – V3 Soybean

V4- V6

Corn 2

AAtrex 0.38 qts E P 0.50 qts E F Clarity 4 fl oz E E

5 fl oz E E

Distinct 1 oz E G

2 oz E E

Hornet 1 oz E F

2 oz E F-G
Widematch 0.25 pt E G

Wheat 2

Bronate Advance d 0.8 pt E E Clarity 4 fl oz E E

5 fl oz E E

Widematch 0.25 pt E G

Cotton 3

Envoke 0.1 oz E E

Rice 4

Grasp 2 oz E NA

Permit 1 oz E NA Regiment 0.4 oz E E

NA denotes “not applicable.”

1 Weed control ratings: E = Excellent (90 to 99% control), G = Good (80 to 90% control), F =

Fair (65 to 80 control), and P = Poor (40 to 65% control).

2 Zollinger, 2005.

3 York et al., 2005.

4 Dillon et al., 2006.

IX.I. Stewardship of MON 87769

Monsanto Company is firmly committed to it s legal, ethical, and moral obligation to ensure that its products and technologies ar e safe and environmentally 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 Program (http://www.excel lencethroughstewardship.org/ )

. These policies and practices include rigorous field complia nce and quality management systems and verification through auditing.Monsanto Company 09-SY-200U Page 163 of 395 As with all of our products, Monsanto is committed to th e rigorous product stewardship of MON 87769. In keeping with past practi ce, Monsanto will seek regulatory approval for MON 87769 in all key soybean import count ries with a functioning regulatory system to assure global compliance an d support the flow of intern ational trade. Monsanto

continues to monitor other coun tries that are key importers of soybean from the U.S., for the development of formal biotechnology a pproval processes. If new functioning regulatory processes are developed, Monsan to will make appropriate and timely regulatory submissions.

Once appropriate approvals are received, as with other value-adde d specialty soybean products, MON 87769 will be grown using an appropriate IDP system based on established practices as desc ribed in Section IX.B.3. IDP practices are implemented for value-added specialty soybean to capture th e enhanced value of the product and ensure that the enduser or processor receives th e soybean with the identity, fatty acid composition of the oil, and desired quality.

Prior to obtaining all key globa l regulatory approvals, Monsanto will conduct seed testing, variety development and production, and o il manufacturing, testing and commercial activities leading up to the commercial in troduction of MON 87769. Monsanto will work in a closed loop system under contract with partners to plant, harvest and process MON 87769 to produce the SDA soybean oil

A closed loop system will utilize appropriate processes for containment, documentation and traceability of seed production, planting, ha rvest, processing and use of the product. Grain production processes wi ll include mass balance and accounting of all planting, harvested seed, secured storage facilities, labeling of all soybean, training of personnel,

identification and audit of fiel d production sites and acreage, spatial isolation of fields, equipment clean out procedures and docum entation. Soybean processing will include segregation, control and traceab ility of handling, processing, packaging, and shipping of products and co-products.

Before implementing a closed loop system, Mo nsanto will dialogue with the appropriate

value chain stakeholders in the countries of production and product use to gain feedback on and confirm the robustness and validity of the closed loop production system. Monsanto will not employ the closed loop syst em without adequate assurance that the system will be effective in containing, pr eventing the escape of , and controlling the disposition of MON 87769, so it is not comingl ed with commodity soybean. As part of this process, Monsanto will continue to provide regular updates on MON 87769 to key members of the soybean industry grain trad e, processing industr y and food industry throughout the regulatory and pr oduct development process.

IX.J. Impact of the Introduction of MON 87769 on Agricultural Practices

Introduction of MON 87769 is expected to ha ve no impact on current cultivation and management practices for soybean. MON 87769 has been shown to be no different from conventional soybean in its agronomic, phenotypic, ecological, and compositional characteristics (refer to Sections VII, VIII, IX), except for the intended change in fattyMonsanto Company 09-SY-200U Page 164 of 395 acid composition (presence of SDA and GL A in MON 87769 seed). MON 87769 is a nutritionally improved specialty soybean product that is expect ed to bring added value to

consumers. The added value of MON 87769 is expected to bri ng higher returns to growers compared to commodity soybean.Monsanto Company 09-SY-200U Page 165 of 395 X. ENVIRONMENTAL CONSEQUENC ES AND IMPACT ON AGRONIMIC

PRACTICES

This section provides a brief review and assessment of the plant pest potential of MON 87769 and its impact on agronomic pr actices. USDA-AHPIS has responsibility, under the Plant Protection Act (7 U.S.C. § 7701-7772), to prevent the introduction and dissemination of plant pests in to the U.S. APHIS regulatio n 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate su bmitted 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 doe s not present a plant pe st risk, the petition is granted, thereby allowing unrestric ted introduction of the article.

The definition of “plant pest” in the Plant Protection Act (PPA) in cludes living organisms that could directly or indirectly injure, da mage, or cause disease in any plant or plant product [7 U.S.C. § 7702(14)]. Information in this petition related to plant pest risk characteristics include disease and pest suscep tibilities, expression a nd characteristics of the gene products (Pj Δ 6D and Nc Δ 15D proteins), impacts to NTOs, changes to plant metabolism, weediness of the regulated ar ticle, impacts on agronomic practices, any impacts on the weediness of any other plant with which it can interbreed, and the transfer of genetic information to organisms with which it cannot interbreed.

The regulatory end-point under the PPA for bi otechnology-derived crop products is not zero risk, but rather a determination that dere gulation of the regulated article is not likely to pose a plant pest risk. The plant pe st risk assessment of MON 87769 is based primarily on eight lines of evidence: (1) m odern soybean has inherently low plant pest potential, (2) insertion of a si ngle functional copy of the inse rted expression cassette, (3) characterization of the Pj Δ 6D and Nc Δ 15D proteins expressed in MON 87769 and the altered soybean oil profile, (4) mode of action and safety of the Pj Δ 6D and Nc Δ 15D proteins and the altered soybean oil pr ofile, (5) compositi onal equivalence of MON 87769 seed as compared to conventiona l soybean, with the exception of intended fatty acid change, (6) phenot ypic and agronomic characte ristics demonstrating no increased plant pest potential, (7) negligible risk to NTOs and thre atened or endangered species, and (8) no greater li kelihood to impact agronomic practices, including land use, cultivation practices, or the management of weeds, diseases, and insects than conventional soybean.

As part of the plant pest risk assessment, the genetic construct inserted into MON 87769 was evaluated to determine if those sequenc es cause plant disease. Morphological characteristics of MON 87769 were analyzed to determine if it will be any more weedy or invasive relative to soybean varieties cu rrently on the market. Agronomic practices

associated with MON 87769 were considered re lative to potential cha nges that could lead to increased plant pest potential. The poten tial for gene flow a nd introgression of the genetic construct into other plant varieties or wild rela tives also was evaluated to determine the potential of increased weedy or invasive characteristics in sexuallyMonsanto Company 09-SY-200U Page 166 of 395 compatible plants. Finally, the propensity of MON 87769 to become a greater reservoir of plant pests (insects or pathogens) compar ed to conventional soybean and the potential for horizontal gene transfer we re evaluated. Using this risk assessment process, the data and analysis presented in this petition lead to a conclusi on that MON 87769 is unlikely to be a plant pest and, therefore, should no longer be subject to regulation under 7 CFR § 340.

The assessment of the impact of MON 87769 on threatened and enda ngered species and

other NTOs concludes that risk to these organisms from the use of MON 87769 is negligible. This risk assessment took into consideration several components, including similarity of Pj Δ 6D and Nc Δ 15D proteins to other desatu rases present in plants, the functional activity of the Pj Δ 6D and Nc Δ 15D proteins, and the expression level of the Pj Δ 6D and Nc Δ 15D proteins in MON 87769. The ev aluation of weediness and gene flow potential concluded that MON 87769 is no more likely to become a weed than conventional soybean, and MON 87769 is expected to be similar to conventional soybean regarding the potential for a nd impact from gene flow. Due to the lack of sexually compatible relatives in the U.S., pollen-mediat ed gene flow is expected to occur only within cultivated soybean. Given the repr oductive biology of soybean, pollen-mediated gene flow is expected to be negligible w ithin cultivated soybean. The probability for horizontal gene flow is exceedingly small. Ev en if it were to occur, the consequences would be negligible because the genes introduced into MON 87769 ( Pj.D6D and

Nc.Fad3 ) are of plant and fungal origin and the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D) and the intended fatty acid change have no meaningful toxicity to humans and other NTOs under the conditions of use.

An assessment of current soybean agronomic practices in the U.S. indicates that the introduction of MON 87769 will not impact cu rrent U.S. soybean cultivation practices and the management of weeds, diseases, and insects (see Section IX). The impact of MON 87769 on agronomic practices associated with production of soybean is addressed in

Sections IX and XI.

APHIS has recently proposed to amend 40 CF R § 340 to include elements derived from the noxious weed authority provided under the Plant Protection Act. Because the data show that MON 87769 has no potential to cause injury or damage to any protected crops, livestock or interests of agri culture, MON 87769 also would not be considered a “noxious weed” as defined by the Pl ant Protection Act.

X.A. Plant Pest Assessment of MON 87769

X.A.1. Characteristics of the Genetic Insert

MON 87769 was developed through Agrobacterium -mediated transformation of meristematic soybean tissue using the bi nary transformation plasmid PV-GMPQ1972 (Section IV: Figure IV-1and Tabl e IV-1). Molecular analys es indicate that MON 87769 contains one copy of the insert at a single inte gration locus. No additional elements from

the transformation vector were detect ed in the genome of MON 87769, including T-DNA II or backbone sequence from plasmi d PV-GMPQ1972. Add itionally, the dataMonsanto Company 09-SY-200U Page 167 of 395 confirm the organization and sequence of the in sert, and demonstrate the stability of the insert over several generations. On the basis of these data, it is concluded that only the expected Pj Δ 6D and Nc Δ 15D proteins are produced from the inserted DNA. Molecular analysis confirmed that MON 87769 does not co ntain elements that promote plant disease (refer to Table IV-1).

X.A.2. Mode of Action and Safety of Pj Δ 6D and Nc Δ 15D Proteins

The Pj Δ 6D and Nc Δ 15D proteins expresse d in MON 87769 are members of a family of integral membrane fatty acid desaturases f ound in all eukaryotic organisms (plants, animals, and fungi) and some prokaryotes, e. g. cyanobacteria (Hashimoto et al., 2008; Los and Murata, 1998). The Pj Δ 6D and Nc Δ 15D proteins expressed in MON 87769 are nearly identical to the native proteins produced by Primula juliae (primrose) and Neurospora crassa , respectively.

The general mode of action of the Pj Δ 6D and Nc Δ 15D proteins are well understood (see Section IV.A). Pj Δ 6D catalyzes the desatu ration of ALA (18:3) to form SDA (18:4) in

the seed of MON 87769 and also conve rts LA (18:2) to GLA (18:3). Nc Δ 15D catalyzes the desaturation of LA to ALA and, theref ore, reduces the substrate pool for GLA production and increases the substrate pool for SDA production (Figure VII-1).

The safety assessment of the Pj Δ 6D and Nc Δ 15D proteins included extensive protein

characterization that demonstrated the lack of similarity to known allergens and toxins and a long history of safe consumption of similar proteins. Expression of the two proteins in leaf and root tissue was below the lim it of detection of 0.1 – 0.2 μ g/g of tissue on a fresh weight (FW) basis for Pj Δ 6D and 0.5 – 1.0 μ g/g on a FW basis for Nc Δ15 D. The mean Pj Δ 6D protein levels in immature seed, ma ture seed, and forage were 100, 1.8

and 16 μ g/g DW, respectively. The mean Nc Δ15 D protein levels in immature seed, mature seed, and forage were 200, 10 and 14 μ g/g DW, respectively. The forage samples, consisting of the above ground portion of plants with developing pods, were harvested at the R6 growth stage and included immature s oybean seed. Thus, it was expected that the Pj Δ 6D and Nc Δ 15D proteins would be detected at low levels in the forage samples due to the presence of immature seed. Data from the safety assessment also confirmed that the full-length Pj Δ 6D and Nc Δ 15D proteins are readily diges tible in simulated gastric and

intestinal fluids and lack acute oral toxici ty in mice (see Sections VI.D and VI.E.). Collectively, these data establish the safety of the Pj Δ 6D and Nc Δ 15D proteins. From these data, it can be concluded with reasonable certainty that the Pj Δ 6D and Nc Δ 15D proteins have no meaningful t oxic potential to exposed organisms in the environment. Furthermore, these integral membrane proteins are structurally and functionally related to

other desaturases that are wide ly distributed in plants and animals and are highly labile ex
vivo (see Section VI). Therefore, it is expected that these pr oteins would neither persist in the environment, nor have any adverse effects on soil-dwelling organisms when postharvest soybean biomass or detr itus decomposes in soil.Monsanto Company 09-SY-200U Page 168 of 395 X.A.3. Environmental Safety of Fatty Acids Produced in MON 87769

Data presented in Section VII summarize the composition of forage and the seed from MON 87769, the conventional control and te n commercial soybean varieties. Compositional analyses compared a total of 75 different analytical components, seven in forage and 68 in seed, between MON 87769 a nd a conventional soyb ean control with genetics comparable to MON 87769, but lacking the in troduced trait. Data presented in

Section VII indicate that there are no mean ingful differences in compositional or nutritional quality of MON 87769 compared to conventional soybean, except for the intended seed fatty acid change (presence of SDA and GLA). Compositional data were statistically analyzed and while there we re some statistical differences between

MON 87769 and the conventional c ontrol, it is concluded that the statistical differences represent the natural variability for these anal ytes in soybean such that they were not regarded as biologically meani ngful. Seed and forage analyt ical component values also were comparable to published scientific lit erature and the ILSI-CCD, further supporting the conclusion that seed and forage from MON 87769 are compositionally equivalent to those of conventional soybean, except for the intended seed fatty acid compositional changes. Thus, except fo r the intended fatty acid change, the composition of MON 87769 is not different from conventional soybean.

X.A.3.1. Biological Role and Metabolism of Polyunsaturated Fatty Acids

Polyunsaturated fatty acids are biologically significant compounds th at play a role in most organisms in metabolic energy storag e, as components of phospholipids, which are

essential for cellular membrane formation and function, and as precursors to the biosynthesis of eicosanoids, which affect numerous biochemical reactions and physiological processes (MacDonald and Sp recher, 1991; Watkins, 1991and 1995). In insects, PUFAs also serve as precursors in the biosynthesis of waxes, sex pheromones, and as components of defens ive secretions (Sta nley-Samuelson et al., 1988). Most animals, including mammals, birds, and most insects, have a dietary requirement for PUFAs, particularly for LA and ALA. These fatty acids often are referred to as “essential fatty acids” (EFA) b ecause of their biological significance, and since they cannot be synthesized de novo , they must be obtained from the diet. The 18-carbon (C18) PUFAs, including GLA and SDA, and C20 PUFAs, are derived from LA and ALA by a sequence of desaturation and elongation processes. One significant difference among insect species, as it relate s to fatty acid biosynthesis, is their ability or inability to biosynthesize LA. Most insects have a diet ary requirement for LA (Canavoso et al., 2001). However, at least 15 species are known to biosynthesize LA from acetate and, therefore, do not require dietary supp lied LA (Stanley-Samuelson et al., 1988).

Linoleic acid is an essential fatty acid in vertebrates. It is the first fatty acid of the n -6

series of PUFAs which includes GLA. Deficien cies of LA in poultry have been reported to include retarded growth, increased wate r consumption, reduced disease resistance, enlarged livers, and reduced testes and delayed development of secondary sexual characteristics in males. Decreased egg si ze and weight and changes in yolk fatty acids

result from LA-deficient la ying hens (Watkins, 1991).Monsanto Company 09-SY-200U Page 169 of 395 ALA is the first fatty acid of the n -3 series of PUFAs which includes SDA. ALA is considered an essential fatty acid; however, an absolute requirement of ALA in mammals, birds, and insects has not been empirically determined (Canavoso et al., 2001; Spector, 1999; Watkins, 1991). Nonetheless, ALA is necessary for normal brain and retina development in mammals and birds (Watkins , 1991) and metamorphosis in Lepidoptera and Hymenoptera (Can avoso et al., 2001).

In most mammals, birds, and insects, the metabolic fate of EFA is similar when they are

either de novo biosynthesized by the organism or deri ved from a dietary source. Primary

routes of metabolism include β -oxidization in the mitoc hondria to generate ATP, incorporation into cellular phospholipids, or further desaturation and elongation to form long chain (LC) PUFAs (Watkins, 1991; Stanley-Samuelson, 1988). The ∆ 6, ∆ 5, and ∆ 4 desaturases along with the elongases, mostly found in animals, are primarily responsible for the conversion of C18 to C20 PUFAs (P an et al., 1994; Watkins, 1991; StanleySamuelson, 1988). Generally, the desaturati on/elongation pathways of vertebrates, including mammals and birds, apply to most insect species in that ALA and LA are the precursors of the n -3 and n -6 PUFAs and are the immediate precursors of SDA and GLA, respectively. SDA is a normal metabolic in termediate in the conversion of ALA to eicosapentaenoic acid (EPA) by the actions of a ∆ 6 desaturase, an elongase, and a ∆ 5 desaturase (Cook, 1991). GLA is an intermed iate in the conversion of LA to dihomo- γ

linolenic acid (DGLA) and arachidonic acid following the same pathway. C20 PUFAs, particularly EPA, DGLA, and AA, play impor tant metabolic roles in most animals as precursors in the biosynthesis of physiol ogically active eicosanoids, which include the prostaglandins (PG), thromboxanes, leukotri enes, and lipoxins (Beare-Rogers et al., 2001).

Thus, PUFAs are important compounds in most animals. They are oxidized in energy production, incorporated in phospholipids a nd are essential for cellular membrane formation and function, desaturated/elonga ted, and oxygenated into physiologically

active eicosanoids. SDA and GLA are PUFA s that are present in MON 87769 seed and

are also consumed from other sources in the environment including pl ants, marine algae, fish/fish oil (see Section X.A.3.2). Ther efore, these PUFAs in MON 87769 seed are expected to be absorbed, transported, and me tabolized in animals in the same manner as they are from other sources. Their lack of toxicity is further supported by ecological

interaction assessments conducted under fi eld conditions (see Section VIII.D.2.2.).

X.A.3.2. Environmental Exposure to Fatty Acids in MON 87769

With the exception of SDA and GLA, the fa tty acids present in MON 87769 are the same as those present in conventional soybean. Thus , the analysis here fo cuses on the potential impact from exposure of SDA and GLA. Environmental exposure to SDA and GLA from MON 87769 is expected to be limited primarily to soybean pests and animals that feed on seeds or potentially th rough trophic interactions. Ho wever, these fatty acids are not new and are already present in the environment. SDA, GLA, and other polyunsaturated fatty acids, such as ALA and LA, exis t in many sources in the environment without known adverse ecologica l effects. SDA and GLA are produced in many plant seed oils. More than 230 species from among 17 plant families areMonsanto Company 09-SY-200U Page 170 of 395 documented to produce SDA in seed oil at con centrations greater than 1% of their total fatty acid composition. 7 Twenty of those species from among three families ( Boraginaceae

, Elaengaceae , and Primulaceae ) are reported to produce greater than 16% SDA, which is comparable to the levels of SDA produced in MON 87769. More than 360 species from 23 plant families are documented to produce GLA in seed oil at concentrations greater than 1% of their total fatty acid composition. 8 Over 230 of those species from 16 families are reported to pr oduce greater than 6% GLA (approximately 6 – 8% GLA is produced in the MON 87769 seed ). The seed oil from many of the commonly recognized plant-based sources of these two fatty acids are known to contain 0.3-22.5% SDA, 0.7-24.4% GLA, and varying con centrations of othe r related PUFAs, such as ALA and LA (Table X-1). The GL A content in many of these species exceeds that found in MON 87769. GLA is also presen t in oats, barley, and human breast milk (Horrobin, 1992). Flax seed ( Linum usitatissimum ) is consumed as a source of dietary fiber and is used to produce lin seed oil. The oil from flaxs eed is not a significant source

of SDA or GLA but is a rich source (approximately 55% in total fatty acid composition) of ALA, an omega-3 fatty acid (Dubois et al., 2007).

Many fish species, particularly marine fish, contain SDA, which the fish obtain in their diet by consuming plankton, algae, and other fish (Table X-2). Fish meal has been used as an animal feed ingredie nt for over a century. The meal is fed to poultry, pigs, ruminants, farmed fish, crustaceans, fur-producing animals, laboratory animals, and household pets (Bimbo and Crowther, 1992; FAO, 1986; Thomson, 1990). In addition,

fish meal also has been used as fert ilizer for crop producti on, although its use in fertilizers has diminished as increasing amounts of the meal are diverted for use in animal feed (FAO, 1986). Fish oils that contain SDA include c od liver (0.94% w/w), herring (2.31% w/w), menhaden (2.74% w/w), salmon (2.80% w/w), and sardine (3.03% w/w) (USDA-ARS National Nutrient Data base for Standard Reference ). 9 Fish oil is used for human consumption and in animal feed a nd industrial applicati ons, including soaps, lubricating greases, and waterproofing agen ts (Bimbo and Crowth er, 1992; FAO, 1986). Oil from some fish species has been approved by the FDA as Generally Recognized as Safe (GRAS) and is used in many dietary s upplements, along with plant-based sources of SDA, such as black currant. GLA may be fou nd in some fish oils but at concentrations generally less than 0.3% (w/w).

A number of species of marine microalgae and macroalgae also contain SDA. Liu and Lin (2001) report that marine microalgae widely used as aquaculture feed contain SDA, including Isochrysis spp. (15.1-24.9% of total fatty acids) and Pavlova spp. (5.9-6.8% of total fatty acids). Edible macroalgae species Ulva pertusa and Undaria pinnatifida contain 16.3 and 26.3% SDA, respec tively (Ishihara et al., 2000).

7 Federal Research Centre for Nutrition and Food – Ins titute for Lipid Research. 2008. Seed Oil Fatty Acids Online Database. http://sofa.bfel.de/ [Accessed on 8 December 2008].

8 Federal Research Centre for Nutrition and Food – Ins titute for Lipid Research. 2008. Seed Oil Fatty Acids Online Database. http://sofa.bfel.de/ [Accessed on 8 December 2008].

9 http://www.ars.usda.gov/nutrientdata [Accessed on 8 December 2008].Monsanto Company 09-SY-200U Page 171 of 395 Additionally, even though conve ntional soybean seed is not a source of SDA and GLA exposure to the environment, conventional s oybean is a well known source of fatty acids used in food and feed. Fatty acids such as ALA and LA are structurally similar to SDA and GLA. These closely related fatty acids have an established hi story of exposure in soybean without known adve rse ecological effects.

Thus, the fatty acids in MON 87769 seed have an established history of exposure through

other routes in the environment and have no known plant pest charac teristics as defined in 7 U.S.C. § 7701-7772.

X.A.3.3. Environmental Fate of Fatty Acids Present in MON 87769

As natural components of the plant and anim al world, SDA, GLA, and other PUFAs are not expected to accumulate, persist or to be detrimental to the environment. A major route for decomposition of PUFAs in the environment is enzymatic oxidation. The enzyme lipoxygenase, which is present in soyb ean, beans, peas and other plants, is a major transformation agent for PUFAs (Gardner, 1995; Feussner and Wasternack, 2002). PUFAs can also undergo non-enzymatic oxidati ve changes such as peroxidation, photooxidation, thermal oxidation, oxidative fissi on, hydrolysis and other reactions that convert the fatty acids into breakdown pr oducts (DeMan, 2000). The decomposition of PUFAs is primarily a result of their react ivity towards oxygen. The more unsaturated fatty acids are more re active, especially the n -3 and n -6 fatty acids, as shown by the following oxygen reactivity index: stearic (18:0) = 1; oleic ( 18:1) = 100; linol eic (18:2) = 1200; and linolenic (18:3) = 2500 (DeMan, 2000). It is expected th at SDA (18:4) and GLA (18:3) are at least as reactive, and likely more re active as their immediate precursors (ALA and LA, respectively).

Based on the oxygen reactivity of n -3 and n -6 fatty acids, as well as the presence of lipoxygenase in soybean, it is very likely that any SDA, GLA, or other PUFAs from MON 87769 seed that reaches soil, water, or other environmental compartments will undergo rapid decomposition via oxidative and enzymatic processes and will not persist

in or be detrimental to the environment or have any adverse effects on soil-dwelling organisms.Monsanto Company 09-SY-200U Page 172 of 395 Table X-1. Plants that Contain Steari donic Acid (SDA), Gamma-Linolenic Acid
(GLA), and Other Polyunsaturated Fatty Acids (PUFAs)

Fatty acid composition of seed oil (%)

Taxa 1 C18:2 n -6 (Linoleic) C18:3 n -6
( γ -Linolenic) C18:3 n -3 ( α -Linolenic) C18:4 n -3 (Stearidonic) Source of

Information

Aleuritia farinosa L. 29.9 1.8 29.2 17.5 Sayanova et al.,

1999

Aleuritia scotica

W.J. Hooker 26.9 2.2 29.0 22.5 Sayanova et al.,

1999 Borago officinalis L. (starflower) 35.0 24.4 0.9 0.3 Velasco and Goffman, 1999

Dodecatheon

meadia L. (pride of Ohio) 27.0 4.5 19.9 11.9 Sayanova et al.,

1999

Dodecatheon

tetrandrum L. (sierra shooting star) 26.8 2.5 27.9 12.2 Sayanova et al.,

1999

Echium italicum L. (Italian viper’s bugloss) 10.6 7.4 43.0 14.6 Özcan, 2008

Echium plantagineum L. (salvation jane) 13.8-15.2 9.2-11.3 33.3-36.7 12.9-13.4 Guil-Guerrero et al., 2000; 2006; Kleiman, 1964; Özcan, 2008
Echium vulgare L. (common viper’s bugloss) 16.9 12.2 36.1 12.9 Özcan, 2008

Lappula

squarrosa (Retz.) Dumort. (European stickseed) 13.6 7.3 27.6 17.1 Velasco and Goffman, 1999

Lithospermum

arvense L. (field gromwell) 10.6 5.2 41.5 17.4 Velasco and Goffman, 1999

Lithospermum officinale L. (European stoneseed) 17.2 12.3 29.1 13.3 Velasco and Goffman, 1999

Primula juliae Kusn. (primrose) 22.1 0.7 35.5 11.5 Sayanova et al.,

1999 Ribes nigrum (black currant) 48.5 15-19 13.5 3.5 Traitler et al., 1984

Ribes uva-crispa L. (European Gooseberry) 40.0 10-12 n.r. n.r. Traitler et al., 1984

1 Common names provided in parentheses. n.r. = not reported.Monsanto Company 09-SY-200U Page 173 of 395Table X-2. Fish that Contain Stearidonic Acid (SDA)Fish % SDA1Fish % SDA Fish % SDAAnchovy 0.055 Mullet 0.082 Shark 0.031Bluefish 0.167 Ocean perch, Atlantic 0.024 Sheepshead 0.027Bass, freshwater 0.039 Perch, mixed species 0.008 Smelt 0.025Carp 0.058 Pollock, Atlantic 0.005 Snapper 0.009Catfish, farmed 0.037 Rockfish, Pacific 0.016 Spot 0.16Catfish, wild 0.013 Roe 0.012 Sturgeon 0.128Cisco 0.029 Roughy, orange 0.001 Sucker, white 0.032Cod, Atlantic 0.001 Sablefish 0.114 Tilapia 0.003Croaker 0.011 Salmon, Atlantic, farmed 0.121 Trout, mixed species 0.064Dolphinfish 0.005 Salmon, Atlantic, wild 0.083 Trout, rainbow, farmed 0.05Flatfish 0.016 Salmon, chinook 0.188 Trout, rainbow, wild 0.066Haddock 0.003 Salmon, chum 0.068 Tuna, fresh, bluefin 0.039Halibut, Atlantic/Pacific 0.039 Salmon, coho, farmed 0.112 Tuna, fresh, skipjack 0.004Halibut, greenland 0.132 Salmon, coho,wild 0.119 Tuna, fresh, yellowfin 0.005Herring, Atlantic 0.214 Salmon, pink 0.09 Whitefish 0.05Herring, Pacific 0.248 Salmon, sockeye 0.102 Whiting 0.041Mackerel, king 0.278 Seabass 0.046 Wolffish 0.061Mackerel, Atlantic 0.09 Seatrout 0.008Mackerel, Pacific 0.125 Shad, American 0.328Source: USDA-ARS National Nutrient Database for Standard Reference (http://www.ars.usda.gov/nutrientdata). [Accessed on 8 December 2008]1Concentration of stearidonic acid (SDA) in raw fish expressed as the % of total mass.Monsanto Company 09-SY-200U Page 174 of 395 X.A.4. Impact on NTOs

An evaluation of the potential risks to (NTOs) is an important component of an overall

environmental risk assessment of a biot echnology-derived crop. An assessment of MON 87769 for potential risks to NTOs was conducted base d on the finding that the presence of the inserted gene s, the expression of the Pj Δ 6D and Nc Δ 15D proteins, and the production of the expected fatty acids (SDA and GLA) in MON 87769 seed were meaningful differences compared to convent ional soybean. The nature of MON 87769 as

a source of SDA and GLA with no pesticidal activity is such that all exposed organisms

are considered to be NTOs. The a ssessment considered pertinent product characterization information, information from the protein safety asse ssments, history of environmental exposure to SDA and GLA (t he additional fatty acids present in MON 87769 seed), and results from the ecologi cal interaction assessment. Based on the weight of evidence of all the information re ported herein, there is no indication that MON 87769 would have an adverse impact on NTOs.

X.A.4.1 Impact Due to introduced Genes and Proteins

Molecular analyses indicate th at MON 87769 contains one copy of the insert at a single integration locus. No additional elements fr om the transformation vector were detected in the genome of MON 87769. Additionally, the data confirm the organization and sequence of the insert and demonstrate the stab ility of the insert over several generations.
On the basis of these data, it is concluded that onl y the expected Pj Δ 6D and Nc Δ 15D proteins are produced from the in serted DNA (Section X.A.1).

From the protein safety assessment data (S ections VI and X.A.2), it can be concluded with reasonable certainty that the Pj Δ 6D and Nc Δ 15D proteins have no meaningful toxic potential to exposed organisms in the envi ronment. Furthermor e, these integral membrane proteins are structurally and functi onally related to other desaturases that are widely distributed in plants a nd animals and are highly labile ex vivo (see Section VI). Therefore, it is expected that these proteins would neither persist in the environment, nor have any adverse effects on NTOs including soil-dwelling organisms when post-harvest soybean biomass or detritus decomposes in soil.

X.A.4.2. Potential Adverse Effects of MON 87769 Fatty Acids on NTOs

With the exception of the intended changes in fatty acid composition (presence of SDA and GLA), MON 87769 seed is compositionally equivalent to conventional soybean (see Section VII). While SDA a nd GLA are not typically pro duced in soybean, they are already present in the environment (Section X. A.3). Importantly, the history of exposure across a wide taxonomic range provides eviden ce that these fatty ac ids when present at

the levels seen in MON 87769 are no more toxic than other fatty acids present in soybean to fish, birds, arthropods, or ot her animals. Their lack of t oxicity is furthe r supported by the known biological role and metabolism of polyunsaturated fatty acids, the ecological

interaction assessments of MON 87769 c onducted under field conditions, and published literature (Hammond et al., 2008; Harris et al., 2008).Monsanto Company 09-SY-200U Page 175 of 395 X.A.4.3. Interactions with NTOs under Field Conditions

In an extensive environmental interaction assessment of MON 87769, more than 700 comparative observations for plant interactions with arthropods a nd diseases under field conditions over two consecutive years, incl uding over 500 observations of plant damage caused by various arthropods and diseases, and 260 observations of the abundance of various pest and beneficial arthropods we re conducted (see Section VIII.D.2.2.). Data support the conclusion that MON 87769 does not have increased susceptibility or

tolerance to the evaluated arthr opods or diseases or altered in teractions with the evaluated pest and beneficial arthropods, as indicated by the abundance data, when compared to conventional soybean. In particular, ther e were no meaningful differences observed between MON 87769 and the control for damage from or abundance of soybean pests that directly feed on soybean seed and, thus, would be exposed to the Pj Δ 6D and

Nc Δ 15D proteins and the additional fatty acids present in MON 87769 immature and mature seed. Thus, the results of the e nvironmental interactions assessment further corroborate the hypothesis that the introduced genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change have no direct adverse or toxic effects on soybean pests or other NTOs.

X.B. Weediness Potential of MON 87769

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 refe rences (Crockett, 1977; Holm et al., 1979; Muenscher, 1980), nor is it iden tified as a noxious weed species on the list distributed by the federal government (7 CFR § 360). Soybe an does not possess any of the attributes commonly associated with weeds (Baker, 1965), su ch as long persistence of seed in the soil, the ability to disperse, invade, and b ecome a dominant species in new or diverse landscapes, or the ability to compete well with native vege tation. Due to a pronounced absence of dormancy, a trait that has b een removed through commercial breeding, soybean seed can germinate quickly under ad equate temperature and moisture and can potentially grow as volunteer plants. Howeve r, volunteer plants likely would be killed by frost in the soybean growing regions during au tumn or winter of the year they were produced. If they did become established, vol unteer plants would not compete well with the succeeding crop, and could be controlled r eadily by either mechanical or chemical means (OECD, 2000). In addition, since the wild populations of Glycine species are not known to exist in the U.S., the potential does not exist for MON 87769 to outcross to wild or weedy relatives and alte r their weediness potential.

In comparative studies between MON 87769 and a conventional soybean control, phenotypic, agronomic and environmental inter action data were eval uated (Section VIII) for changes that would impact the plant pest potential and, in particular, plant weediness potential. Results of these ev aluations indicate that ther e is no fundamental difference

between MON 87769 and the conventional control fo r traits associated with weediness. Collectively, these findings support the conclusion that MON 87769 has no increased weediness potential compared to conven tional soybean. Data on environmentalMonsanto Company 09-SY-200U Page 176 of 395 interactions also indicate that MON 87769 does not confer a ny biologically meaningful increased susceptibility or to lerance to specific disease, arthropod, or abiotic stressors.

X.C. Potential for Pollen-mediated Gene Flow

Gene flow and gene introgre ssion are processes whereby one or more genes successfully integrate into the genome of a recipient plant. Introgression is a ffected 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. Since gene introgression is a natural biol ogical process, it does not constitute an environmental risk in and of itself (Sunde rland 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 tran sgenes and their subsequent transfer to recipient plants will resu lt in increased plant pest potential.

X.C.1. Vertical Gene Flow

X.C.1.1. Hybridization with Cultivated Soybean Glycine max

Although soybean is a largely self-pollinate d species, low levels of natural crosspollination can occur (Caviness, 1966; OECD, 2000). In studies with cultivated soybean where conditions have been optimized to ensure close proximity and flowering synchrony, natural cross-pollina tion has been found to be ge nerally very low. Most outcrossing occurred with surrounding plants and cross-pollination frequencies vary, depending on growing season and genotype. Ins ect activity does increase the outcrossing rate, but soybean generally is not a prefe rred plant for pollinators (Erickson, 1975 and 1984).

Numerous studies on soybean cross-pollina tion have been conducted, and the published results (with and without supplemental pollina tors) are summarized in Table X-3. Under natural conditions, cross pollination among adj acent plants in a row or among plants in adjacent rows ranged from 0.3 to 3.62% (Beard and Knowles, 1971 ) . In experiments where supplemental pollinators (primarily bees) were added to the experimental area, cross-pollination range d from 0.5 to 7.74% in adjacent plants or adjacent rows. However, cross-pollination does not occur at these levels over long distances. Crosspollination rates decrease to less than 1.5% beyond one meter from the pollen source and rapidly decrease with greater distances from the source. The following cross-pollination rates at extended distances have been repor ted: 0.02% at 8.2 m (Caviness, 1966), 0.05% at 5.4 m (Ray et al., 2003), and 0% at 6.5 m (Abud et al., 2003).

The potential for cross-pollination is limite d. This is recognized in certified seed regulations for Foundation seed in the U.S., which permit any distance between different soybean cultivars in the field so long as th e distance is adequate to prevent mechanical mixing (USDA-APHIS, 2006).

In the event when cross-pollination may occur, MON 87769 and its progeny are not expected to have a significant environmental impact because evaluati ons have shown thatMonsanto Company 09-SY-200U Page 177 of 395 the introduced genes ( Pj.D6D and Nc.Fad ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D) and the intended fatty acid chan ge are not likely to enhance weediness or plant pest potential. Therefor e, the environmental consequence of pollenmediated gene flow from MON 87769 to other soybean plants is considered negligible.Monsanto Company 09-SY-200U Page 178 of 395 Table X-3. Summary of Published Litera ture on Soybean Cross-Pollination

Distance

from Pollen

Source % CrossPollination Comments Reference

0.3 m 0.04% (estimated per pod) Interspaced plants within a row. Experiment conducted in a single year. Single male and female parental varieties. Percent outcrossing calculated per pod rather than per seed. Woodworth,

1922

0.8 m 0.07 to 0.18% Adjacent rows. Experiment conducted over two years. Several male and female parental

varieties. Garber and Odland, 1926

0.1 m 0.38 to 2.43% Adjacent plants within a row. Experiment conducted in a single year. Several male and female parental varieties. Cutler, 1934

0.1 m 0.2 to 1% Adjacent plants within a row. Experiment conducted in single year at two locations. Several male and female parental varieties. Weber and Hanson, 1961

0.9 m

2.7 – 4.6 m

6.4 – 8.2 m

10 – 15.5 m 0.03 to 0.44 %

0.007 to 0.04%

0 to 0.02%

0 to 0.01% Frequency by distance was investigated. Experiment conducted over three years. Single male and female parental varieties. Caviness, 1966

0.8 m 0.3 to 3.62% Various arrangements within and among adjacent rows. Experiment conducted over three years. Several male and female parental varieties. Beard and Knowles, 1971

One row (undefined) 1.15 to 7.74% Bee pollination of single-row, small-plots of pollen receptor surrounded by large fields (several acres) of pollen donor soybean. Soybean is not a preferred flower for honeybee. Abrams et al.,

1978

0.1 – 0.6 m 0.5 to 1.03% (depending on planting design) Bee pollination of soybean grown in various spatial arrangements. Experiment conducted over four years. Several soybean cultivars. Chiang and Kiang, 1987

1.0 m 0.09 to 1.63% Adjacent rows. Experiment conducted over two years. Several male and female parental

varieties. Ahrent and Caviness, 1994

0.5 m

1.0 m

6.5 m 0.44 to 0.45%

0.04 to 1.4%

none detected Frequency by distance was investigated. Experiment conducted in a single year. Single male and female parental varieties. Abud et al.,

2003

0.9 m

5.4 m 0.29 to 0.41%

0.03 to 0.05% Frequency by distance was investigated. Experiment conducted in a single year. Single male and female parental varieties. Ray et al., 2003

0.15 m 1.8% Interspaced plants within a row. Experiment conducted in a single year. Single male and female parental varieties. Ray et al., 2003Monsanto Company 09-SY-200U Page 179 of 395 X.C.1.2. Hybridization with the W ild Annual Species within Subgenus Soja

The subgenus Soja includes the cultivated soybean G. max and the wild annual species G. soja. G. soja is found in China, Taiwan, Japan, Korea, and Russia and can hybridize naturally with the cultivated soybean, G. max (Hymowitz, 2004). Hybridization between

female G. soja and male G. max was less successful than hybridization in the opposing direction (Dorokhov et al., 2004), where frequency of spontaneous cross pollination in reciprocal combinations of G. max and G. soja varied from 0.73 ( ♀ G. soja × ♂ G. max ) to 12.8% ( ♀ G. max × ♂ G. soja ). Species relationships in the subgenus Soja indicated that F1 hybrids of G. max and G. soja carry similar genomes and are fertile (Singh and Hymowitz, 1989). Abe et al. (1999) notes that “n atural hybrids between G. max and G.
soja are rare ... and hybrid sw arms involving both species ha ve never been reported.” Many barriers exist to natura l hybridization between soybean and wild relatives including the highly selfing nature of both plants, requi red proximity of wild soybean to cultivated soybean, synchrony of flowering, and presence of pollinators. As such, it is unlikely that naturally occurring, pollen-mediated gene fl ow and transgene introgression into wild soybean relatives from incidentally releas ed biotechnology-derived soybean will occur at any meaningful frequency.

As described earlie r, the subgenus Soja also contains an unofficial species, G. gracilis (Hymowitz, 2004). G. gracilis is known only from Northeast China, and is considered a weedy or semi-wild form of G. max , with some phenotypic characteristics intermediate to

those of G. max and G. soja

. G. gracilis may be a hybrid between G. soja and G. max (Hymowitz, 1970). Interspecific fertile hybrid s formed by intentional crosses between G.

max and G. soja and between G. max and G. gracilis have been easily obtained (Dorokhov et al., 2004). Although hybridization between G. max and members of the subgenus G. soja can take place, G. soja is not found in North or South America, and it is highly unlikely that gene transfer will occur.

X.C.1.3. Hybridization with Wild Perennial Species of Subgenus Glycine

The wild perennial species of the Glycine subgenus occur in Aust ralia, West Central and South Pacific Islands, China, Papua New Guinea, Philippines, and Taiwan. Therefore, the only opportunities for inter-subgeneric hybridization would occur in areas where those species are endemic (Hymowitz et al., 1992; Hymowitz and Singh, 1992). Nonetheless, the likelihood of in terspecific hybridization between G. max and the wild perennial Glycine species is extremely low because th ey are genomically dissimilar (see Table II-2) and pod abortion is common. From time to time, immature seeds of the crosses could be germinated aseptically in vitro , but the resulting F1 hybrids are slowgrowing, morphologically weak, and completely sterile. Their steril ity is due to poor chromosome pairing. Furthermore, specie s distantly related usually produce nonviable F1 seeds that either have premature death of the germinating seedlings or suffer from seedling and vegetative lethality (Kollipara et al., 1993; Singh and Hymowitz, 1989). In North and South America, it is not possible fo r gene transfer between cultivated soybean and wild perennial species of Glycine subgenera, because these wild species do not exist in these regions.Monsanto Company 09-SY-200U Page 180 of 395 X.C.2. Transfer of Genetic information to Species with which Soybean Cannot Interbreed (Horizontal Gene Flow)

Monsanto is not aware of any reports regarding the unaided transfer of genetic material from soybean species to ot her sexually incompatible species. The probability for horizontal gene flow to occur is judged to be exceedingly small. Even if it were to occur, the consequences would be negligible since the genes introduced into MON 87769 are of plant and fungal origin and the Pj Δ 6D and Nc Δ 15D proteins as well as the two fatty acids not previously produced in soybean (SDA an d GLA) have no meaningful toxicity to animals, including humans, and other NTOs under the conditions of use.

X.D. Summary of Plant Pest Assessments

Plant pests are defined in the Plant Protection Act as any 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)]. Characterizatio n data presented in Sections III through X of this petition confirm that except for the introduced genes ( Pj.D6D and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and the intended fatty acid change, MON 87769 is not differe nt from conventional soybean in its phenotype, environmental interactions, or susceptib ility to pest or disease. A plant pest assessment was conducted to assess the poten tial impact of the introduced genes ( Pj.D6D

and Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and intended fatty acid change (SDA and GLA) on NTOs and the weediness potential of

MON 87769. Based on the results of this assessment, it is c oncluded that the potential

risk of MON 87769 to cause adverse effect s on NTOs and endangered species is negligible. MON 87769 is no more likely to become a weed than conventional soybean. The environmental risk associated with po llen-mediated gene flow from MON 87769 to other sexually compatible plants is considered negligible because the potential for gene

flow is low. Evaluations have show n that the introduct ion of the genes ( Pj.D6D and

Nc.Fad3 ), the proteins produced from the introduced genes (Pj Δ 6D and Nc Δ 15D), and intended fatty acid change is not likely to en hance weediness or plant pest potential. There are no changes expected in ag ronomic practices for MON 87769 and the production systems currently used with sp ecialty soybean. Thus, compared to conventional soybean, there are no increased pl ant pest characterist ics associated with

MON 87769.

Based on the data and information presented in this petition, it is concluded that MON 87769 is not likely to be a plant pest. Therefore, M onsanto Company requests a

determination from APHIS that MON 87769 and any progeny derived from crosses between MON 87769 and conventional soybean or deregulated biotechnology-derived soybean be granted nonregulated status under 7 CFR Part 340.Monsanto Company 09-SY-200U Page 181 of 395 XI. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto knows of no study results or obs ervations associated with MON 87769 that

indicate there would be an adverse environmental conseque nce from the introduction of

MON 87769. MON 87769 is an altern ate source of omega-3 fatty acid. As demonstrated by field results and laborato ry tests, the only phenotypic difference between MON 87769 and conventional soybean is the intended cha nge in seed fatty acid composition and the presence of the two desaturase proteins (Pj Δ 6D and Nc Δ 15D).

The data and information presented in th is petition demonstrate that MON 87769 is unlikely to pose an increased plant pest poten tial or to have an adverse environmental consequence compared to conventional soyb ean. This conclusion is reached based on multiple lines of evidence developed from a detailed characterization of the product compared to conventional soybean, followed by ri sk assessment on detected differences.

The characterization evalua tions included molecular an d protein analyses, which

confirmed the insertion of a single functional copy of the Pj.D6D and Nc.Fad3 expression cassette at a single locus w ithin the soybean genome and that Pj Δ 6D and

Nc Δ 15D proteins were detected only in seed at low levels. Extensive characterization of the plant phenotype, including compositional analysis of key nutrient and antinutrients also indicated that MON 87769, with the exception of intended modification, was unchanged compared to conventional soybean. Allergenicity assessment and history of safe use of the Pj Δ 6D and Nc Δ 15D proteins concluded that the Pj Δ 6D and Nc Δ 15D proteins are unlikely to be an allergen for humans. Toxicity tests, including an acute mouse oral gavage with proteins produced in MON 87769 immature seed, showed no signs of adverse effects at high doses. An endangered species risk assessment also concluded that MON 87769 is unlikely to have adverse effects on these organisms. Therefore, the risks for humans, animal s, and other NTOs from MON 87769 are negligible under the co nditions of use.

The introduction of MON 87769 will not im pact cultivation practices and the management of weeds, diseases, and insects in soybean production systems. Successful adaptation of MON 87769 will provide grow ers with an opportunity to produce this value-added speciality soybean containing SDA, which is a sustainable source of omega-3 fatty acid.Monsanto Company 09-SY-200U Page 182 of 395 REFERENCES

Abe, J., A. Hasegawa, H. Fukushi, T. Mikami, M. Ohara, and Y. Shimamoto. 1999. Introgression between and cultivated soybeans of Japan revealed by RFLP analysis for chloroplast DNAs. Economic Botany 53:285-291.

Abrams, R.I., C.R. Edwards, and T. Harri s. 1978. Yields and cr oss-pollination of soybeans as affected by honey bees and alfa lfa leafcutting bees. American Bee Journal

118:555-560.

Abud, S., P.I. Mello de Souza, C.T. Moreir a, S.R.M. Andrade, A.V. Ulbrich, G.R. Vianna, E.L. Rech, and F.J. Lima Aragao. 2003. Pollen dispersal in transgenic soybean plants in the Verrado region. Pes quisa Agropecuaria Brasilera 38:1229-1235.

Aguilar, P.S. and D. de Mendoza, D. 2006. Control of fatty acid desaturation: A

mechanism conserved from bacteria to hum ans. Molecular Micr obiology. 62:1507-1514.

Ahrent, D.K., and C.E. Caviness. 1994. Natu ral cross-pollination of twelve soybean cultivars in Arkansas. Crop Sci. 34:376-378.

Alexander, M.P. 1980. A versatile stain for po llen fungi, yeat and bacteria. Stain technol. 55(1): 13-18.

Al-Kaisi, M. 2001. Value of crop rotation in ni trogen management. Iowa State University. http://www.ipm.iastate.edu/ipm/icm/2001/4-23-2001/valuen.html

.

Al-Kaisi, M., M.H. Hanna, and M. Tidma n. 2003. Crop rotation considerations for 2004 management season rotation. Iowa State university, Department of Entomology. http://www.ent.iastate.edu/ipm/icm/2003/croprotation.html [Accessed June 24, 2009].

Anderson, W.P. 1996. Weed Ecology. Pages 27- 38 in Weed Science Principles and Applications, Third Edition West P ublishing Company, St. Paul, Minnesota.

AOSA. 2000. Tetrazolium Testing Handbook -C ontribution No. 29 to the Handbook on Seed Testing. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2007. Rules for Testing Seeds. Associa tion of Official Seed Analysts, Lincoln,

Nebraska.

AOSCA. 2003. "Yellow Books" 2003 Operatio nal Procedures, Crop Standards and Service Programs Publication (Genetic and Crop Standards). Association of Official Seed Certifying Agencies, Meridian, Idaho.

AOSCA. 2009. Seed Certification Handbook. Asso ciation of Official Seed Certifying Agencies, Moline, Illinois.Monsanto Company 09-SY-200U Page 183 of 395 Appunu, C. and B. Dhar. 2006. Differential symbio tic response of pha ge-typed strains of Bradyrhizobium japonicum with soybean cultivars. Journal of Microbiology 44(3):363-

368.

Aref, S., and D.R. Pike. 1998. Midwest farmer's perceptions of crop pest infestations. Agronomy. Journal 90:819-825.

Armstrong, T., K. Barton, L.J. Crow, L.A. Gilbertson, S. Huang, Y. Huang, B.J. Martinell, M.W. Petersen, K.H. Piller, Q. Wang, and X. Ye. 2007. Methods for enhancing segregation of transgenes in plan ts and compositions thereof. US Patent

7288694.

ASA. 2008. Soy Stats 2008. American Soybean Association, St. Louis, Missouri.

ASA. 2009. Grower Opportunities for Ident ity Preserved Value-Added Soybeans. American Soybean Associat ion, St. Louis, Missouri.

Baker, H.G. 1965. Characteristics and modes of origin of weeds. Page 147-172 in The Genetics of Colonizing Species. Baker, H.G. a nd G.L. Stebbins (eds.). Academic Press,

New York.

Barker, K., I. Chibata, K. Nakayama, K. Takinami, and H. Yamada. 1983. Nucleotide sequence of the T-DNA Region from the Agrobacterium tumefaciens Octopine Ti Plasmid pTi15955. Plant Molecular Biology 2:335-350.

Barry, G.F., G.M. Kishore, S.R. Padgette, and W.C. Stallings. 1997. Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases. U.S.A. Patent 5,633,435.

Baumann, T.T., A.F. Dobbels, W.G. Johnson, M.M. Loux, G.R.M. Nice, and S. J.M. 2008. Weed control guide for Ohio and Indian a. Purdue University and Ohio State University Extension Bulletin 789.

Beard, B.H., and P.F. Knowles. 1971. Frequenc y of cross-pollination of soybeans after seed irradiation. Crop Science 11:489-492.

Beare-Rogers, J., A. Dieffenbacher, and J. V. Holm. 2001. Lexicon of lipid nutrition. Pure and Applied Chemistry 73(4):685-744.

Bevan, M. 1984. Binary Agrobacterium vectors for plant tran sformation. Nucleic Acid

Research 12:8711-8721.

Bimbo, A.P. and J. B. Crowther. 1992. Fish Meal and Oil: Current Uses JAOCS, Vol.

69:221-227.Monsanto Company 09-SY-200U Page 184 of 395 Boerma, H.R., and J.E. Specht. 2004. Soyb ean production in the U.S.A. pp. 3. Managing inputs. pp. 502 and 507. Harvesting. pp. 522. in Soybeans: Improvement, Production, and Uses. Boerma. H.R., and J.E. Sprecht (eds ). American Society of Agronomy, Crop Science Society of America, Soil Scien ce Society of America, Madison, Wisconsin.

Bowers, G.R. and J.S. Russin. 1999. Soybean disease management. Pages 233-271 in Soybean Production in the Mids outh. Heatherly, L.G. and H. F. Hodges (eds.). CRC Press, Boca Raton, Florida.

Bradford, K.J. 2006. Methods to Maintain Gene tic Purity of Seed Stocks. Agricultural Biotechnology in California Series, Publica tion 8189. Regents of the University of

California.

Breitler, J.C., D. Meynard, J. Van Boxtel, M. Royer, F. Bonnot, L. Cambillau, and E. Guiderdoni. 2004. A novel two T-DNA binary vect or allows efficient generation of marker-free transgenic plants in three elite cultivars of rice ( Oryza sativa L.). Transgenic

Research 13:271-287.

Cahoon, E.B. 2003. Genetic enhancement of soyb ean oil for industrial uses: prospects and challenges. AgBioForum 6:11-13.

Canavoso, L.E., Z.E. Jouni, K.J. Karnas, J.E. Pennington, and M.A. Wells. 2001. Fat

metabolism in insects. Annual Review of Nutrition 21:23-46.

Carpenter, J, A. Felsot, T. Goode, M. Hammig, D. Onstad, and S. Sankula. 2002. Comparative Environmental Impacts of Bi otechnology-derived and Traditional Soybean, Corn, and Cotton Crops. Council for Agricult ural Science and Technology, Ames, Iowa.

CAST. 2007. Implications of Gene Flow in the Scale-up and Commercial Use of Biotechnology-derived Crops: Economic and Policy Considerations. Issue Paper 37. Council for Agricultural Science and Technology, Ames, Iowa.

Caviness, C.E. 1966. Estimates of natural cr oss-pollination in Jackson soybeans in Arkansas. Crop Science 6:211-212.

CFIA. 1996. The biology of Glycine max (L.) merr. (soybean). Biology Document BIO1996-10. Canadian Food Inspection Agency, Ontario, Canada.

Chardigny J.M., J.L. Sébédio, and O. Berde ux. 1996. Trans Polyunsaturated Fatty Acids:

Occurrence and Nutritio nal Implications. Annals of A pplied Lipid Research 2:1-33.

Chiang, Y.C. and Y.T. Kiang. 1987. Geometric position of genotypes, honeybee foraging patterns,a nd outcrossing in soybean. Bot. Bull. Academia Sinica 28:1-11.Monsanto Company 09-SY-200U Page 185 of 395 Choowongkomon, K., C.R. Carlin, and F.D. Sonn ichsen. 2005. A structural model for the membrane-bound form of the juxtamembrane domain of the epidermal growth factor receptor. Journal of Biological Chemistry. 280:24043-24052.

Codex. 2003. Guideline For The Conduct Of Food Safety Assessment Of Foods Derived From Recombinant-DNA Plants. ftp://ft p.fao.org/es/esn/foo d/guide_plants_en.pdf [Accessed June 24, 2009].

Cook, H.W. 1991. Fatty acid desa turation and chain elongati on in eukaryotes. Pages 141-169 in D.E. Biochemistry of Lipids, Lipopr oteins and Membranes. Vance, J.E. (ed.). Elsevier Science Publishers, B. V., Amsterdam.

Coruzzi, G., R. Broglie, C. Edwards, a nd N. Chua. 1984. Tissue-specific and lightregulated expression of a pea nuclear gene encoding the small subunit of ribulose-1,5- bisphosphate carboxylase. EMBO 3:1671-1679.

Crockett, L. 1977. Wildly Successful Plants: North American Weeds. University of Hawaii Press, Honolulu, Hawaii.

CTIC. 2004. Crop residue management. Cons ervation Technology Information Center, West Lafayette, Indiana.

Cutler, G.H. 1934. A simple method for ma king soybean hybrids. Agronomy Journal

26:252-254.

Dalley, C.D., K.A. Renner, and J.J. Ke lls. 2001. Weed competition in Roundup Ready soybean and corn. Department of Crop and Soil Science, Michigan State University.

Debeaujon, I., L. Lepiniec, L. Pource l, and J.-M. Routaboul. 2007. Seed coat development and dormancy. Pages 23-49 in Seed Development, Dormancy, and Germination, K.J. Bradford and H. Nonaga ki, (eds.) Blackwell Publishing, Ltd., Ames,

Iowa

Della-Cioppa, G., S.C. Bauer, B.K. Klein, D. M. Shah, R.T. Frayley, and G.M. Kishore. 1986. Translocation of the precu rsor of 5-enolpyruvylshikim ate-3-phosphate synthase into chloroplasts of higher plants in vitro. Proceedings of the National Academy of

Sciences 83:6873-6877.

DeMan, J.M. 2000. Chemical and Physical Pr operties of Fatty Acids. Pages 17-46 in Fatty Acids in Foods and their Health Imp lications. Chow, C.K. (ed.). Marcel Decker,

New York.

Depicker, A., S. Stachel, P. Dhaese, P. Za mbryski, and H.M. Goodman. 1982. Nopaline synthase: transcript mapping and DNA sequen ce. Journal of Molecular Applied Genetics

1:561-573.Monsanto Company 09-SY-200U Page 186 of 395 Dillon, T.W., R.C. Scott, N.D. Pearrow, a nd K.A. Meins. 2006. Effect of sulfonylurea rice herbicides on soybeans. Sout hern Weed Science Society.

Dorokhov, D., A. Igantov, E. Deineko, A. Serjapin, A. Ala, and K. Skryabin. 2004. Potential for gene flow from herbicide-resistant gm soybeans to wild soya in the Russian Far East. Pages 151-161 in Introgression from Genetically Modified Plants into Wild Relatives. Nijs, H.C.M., D. Bartsch, and J. Sweet (eds.). CAB International, Wallingford.

Dorrance, A.E., M.A. Draper, and D. Hers hman. 2007. Using foliar fungicides to manage soybean rust. Ohio State Univer sity and Uiversity of Kentucky.

Doyle, J., M. Schuler, W. Godette, W. Zenger, R. Beachy, and J. Slightom. 1986. The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris : Structural homologies of genes and proteins. Journal of Biol ogical Chemistry 261:9228-9238.

Dubois, V., S. Breton, M. Linder, J. Fanni, and M. Parmentier. 2007. Fatty acid profiles of 80 vegetable oils with rega rd to their nutritional potentia l. European Journal of Lipid Science Technology 107:710-732.

Elberhi, A. 2007. The Changing Face of th e U.S. Grain System: Differentiation and Identity Preservation Trends. United Stat es Department of Agriculture - Economic Research Department, Washington, D.C. (http://www.ers.usda.gov/publications/err35/ ) [Accessed May 15, 2009].

Erickson, E.H. 1975. Variability of floral characteristics in fluences honey bee visitation to soybean blossom. Crop Science 15:767-771.

Erickson, E.H. 1984. Soybean pollination and honey production: A research progress report. American Bee Journal 124:775-779.

FAO. 1986. The production of fish meal and o il. FAO Fisheries Technical Paper 142. Food and Agricultural Organiza tion of the United Nations.

Farès, C., D. Libich, and G. Hara uz. 2006. Solution NMR structure of an immunodominant epitope of myelin ba sic protein. FEBS Journal 273:601-614.

FDA. 1992. Satatement of policy: Food derive d from new plant varieties. Food and Drug

Administration. 57 FR 22984-23005.

Fehr, W.R. 2007. Breeding for modified fatty acid composition in soybean. Crop Science 47(S3):S73-S87.

Feussner, I and C. Wasternack. 2002. The lipoxygenase pathway. Annual Review of Plant Biology 53:275-297.Monsanto Company 09-SY-200U Page 187 of 395 Fling, M.E., J. Kopf, and C. Richards. 1985. Nucleotide sequence of the transposon Tn 7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase.

Nucleic Acids Research 13:7095-7106.

Funderburk, J., R. McPherson, and D. Buntin. 1999. Soybean insect management. Pages 273-290 in Soybean Production in the Midsouth. Heatherly, L.G. and H.F. Hodges (eds.). CRC Press, Boca Raton, Florida.

Gage, D.J. 2004. Infection and invasion of r oots by symbiotic, nitrogen-fixing Rhizobia during nodulation of temperate legumes. Microbiology and Molecular Biology Reviews

68:280-300.

Garber, R.J. and T.E. Odland. 1926. Natural crossing in soybeans. Agronomy Journal

18:967-970.

Gardner, H.W. 1995. Biological roles and bi ochemistry of the lipoxygenase pathway.

Horticultural Science 30: 197–205.

Giza, P.E., and R.C. Huang. 1989. A self -inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene 78:73-84.

Gorzelle, B.M., J.K. Nagy, K. Oxenoid, W.L. Lonzer, D.S. Cafiso, and C.R. Sanders. 1999. Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein. Biochemistry 38:16373-16382.

Graham, P. H., Hungria, M., and Tlusty, B. 2004. Breeding for better nitrogen fixation in grain legumes: Where do the rhizobia fit in? Online. Cr op Management doi:10.1094/CM2004-0301-02-RV.

Guil-Guerrero, J.L., J.C. Lopez-Martinez, F. Gomez-Mercado, P. Campra-Madrid. 2006.

Gamma-linolenic and stearidonic acids from Moroccan Boraginaceae. European Journal of Lipid Science and Technology 108:43-47.

Guil-Guerrero, J.L., F. Gomez-Mercado, F. Garcia-Maroto, P. Campra-Madrid. 2000.

Occurrence and characteriza tion of oils rich in γ -linolenic acid. Part I: Echium seeds from Macaronesia. Phytochemistry 53: 451-456.

Gupta, M., W. Nirunsuksiri, G. Schulenberg, T. Hartl, S. Novak, J. Bryan, N. Vanopdorp, J. Bing, and S. Thompson. 2008. A non-PCR-base d Invader assay quantitatively detects single-copy genes in complex plant genomes. Molecular Breeding 21:173-181.

Gurley. 1979. Sequence organization of the soybean genome. Biochimica et Biophysica

Acta 561:167-183.Monsanto Company 09-SY-200U Page 188 of 395 Hammond, B.G., J.K. Lemen, G. Ahmed, K.D. Miller, J. Kirkpatrick, and T. Fleeman. 2008. Safety assessment of SDA soybean oil: Results of a 28-day gavage study and a 90- day/one generation reproduction feeding st udy in rats. Regulatory Toxicology and Pharmacology 52:311-323.

Hammond, R.B., R.A. Higgins, T.P. Mack, L.P. Pedigo, and E.J. Bechinski. 1991. Soybean pest management. Pages 341-350 in CRC Handbook of Pest Management in Agriculture. Volume III. CRC Press. Boca Raton, Florida.

Harris, W.S., S.L. Lemke, S.N. Hansen, D. A.a Goldstein, M.A. DiRienzo, H. Su, M.A. Nemeth, M.L. Taylor, G. Ahmed, and C. George. 2008. Stearidonic acid-enriched soybean oil increased the omega-3 index, an em erging cardiovascular risk marker. Lipids

43:805-811.

Hashimoto, K., A.C. Yoshizawa, S. Okuda, K. Kuma, S. Goto, and M. Kanehisa. 2008. The repertoire of desaturases and elongases re veals fatty acid varia tions in 56 eukaryotic genomes. Journal of Li pid Research 49:183-191.

Helsel, Z.R. and H.C. Minor. 1993. Soybean production in Missour i. University of Missouri Department of Ag ronomy. Publication G4410. http://extension.missouri.edu/explore/ agguides/crops/g04410.htm [Accessed Jan. 7, 2009].

Hermann, F.J. 1962. A revision of the genus glycine and its immediate allies. United States Department of Agri culture Technical Bulletin 1268:1-79.

Ho, T. 1969. The loess and the origin of Chinese agriculture. Am erican Historical

Review 75:1-36.

Hoeft, R.G., E.D. Nafziger, R.R. Johnson, and S.R. Aldrich. 2000. Soybean as a crop. Pages 31,36,38,39,41,43,47,86,89,93,96,120,208 in Modern Corn and Soybean Production MCSP Publications, Champaign, Illinois.

Holm, L., J.V. Pancho, J.P. Herberger, and D.L. Plucknett. 1979. In troduction. Pages i-vii in A Geographical Atlas of World W eeds. John Wiley and Sons, New York.

Horrobin, D. F. 1992. Nutritional and medi cal importance of gamma-linolenic acid. Progress in Lipid Re search 31(2):163-194.

Hymowitz, T. 1970. On domestica tion of soybean. Economic Botany 24:408-421.

Hymowitz, T. 2004. Speciation and cytoge netics. Pages 97-236 in Soybeans: Improvement, Production, and Uses. Boerma. H. R., and J.E. Sprecht (eds). American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, Wisconsin.Monsanto Company 09-SY-200U Page 189 of 395 Hymowitz, T., and C.A. Newell. 1981. Taxonomy of the genus Glycine , domestication and uses of soybeans. Economic Botany 35:272-288.

Hymowitz, T., R.G. Palmer, and R.J. Singh. 1992. Cytogenetics of the genus Glycine

. Pages 53-63 in Chromosome Engineering in Plants: Genetics, Breeding, Evolution, Part B. Tsuchiya, T. and P.K. Gupta, (eds.). Elsevier Science Publishers, Amsterdam.

Hymowitz, T., and R.J. Singh. 1992. Biosystems of the genus Glycine , 1991. Soybean

Genetics Newsletter 19:184-185.

Hymowitz, T., R.J. Singh, and R.P. Larkin. 1990. Long distance dispersal: The case for the allopolyploid glycine tabacina Benth. and G. tomentella Hayata in the west-central pacific. Micronesia 23:5-13.

Iowa State University. 2004. Soybean Grow th and Development, PM 1945, Ames, IA.

Ishihara, K., M. Murata, M. Kaneniwa, H. Saito, W. Komatsu, and K. Shinohara. 2000. Purification of stearidonic [ 18:4(n-3)] and hexadecatetraenoi c acid [16:4(n-3)] from algal fatty acid with lipase and medium pre ssure liquid chromatography. Bioscience Biotechnology and Biochemistry 64:2454-2457.

ISO. 2009. International Organi zation for Standardization. Geneva. http://www.iso.org [Accessed Jan. 9, 2009].

Israel D.W., J.N. Mathis, W.M. Barbour, G.H. Elkan. 1986. Symbiotic effectiveness and

host-strain interactions of Rhizobium fredii USDA 191 on different soybean cultivars. Applied and Environmental Microbiology 51(5):898-903.

James, C. 2008. Global Status of Co mmercialized Biotech/GM Crops: 2008. ISAAA Brief No. 39. ISAAA: Ithaca, NY.

James, M.J., V.M. Ursin, and L.G. Clela nd. 2003. Metabolism of st earidonic acid in human subjects: comparison with the metabo lism of other n-3 fatty acids. American

Journal of Clinical Nutrition 77:1140-1145.

Kemp, J.D., R.F. Barker, and M.J. Adang. 2000. Octopine T-DNA struct ural genes. U.S.

Patent 6090627.

Kishore, G., D. Shah, S. Padgette, G. Della-C ioppa, C. Gasser, D. Re, C. Hironaka, M. Taylor, J. Wibbenmeyer, D. Eichholtz, M. Hayford, N. Hoffmann, X. Delannay, R. Horsch, H. Klee, S. Rogers, D. Rochester, L. Brundage, P. Sanders, and R.T. Fraley. 1988. 5-enolpyruvylshikimate 3-phosphate syntha se: from biochemistry to genetic engineering of glyphosate tolerance. Pages 37-48 in Biotechnology for Crop Protection. Hedin, P.A., J.J. Menn, and R.M. Hollingwor th (eds.). American Chemical Society. Division of Agrochemicals, Snowbird, Utah.Monsanto Company 09-SY-200U Page 190 of 395 Klee, H.J., Y.M. Muskopf, and C.S. Gasser. 1987. Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate- 3- phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Molecular Ge netics and Genomics

210:437-442.

Kleiman, R., F.R. Earle, I.A. Wolff, and Q. Jones. 1964. Search for new industrial oils. XI. Oils of boraginaceae. Journal of Am erican Oil Chemists Soc. 41(7):459-460.

Kollipara, K.P., R.J. Singh, and T. Hymow itz. 1993. Genomic diversity in aneudiploid (2n=38) and diploid (2n=40) Glycine tomentella revealed by cytogenetic and biochemical

methods. Genome 36:391-396.

Komari, T., Y. Hiei, Y. Saito, N. Murai, and T. Kumashiro. 1996. Vectors carrying two separate T-DNAs for co-transformati on of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. The Plant

Journal 10:165-174.

Koncz, C. and J. Schell. 1986. The promoter of T L -DNA gene 5 controls the tissuespecific expression of chimaeric ge nes carried by a novel type of Agrobacterium binary

vector. Molecular Genetics and Genomics 204:383-396.

Lackey, J.A. 1981. Phaseoleau DC. Pages 301-327 in Advances in legume systematics, Part I. Polhill, R.M. and R.H. Raven, (eds.). Royal Botanic Gardens, London.

Lee, C, and J. Herbek. 2004. Specialty Soybean Production and Management in Kentucky. Cooperative Extension Service. University of Kentucky-College of Agriculture.

Lee, J.S., and D.P.S. Verma. 1984. Stru cture and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetraploid ization. EMBO Journal 3:2745-2752.

Lersten, N.R. and J.B. Carlson. 2004. Vegetative Morphology. Pages 15-57 in Soybeans:Improvement, Production, and Uses. Boerma, H.R. and J.E. Sprecht (eds). American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, Wisconsin.

Li, L., A.F. Tutone, R.S.M. Drummond, R. C. Gardner, and S. Luan. 2001. A Novel Family of Magnesium Transport Gene s in Arabidopsis. Plant Cell 13:2761-2775.

Liener, I.E. 1994. Implications of antinutriti onal components in soybean foods. Critical

Reviews in Food Science Nutrition 34:31-67.

Liener, I.E. 2000. Non-nutritive factors and bioa ctive components in soy. Pages 13-45 in Soy in Animal Nutrition. J.K. Drackley (ed.) . Federation of Animal Science Societies, Savoy, IL.Monsanto Company 09-SY-200U Page 191 of 395 Lingenfelter, D.D., and N.L. Hartwig. 2003. Wh at are weeds and their impacts? Pages 2-

20 in Introduction to Weeds a nd Herbicides. Pennsylvania St ate University College of Agricultural Sciences, Univ ersity Park, Pennsylvania.

Liu, C.P. and L.P. Lin. 2001. Ultrastr uctural study and lipid formation of Isochrysis sp. CCMP1324. Botanical Bulletin of Academia Sinica 42:207-214.

Los, D.A. and N. Murata. 1998. Structure a nd expression of fatty acid desaturases. Biochimica et Biophysica Acta 1394(1):1403-1415.

Lu, B.-R. 2004. Conserving biodiversity of s oybean gene pool in the biotechnology era. Plant Species Biology 19:115-125.

MacDonald, J.I.S. and H. Sprecher. 1991. Phospholipid fatty acid remodelling in

mammalian cell. Biochimica et Biophysica Acta 1084:105-121.

Massey, R.E. 2002. Identity preserved crops . Iowa State University Extension. http://www.extension.iastate.edu/ agdm [Accessed June 1, 2009].

Martinell, B.J., L.S. Julson, C.A. Emler, Y. Huang, D.E. McCabe, and E.J. Williams. 2002. Soybean agrobacterium transfor mation method. U.S. Patent 6,384,301.

Matthews, P.R., M.B. Wang, P.M. Waterhouse, S. Thornton, S.J. Fieg, F. Gubler, and J.V. Jacobsen. 2001. Marker gene elimination from transgenic barley, using co-transformation with adjacent “twin T-DNAs” on a standard Agrobacterium transformation vector. Molecular Breeding 7:195-202.

Messina, M. 2001. Soy a nd health: Isoflavones http://www.soyconnection.com/health_nutriti on/pdf/IsofavonesFactSh eet.pdf [Accessed June 24, 2009].

Miller, M., L. Tagliani, N. Wang, B. Be rka, D. Bidney, and Z.Y. Zhao. 2002. High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Tran sgenic Research 11:381-396.

Montgomery, R.F., R.M. Hyayes, C.H. Tingle, and J.A. Kendig. 2002. Control of glyphosate-tolerant soybean ( Glycine max ) in no-till Roundup Ready cotton ( Gossypium

hirsutum L.) in Proceedings of the Beltwide Cotton Conference National Cotton Council of America, Memphis, Tennessee.

MSA. 2009. Market Trends for IP Crops . Midwest Shippers Association. http://www.mnshippers.com/html/news.cfm?ID=44 [Accessed June 24, 2009].

Muenscher, W.C. 1980. Weeds. 2nd ed. Co rnell University Press, New York.Monsanto Company 09-SY-200U Page 192 of 395 Mullin, J.W. and W. Xu. 2001. Study of soyb ean seed coat co mponents and their relationship with water absorption. Journal of Agriculture and Food Chemistry 49:5331-

5335.

Murdock, E.C., M.A. Jones, and R.F. Gr aham. 2002. Control of volunteer glyphosate (Roundup)-tolerant cotton and soybean in R oundup Ready cotton in Proceedings of the 2002 Beltwide Cotton Conferences, Memphis, Tennessee.

Muth, M.K., D. Mancini, and C. Viator. 2003. The role of identity -preservation in foodmanufacturer responses to bioengineered f oods. Journal of Food Distribution Research 34(1):43-49.

Nakamura, M.T. and T.Y. Nara. 2004. Struct ure, junction, and di etary regulation of Delta 6, Delta 5, and Delta 9 desaturases. Annual Review of Nutrition 24:345-376.

Narayanan, S., T. Sato, and M.S. Wolfe. 2007. A C-terminal region of signal peptide peptidase defines a functional domain for in tramembrane aspartic protease catalysis. Journal of Biological Chemistry 282:20172-20179.

NDSU. 2002. Soybean production guide for Nort h Dakota and Northwestern Minnesota. A-1172. North Dakota State Univeristy Extension Service. http://www.ag.ndsu.edu/pubs/plantsci/row crops/a1172.pdf [Accessed June 24, 2009].

Nelson, R., V. Lozovaya, A. Lygin, and J. Widholm. 2001. Variations in isoflavones in seeds of domestic and exotic soybean germplasm. Abstract of presentation at ASACSSA-SSSA annual meeting in Charlotte, North Carolina, October 21-25, 2001.

Nice, G., and B. Johnson. 2005. Indiana's Top Ten Most Problematic Weeds. Purdue

Extension Weed Science.

OECD. 1993. Safety considerations for biotechnology: Scale-up of crop plants. Organization for Economic Co-operation and Development, Paris. http://www.biosafety.be/CU/BSL_Ressour ces/PDF/M00034525.pdf [Accessed January 9, 2009].

OECD. 2000. Consensus document on the biology of glycine max (L.) merr. (soybean)..

OECD ENV/JM/MONO(2000)9. Organization for Economic Co-operation and Development, Paris.

OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean: key food and feed nutrients and anti-nutrients. OECD ENV/JM/MONO(2001)15. Organization for Econo mic Co-operation and Development,

Paris.Monsanto Company 09-SY-200U Page 193 of 395 Oxenoid, K. and J.J. Chou. 2005. The struct ure of phospholamban pentamer reveals a

channel-like architecture in membranes. Proceedings of the National Academy of

Sciences of the U.S.A 102:10870-10875.

Özcan, T. 2008. Analysis of the total oil and fatty acid composition of seeds of some
Boraginaceae taxa from Turkey. Plant Systematics and Evolution 274:143-153.

Padgette, S.R., D. Re, G. Barry, D. Eichholtz, X. Delannay, R.L. Fuchs, G. Kishore, and R.T. Fraley. 1996. New weed control opportuni ties: Development of soybeans with a Roundup Ready gene. Pages 53–84 in Herbicid e Resistant Crops: Agricultural, Environmental, Economic, Regulatory, and Tech nical Aspects. Duke S.O. (ed.). Lewis,

New York.

Pazdernik, D.I., Ph.H. Graham, C. P. Vance, and J.H. Orf. 1996. Host genetic variation in early nodulation and dinitrogen fixati on of soybean. Crop Science 36:1102-1107.

Pan, D.A., A.J. Hulbert, and L.H. Storli en. 1994. Dietary fats, membrane phospholipids and obesity. Journal of Nutrition 124:1555-1565.

Pedersen, P. 2008a. Soybean production. Iowa State University Extension Service.

Pedersen, P. 2008b. Managing soybean cyst nema tode. Iowa State University Extension. http://extension.agron.iastate. edu/soybean/documents/SCN. pdf. [Accessed May 9, 2008].

Potts, H.C., J. Duangpatra, W.G. Hairston, and J.C. Delouche. 1978. Some influences of hardseededness on soybean seed quality. Crop Science 18:221-224.

Pritchett, J.J., J. Fulton, R. Beyers, L. Pederson, and L. Lawson. 2002. Specialty Corn and Soybeans: Production and Marketing in Indiana. EC-714. Purdue University Cooperative Extension Service.

Ray, J.D., T.C. Kilen, A.C. Abel, and R.L. Paris. 2003. Soybean natural cross-pollination

rates under field conditions. E nvironmental Biosafety Research 2:133-138.

Reed, D.W., U.A. Schafer, and P.S. Covello. 2000. Characterization of the Brassica

napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae. Plant Physiology 122:715-720.

Rogers, S.G. 2000. Promoter for transgenic plants. U.S. Patent 6018100.

Rosenberg, I.M. 1996. Membrane and particulat e-associated proteins . Pages 135-152 in Protein Analysis and Purification: Be nchtop Techniques. Birkhauser, Boston.

Sambrook, J. and D.W. Russell. 2001. Agar ose Gel Electrophoresis. Pages 5.4-5.13 in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Monsanto Company 09-SY-200U Page 194 of 395 Sayanova, O., F. Beaudoin, B. Libisch, A. Ca stel, P.R. Shewry, and J.A. Napier. 2001. Mutagenesis and heterologous expression in yeast of a plant Delta(6)-fatty acid

desaturase. Journal of E xperimental Botany 52:1581-1585.

Sayanova, O., Beaudoin, F., Libisch, B., Shewr y, P., and Napier, J. 2000. Mutagenesis of the borage Delta(6) fatty acid desaturase. Biochemical Society Transactions 28:636-638.

Sayanova, O., J.A. Napier, and P.R. Shewr y. 1999. Delta-6 unsaturated fatty acids in species and tissues of the Pr imulaceae. Phytochemistry 52:419-422.

Scott, O.S., and S.R. Aldrich. 1970. M odern Soybean production. Pages 16, 18, 67, 151, 152 in The Farm Quarterly, Cincinnati, Ohio.

Shanklin, J. and E.B. Cahoon. 1998. Desaturatio n and Related Modifications of Fatty Acids1. Annual Review of Plant Physiol ogy and Plant Molecular Biology 49:611-641.

Shanklin, J., E. Whittle, and B. Fox. 1994. Ei ght histidine residues are catalytically

essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xyl ene monooxygenase. Biochemistry 33:12787-

12794.

Sinclair, T.R., A.R. Soffes, K. Hinson, S.L. Albrecht, and P.L. Pfahler. 1991. Genotypic variation in soybean nodule number and weight. Crop Science 31:301-304.

Singh, R.J., and T. Hymowitz. 1989. The genomic relationships between Glycine soja Sieb. and Zucc., G. max (L.) Merr., and G. gracilis Skvortz. Plant Breed 103:171-173.

Singh, R.J., H.H. Kim, and T. Hymowitz. 2001. Distribution of rDNA loci in the genus Glycine Willd. Theoretical and Applied Genetics 103:212-218.

Skorupska. 1989. Detection of ri bosomal RNA genes in soybean, glycine max (L.) Merr, by in situ hybridization. Genome 32:1091-1095.

Smyth, S. and P.W.B. Phillips. 2002. Pr oduct differentiation alte rnatives: Identify preservation, segregation, and tr aceability. AgBioForum 5(2):30-42.

Somerville, C. and J. Browse. 2000. Lipi ds. Pages 456-527 in Biochemistry and Molecular Biology of Plants. Buchanan, B., W. Gruissem, and R. Jones (eds.). American Society of Plant Physiologists, Waldorf, Maryland.

Sonka, S., Bender, K.l. and Fisher, D.K. 2004 Economics and Marketing. Pages 919- 948 in Soybeans: improvement, production, and uses. Boerma. H.R., and J.E. Sprecht (eds). American Society of Agronomy, Crop Science Societ y of America, Soil Science Society of America, Madison, Wisconsin.Monsanto Company 09-SY-200U Page 195 of 395 Soyatech. 2008. Statistics. Soya an d Oilseed Bluebook, Manitoba, Canada.

Spector, A.A. 1999. Essentiality of fatty acids. Lipids 34:S1-S3.

Stalker, D.M., C.M. Thomas, and D.R. Helinsk. 1981. Nucleotide sequence of the region of the origin of replicatio n of the broad host range plas mid RK2. Molecular Genetics and

Genomics 181:8-12.

Stanley-Samuelson, D.W., R.A. Jurenka, C. Cr ipps, G.J. Blomquist, and M. de Renobales. 1988. Fatty acids in insects: Composition, me tabolism, and biological significance. Archives of Insect Biochemistry and Physiology 9:1-33.

Steffen, D. 2004. Soybean. U.S. Patent 200400321.

Stumpf, P.K. 1980. Biosynthesis of saturated and unsaturated fatty-a cids. Pages 177-204 in The Biochemistry of Plants: A Comprehens ive Treatise, Vol. 4. Lipids: Structure and Function. Stumpf, P.K. and E.E. Conn. (e ds.). Academic Press Inc., New York.

Sundstrom, F.J., J. Williams, A. Van De ynze, and K.J. Bradford. 2002. Identity preservation of agricultural commodities. Division of Agricultural and Natural Resources Publication 8077. University of California-Oakland.

Sutcliffe, J.G. 1978. Nucleotide sequence of the ampicillin resistance gene of Escherichia

coli plasmid pBR322. Proceedings of the National Academy of Sciences USA 75:3737-

3741.

Sunderland, J.P. and G.M. Poppy. 2005. Quantif ying exposure. Pages 186-208 in Gene Flow from GM Plants. Poppy, G.M. and M. J. Wilkinson (eds.). Blackwell Publishing, Ames, Iowa.

TCM. 2009. Nightshade: Threat to ha rvest and export. Top Crop Manager. http://www.topcropmanager.com/index.php?op tion=com_content&task=view&id=2507 &Itemid=182 [Acacessed May 28, 2009].

Thomson, C.J. 1990. The market for fish meal and oil in the United States: 1960-1988 and future prospects. CalCOFl Rep 31:124-131.

Traitler H., H. Winter, U. Richli, Y. Ingenbleek. 1984. Characterization of gammalinolenic acid in Ribes seed. Lipids 19:923-928.

UK. 2009. Specialty soybeans. University of Kentucky College of Agriculture Cooperative Extension Service.

Ursin, V., B. Froman, J. Gonzales, S.E. Scr een, F. Dong, and T. Larosa. 2008. Fatty acid desaturases from primula. U.S. Patent Application.Monsanto Company 09-SY-200U Page 196 of 395 Ursin, V., T. Voelker, and B. Froman. 2006. Fatty acid desaturases from fungi. U.S. Patent Application.

USDA. 1986. Coordinated Framework for Re gulation of Biotechnology. United States Department of Agriculture, Washington, D.C.

USDA-APHIS. 2006. Soybean. United States De partment of Agriculture, Washington,

D.C.

USDA-APHIS. 2008. Monsanto Company: Availab ility of determination of nonregulated status for corn genetically engineered for insect resistance. 73 FR 43202-43203.

USDA-ERS. 2006. Forage soybean cultivars: a source of high protein livestock feed. United States Department of Agriculture, Washington, D.C.

USDA-ERS. 2006. Soil Management and Conser vation: Crop rotation system use for major crops. http://www.ers.usda.gov/publi cations/arei/eib16/chap ter4/4.2 [Accessed Mar. 16, 2009].

USDA-ERS. 2008. Agricultural Biotechnology: Adoption of Biotechnology and its Production Impacts. http://www.ers.usda.gov/ briefing/biotechnology/chapter1.htm [Accessed Feb. 10, 2009]

USDA-NASS. 2006a. Crop production 2005 summ ary, United States Department of Agriculture, Washington, D.C.

USDA-NASS. 2006b. Quick Stats (Soybean): 1996-2005 Reports. United States Department of Agriculture National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2007a. Acreage 2007 (June repo rt). United States Department of Agriculture National Agricultural St atistics Service, Washington, D.C.

USDA-NASS. 2007b. Agricultural chemical usage: 2006 field crops summary. United States Department of Agricu lture National Agricultural Statistics Service, Washington,

D.C.

USDA-NASS. 2009. Soybeans: State and Country Statistics. United States Department of Agriculture National Agricultural St atistics Service, Washington, D.C. http://www.nass.usda.gov/QuickStats/inde xbysubject.jsp?Text1=&site=NASS_MAIN&s
elect=Select+a+State&Pass_name=&Pass _group=Crops+%26+Plants&Pass_subgroup=F
ield+Crops.

Velasco, L. and F.D. Goff man. 1999. Chemotaxonomic signi ficance of fatty acids and tocopherols in Boraginaceae. Phytochemistry 52:423-426.Monsanto Company 09-SY-200U Page 197 of 395 Voet, D. and J. Voet. 1995. Fatty Acid Bios ynthesis. Pages 680-687 in Biochemistry. John Wiley & Sons, Inc., New York.

Wang, Q. and P. Dubois. 2004. Seed specific 7S α promoter for expressi ng genes in plants.

U.S. Patent.

Warude, D., K. Joshi, and A. Harsul kar. 2006. Polyunsaturated fatty acids: Biotechnology. Critical Review s in Biotechnology 26:83-93.

Watkins, B.A. 1991. Importance of essential fatty acids and their derivatives in poultry.

Journal of Nutrition 121:1475-1485.

Watkins, B.A. 1995. Biochemical and physio logical aspects of pol yunsaturates. Poultry

and Avian Review 6:1-18.

Way, M.O. 1994. Status of soybean insect pe sts in the United States. Pages 15-18 in Handbook of Soybean Insect Pests. Higley, L.G. and D.J. Boethel (eds.). The Entomological Society of America, Lanham, Maryland.

Weber, C.R. and W.D. Hanson. 1961. Natural hybridization with and without ionizing radiation in soybeans. Crop Science 1:389-392.

Webster, T.M., M. Patterson, J. Everest, J. Ferrell, B. Brecke, A.S. Culpepper, E.P. Prostko, J.D. Green, J.R. Martin, E. Webster, S. Kelly, J. Griffin, D. Sanders, J. Byrd, A. Kendig, A. York, D. Jordan, L. Fisher, C. Medlin, D. Murray, J. Norsworthy, J. Chapin, L. Nelson, and L. Steckel. 2005. Weed survey : Southern states 2005: Broadleaf crops section (cotton, peanut, soybean, tobacco, and forestry). Pages 291-306 in Southern Weed Science Society Proceedings.

Wiener, M.C. 2004. A pedestrian guide to me mbrane protein crystallization. Methods

34:364-372.

Woodworth, C.M. 1922. The extent of natura l cross-pollination in soybeans. Agronomy

Journal 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. Canadian Journal of Plant Pathology 23:122-131.

Xing, A.Q., Z.Y. Zhang, S. Sa to, P. Staswick, and T. Clemente. 2000. The use of the two T-DNA binary system to derive ma rker-free transgenic soybeans. In Vitro Cellular & Developmenal Biology - Plant. 36:456-463.

Yamazaki, K.F.M., T. Hamazaki, S. Yano, and T. Shono. 1992. Comparison of the Conversion Rates of alpha-linol enic aid [18:3(n-3)] to longe r polyunsaturated fatty acids

in rats. Biochimica et Biophysica Acta 1123:18-26.Monsanto Company 09-SY-200U Page 198 of 395 York, A.C., J.B. Beam, and A.S. Culpe pper. 2005. Control of volunteer slyphosateresistant soybeans in cotton. Journal of Cotton Science 9:102-109.

Zhang, L.X., S. Kye-boahen, J. Zhang, a nd C.E. Watson. 2004. Redefining zones of adaptation of soybean maturity groups in the U.S. 2004 An nual Meeting Abstracts (CDRom). ASA, CSSA, and SSSA, Madison, Wisconsin.

Zollinger, R.K. 2005. North Dakota Weed Control Guide. http://www.ag.ndsu.edu/weeds/w253/w253w.htm [Accessed June 28, 2006].Monsanto Company 09-SY-200U Page 199 of 395 APPENDICES

Appendix A. USDA Notifications

Field trials of MON 87769 we re conducted in the U.S. since 2001. The protocols for these trials include field performance, breeding and observation, agronomics, and generation of field materials and data necessary for this petition. In addition to the phenotypic assessment data provided for M ON 87769, observational data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2007-2008 seasons, are still in prepar ation. A list of tr ials conducted under

USDA notification and the status of the final reports for these trials are provided in

Table A-1.Monsanto Company 09-SY-200U Page 200 of 395 Table A-1. USDA Notification Approve d for MON 87769 and Status of Trials

Conducted under These Notifications

USDA # Effective Date Release Site (State) Trial Status

2003 Field Trials

03-225-02n 9/12/2003 PR(2) Submitted to USDA

03-325-02n 1/21/2004 HI(1) Submitted to USDA

2004 Field Trials

04-035-02n 3/12/2004 IA(3), IL(9), KS(3) Submitted to USDA

04-035-09n 3/26/2004 IA(2), IL(2) Submitted to USDA

04-090-02n 4/29/2004 IA(2), IL(3) Submitted to USDA

04-174-01n 8/11/2004 PR(2) Submitted to USDA

2005 Field Trials

05-035-01n 3/10/2005 IA(2), IL(10) Submitted to USDA

05-035-02n 3/10/2005 IL(5), IN(2), KS(8) Submitted to USDA

05-035-03n 3/10/2005 IA(12) Submitted to USDA

05-035-04n 3/10/2005 IA(2), IL(1 1), IN(2) Submitted to USDA

05-046-02n 3/28/2005 IL(1) Submitted to USDA

05-073-15n 5/11/2005 IA(2), IL(10), IN(2), WI(1) Submitted to USDA

05-131-05n 6/15/2005 PR(2) Submitted to USDA

05-196-01n 8/17/2005 HI(1) Submitted to USDA

05-217-03n 9/28/2005 PR(3) Submitted to USDA

05-245-01n 10/3/2005 PR(1) Submitted to USDA

05-299-09n 1/4/2006 HI(3) Submitted to USDA

2006 Field Trials

06-026-05n 2/27/2006 PR(2) Submitted to USDA

06-027-03n 4/5/2006 IA(7), IL(5 ), IN(2) Submitted to USDA

06-027-05n 2/27/2006 IL(8), KS(5) Submitted to USDA

06-037-06n 3/14/2006 IL(3) Submitted to USDA

06-045-12n 5/18/2006 HI(5) Submitted to USDA

06-045-19n 5/18/2006 PR(3) Submitted to USDAMonsanto Company 09-SY-200U Page 201 of 395 Table A-1 (continued). USDA Notificati on Approved for MON 87769 and Status of

Trials Conducted under These Notifications

USDA # Effective Date Release Site (State) Trial Status

06-045-21n 3/22/2006 IL(1) Submitted to USDA

06-045-22n 4/26/2006 IA(2), IL(3), IN(3), KS(1), MI(1), MO(3), NE(1), WI(1) Submitted to USDA

06-069-10n 4/24/2006 PR(4) Submitted to USDA

06-074-05n 5/15/2006 AR(1), IA(2), IL(3), MI(1), NE(1), OH(1), WI(1) Submitted to USDA

06-076-12n 5/5/2006 IA(1), IL(2), KS(1), KY(1), MN(1), NE(1), OH(1), SD(1) Submitted to USDA

06-101-02n 5/30/2006 IA(1), IL(3) Submitted to USDA

06-118-04n 5/9/2006 IA(2) Submitted to USDA

06-137-116n 6/7/2006 IA(1) Submitted to USDA

06-195-102n 9/11/2006 PR(3) Submitted to USDA

06-271-101n 10/28/2006 IA(2), IL(1), IN(2), MO(3) Submitted to USDA

06-319-101n 12/15/2006 PR(4) Submitted to USDA

2007 Field Trials

07-019-101n 2/18/2007 IA(1), IL(10), IN(3), MO(1) Submitted to USDA

07-019-104n 3/17/2007 IA(7), KS(6) Submitted to USDA

07-029-103n 3/18/2007 IA(1), MN(2), PR(4) Submitted to USDA

07-031-115n 3/21/2007 IL(2) Submitted to USDA

07-039-122n 4/5/2007 IA(1), IL(2), IN(1), KS(1), KY(1), MN(1), NE(1), SD(1) Submitted to USDA

07-046-108n 4/1/2007 IL(2) Submitted to USDA

07-052-104n 4/10/2007 IL(1), IN(2), MI(1), MN(1), WI(2) Submitted to USDA

07-058-101n 4/4/2007 AR(1), IA(1), IL(2), IN(1), MI(1), NE(1), OH(1), PA(1), WI(2) Submitted to USDA

07-094-108n 5/4/2007 IL(1) Submitted to USDA

07-095-101n 5/5/2007 IA(1) Submitted to USDAMonsanto Company 09-SY-200U Page 202 of 395 Table A-1 (continued). USDA Notificati on Approved for MON 87769 and Status of

Trials Conducted under These Notifications

USDA # Effective Date Release Site (State) Trial Status

07-099-102n 5/10/2007 IA(1), IN(1), MN(3), NE(1) Submitted to USDA

07-159-101n 7/8/2007 PR(1) Submitted to USDA

07-283-101n 12/3/2007 PR(2) Submitted to USDA

07-296-101n 12/3/2007 PR(1) Submitted to USDA

2008 Field Trials

08-038-101n 3/25/2008 IA(5), KS(2) Submitted to USDA

08-039-108n 3/9/2008 IA(2), IL(6), IN(2) Submitted to USDA

08-045-102n 3/15/2008 IL(2) Submitted to USDA

08-059-106n 4/1/2008 IA(2), MN(4), ND(1), SD(1), WI(2) Submitted to USDA

08-065-102n 4/4/2008 MI(1), WI(1) Submitted to USDA

08-078-108n 4/17/2008 IL(1) Submitted to USDA

08-079-101n 4/17/2008 IA(3) Submitted to USDA

08-084-102n 4/24/2008 IA(1), NE(1) Submitted to USDA

08-084-107n 4/24/2008 IL(5) Submitted to USDA

08-086-103n 4/25/2008 IL(2), IN(1) Submitted to USDA

08-098-101n 5/7/2008 IA(1), IL(1), IN(1), NE(1) Submitted to USDAMonsanto Company 09-SY-200U Page 203 of 395 Appendix B. Materials and Methods Us ed for Molecular Analyses of MON 87769

B.1. Materials

The DNA used in the molecular analyses wa s isolated from leaf tissue grown from MON 87769 seed (seed lot number GLP-0509-16624-S). Additional DNA was extracted from leaf tissue grown from various ge nerations of MON 87769 seed and used in generational stability analyses. The control DNA was is olated from leaf tissue from conventional soybean with the same ge netic background (seed lot number GLP-0509- 16625-S). The reference substances incl uded the PV-GMPQ1972 plasmid, probe templates generated from this plasmid, and the size estimation molecular weight standards. As a positive control on Sout hern blots, PV-GMPQ1972 plasmid DNA was digested with a combination of enzymes to produce the banding patterns that were most

relevant to the assessment of the test substa nce digested with appropriate enzyme(s). Probe templates generated from this plas mid also served as positive hybridization controls. Digested plasmi d DNA or probe templates we re added to predigested conventional soybean genomic DNA. The molecular weight standards include the 1 kb DNA Extension Ladder (Invitrogen, Carlsbad, CA) and λ DNA/ Hin d III fragments (Invitrogen) for size estimations on Southern blots. The 100 bp and 500 bp DNA ladders (Invitrogen) were used for size estimations for PCR analyses

.

B.2. Characterization of the Materials

The test and control materials identity were verified by PCR analysis to confirm the presence or absence of MON 87769, except for the materials used in the generational stability analyses. In this analysis the id entity of the materials was confirmed by the generation stability Southern blots themselv es. The stability of the genomic DNA was confirmed in each Southern analysis by obser vation of the digested DNA sample on an ethidium bromide-stained agarose gel.

B.3. DNA Isolation for Southern Blot and PCR Analyses

Genomic DNA samples from MON 87769 and conventional soybean control were isolated from leaf tissue that was processed in to a fine powder using a mortar and pestle under liquid nitrogen. DNA was extracted from the processed seed using a hexadecyltrimethylammonium bromide (CTA B) based method as follows: 2-4 mL equivalent of processed leaf tissue was placed in a 13 mL Sarstedt tube or 50 ml centrifuge tube and ~10 ml of CTAB extrac tion buffer (1.5% CTAB, 75 mM Tris pH 8.0, 100 mM EDTA, 1.05 M NaCl, 0.75% PVP [40K]) and 5-10 μ L of 10 mg/mL RNase was added. The samples were incubated at 65 o C for 40-50 minutes and mixed halfway through the incubation. The samples were co oled to room temperature, and divided

between two tubes. 20 mL of 24:1 chlo roform: isoamyl alcohol was added and the samples were mixed for 2-3 minutes a nd centrifuged for 8-10 minutes at 10,300 x g and

20-25 ° C to separate the aqueous and organic phases. The upper aqueous phase was

transferred to a clean 50 mL centrifuge tube and 20 mL of 100% ethanol was added. The samples were then inverted to mix the samples until the DNA precipitated. The samplesMonsanto Company 09-SY-200U Page 204 of 395 were then centrifuged at 20-25 o C to pellet the DNA, and the supernatant was discarded. The DNA pellet was air dried for ≤ 2 hours, and resuspended in an appropriate volume (250 – 750 mL) of TE buffer (10 mM Tris HC l, pH 8.0, 1 mM EDTA, pH 8.0). To facilitate resuspension of the DNA, additiona l TE buffer was added and/or the solution was heated up to 70 ° C for 1-4 hours. The DNA was stored in a 4 ° C refrigerator, a -20 ° C,

or a -80 ° C freezer.

B.4. Quantification of Genomic DNA

Quantification of DNA samples was perf ormed using a Hoefer DyNA Quant 200

Fluorometer with Roche molecular size ma rker IX as a DNA calibration standard.

B.5. Restriction Enzyme Digestion of Genomic DNA

Approximately 10 or 20 μ g of genomic DNA extracted from the test and control substances were used for restriction enzyme digestions. When digesting genomic DNA

with Lgu I (Fermentas, Hanover, MD) or the enzyme combination Bsr G I and Psh A I (New England BioLabs, Ipswich, MA), Ta ngo Buffer (Fermentas) was used. When digesting genomic DNA with the enzyme combination Lgu I and Bst X I (New England BioLabs), NEbuffer 2 (New England BioLabs) was used. All digests were performed at

55 °C or 37 ° C according to SOP BR-ME-0316-01 in a total volume of approximately

500 μ l using approximately 50-100 units of the appropriate restriction enzyme(s).

B.6. DNA Probe Preparation for Southern Blot Analyses

Probe template DNA containing sequences of plasmid PV-GMPQ1972 was prepared by PCR amplification according to SOP BR-ME-0486-01. Approximately 18-25 ng of each probe template were radiolabeled with 32 P-deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) using the random priming method (RadPrime DNA Labeling System, Invitrogen). Probe locations relative to the genetic elements in plasmid PV-GMPQ1972 are depicted in Figures V-1 and V-2.

B.7. Southern Blot Analyses of Genomic DNA

Digested DNA was separated using 0.8% (w/v) agarose gel electrophore sis. Except for generational stability analyses, DNA samples we re loaded on the gels for a long run and a short run in an effort to provide better reso lution of larger DNA fragments while retaining smaller DNA fragments on the gel. After transferring the DNA to the membrane, Southern blots were hybridized at 65 ° C with the exception of the E9 3 ′ nontranslated /Left Border regions and the T-DNA II Southe rn blots which were hybridized at 60 °C. Multiple exposures of each blot were then generated using Kodak Biomax MS film in conjunction with one Kodak Biomax MS intensifying screen in a -80 ° C freezer.

B.8. DNA Sequence Analyses of the Insert

Overlapping PCR products were generated that span the insert in MON 87769 as well as genomic DNA directly adjacent to the in sert. The PCR analyses were conductedMonsanto Company 09-SY-200U Page 205 of 395 according to SOP BR-ME-0486-01 using 60 ng of genomic DNA template or approximately 0.9 ng plasmid DNA in a 50 μ l reaction volume containing a final concentration of 1.8 mM MgCl2, 0.2 μ M of each primer, 0.2 mM each dNTP, and 1 unit of DNA polymerase mix. The specific DNA polymerase mix used to generate the products was Platinum Taq (Invitrogen, Prod uct A) or Platinum High Fidelity Taq (Invitrogen, Products B, C, D, and E). The amplification of Products A, B and E was performed under the follow ing cycling conditions: 97 ° C for one minute; 34 cycles at

96 ° C for 20 seconds, 55 ° C for 20 seconds, and 68 ° C for 2 minutes; one cycle at 68 ° C for 5 minutes. The amplification of Product C was performed under the following cycling

conditions: 97 ° C for 1 minute; 32 cycles at 96 ° C for 20 seconds, 55 ° C for 20 seconds,

and 68 ° C for 3 minutes and 20 seconds; one cycle at 68 ° C for 5 minutes. The amplification of Products D was performe d under the following cycling conditions: 97 ° C for 1 minute; 36 cycles at 96 ° C for 20 seconds, 55 ° C for 20 seconds, and 68 ° C for 2 minutes and 30 seconds; one cycle at 68 ° C for 5 minutes.

Aliquots of each PCR reaction we re separated on a 1.0 % (w/v ) agarose gel according to

SOP BR-ME-0315-02 and visualized by ethidium bromide staining to verify that the products were of the expected size prior to sequencing. The PCR product was sequenced with the multiple primers used for PCR amplification. In addition, primers internal to the PCR primers were used to sequence other regions of the amplified product. All sequencing was performed by the Monsanto Genomics Sequencing Center using dyeterminator chemistry.Monsanto Company 09-SY-200U Page 206 of 395 Appendix C. Characterization of Pj Δ 6D and Nc Δ 15D Proteins Produced in MON 87769 – Materials, Methods and the Results

C.1. Materials and Methods Used for the Characterization of Pj Δ 6D Protein

C.1.1. Protein Purification

The Pj Δ 6D protein was purified from immature MON 87769 seed prior to initiation of this characterization pla n. The purification of Pj Δ 6D from immature MON 87769 seed was not performed under a GLP study or plan; however, all procedures were documented on worksheets and, where applicable, SOPs we re followed. All oper ations were carried

out at 4 °C unless otherwise indicated.

A total of 11 kg of immature MON 87769 s eed (Orion Lot No. 10002214) was used for the isolation of Pj Δ 6D. The seed was homogenized for me mbrane isolation at a scale of ~1000-2500 g seed per extraction (a total of six runs). The isolated membrane was

solubilized with 2% Fos-choline 12 a nd separated on a 3,000-mL cation exchange chromatography column (a total of three r uns) followed by immuno- affinity purification (a total of 30 runs). The MON 87769-produced Pj Δ 6D protein isolated from the different batches were combined as a final preparati on. After preliminary pur ity and concentration determination, the protein preparation was s ubmitted to Monsanto’s Analytical Protein Standards (APS) program under Orion lot # 10001532.

Each run included the following series of purification steps:

Homogenization and membrane isolation - Approximately 400 g of immature MON 87769 seed was homogenized with a Viking blender (W aring Commercial, Torrington, CT) for 3 min in 4 L of ice co ld homogenization buffer consisting of 100 mM Tris-HCl, pH 8.0, 350 mM NaCl, 5.0 mM DTT, 0.5 mM PMSF, 1 μM leupeptin, and 1.0 mM sodium benzamidine hydrochloride. Afte r filtration of the homogenate through four layers of cheese cloth, the filtrate was centrifuged at 2,000 x g to remove cell debris. The supernatant was centrifuged at 37,000 x g for 1 hr. After the centrifugation, the supernatant was discarded and the pellet re-s uspended in ice cold de-ionized water (DI

H 2 O) at a total protein con centration of approximately 40 mg/mL. The membrane suspension was mixed with an equal volume of 2X Carbonate Wash Buffer (200 mM sodium carbonate, pH 11.5, 2 mM MgCl 2, 0.5 mM PMSF, 1 μM leupeptin, and 1.0 mM benzamidine hydrochloride). Immediately af ter mixing, the mixture was centrifuged at 37,000 x g at 4 °C for 30 min. The supernatant was discarded and the membrane pellets

were stored at -80 °C until use. Extraction was repeated until all the remaining seed was homogenized.

Solubilization and cation exchange chromatography – The frozen membrane pellets (~100 g fresh weight) were thawed and re-sus pended in 5 L of the solubilization buffer [50 mM sodium acetate, pH 5.6, 100 mM NaCl, 1.0 mM DTT

, 10% Glycerol (v/v), 0.5 mM PMSF, and 1 μM leupeptin]. Soli d Fos-choline 12 was added to a final concentration of 2% and the membrane/deterge nt solution was incubated at 4 °C for 2 hr with stirring. The mixtur e was centrifuged at 37,000 x g at 4 °C for 30 min to remove the insoluble fractions. The supernatant was applied onto a SP-Sepha rose column (bedMonsanto Company 09-SY-200U Page 207 of 395 volume ~3.0 L, GE Healthcare, Piscataway, NJ ) that had been previously equilibrated with SP-Buffer A consisting of 50 mM sodium acetate, pH 5.6, 10% gl ycerol (v/v), and 0.1% Fos-choline 12 (w/v). After washing the column with SP-Buffer A, the column was

further washed with 0.1 M NaCl in SP-Buffer A. The bound proteins were then eluted stepwise with 0.5 M NaCl in SP-Buffer A. Fr actions containing the protein of interest were pooled, and stored at -80 ° C.

Immuno-affinity chromatography - Immuno-affinity resin us ed in the purification was prepared by immobilizing anti-Pj Δ 6D peptide IgG to agarose resin using an IgGorientation kit from Pierce (Cat. No. 44990, Rockford, IL), according to the

manufacturer’s instru ctions. The anti-Pj Δ 6D antibody (Lot Numbers: 7580967 and 7580957) was used for immunoaffinity resin prepar ation (total vol. of resin ~28 mL). The immuno-affinity column was equilib rated with Immunoaffinity Binding and Washing (IABW) Buffer consisti ng of 1X PBS, pH7.2, 1mM MgCl 2, 10% glycerol (v/v) and 0.1% Fos-choline 12 (w/v). Before a pplying the samples to the immuno-affinity column, the pH of the SP-Sepharose-purified protein preparation was adjusted to pH 7.4 with 2.0 M potassium phosphate, pH 8.5. The flow through fraction was re-applied to the column and the re-application of the flow th rough was repeated three times. The column was washed extensively with the IA washing buffer and Pj Δ 6D was eluted with 0.1 M Glycine, pH 2.8 containing 1 mM MgCl 2, 10% glycerol (v/v) a nd 0.1% Fos-choline 12 (w/v). The pooled protein fraction (~140 mL ) was neutralized to pH 5.5 with 2.0 M MES, pH 10, and stored at -80 ° C.

Protein concentration - To concentrate the IA-purifie d protein preparation, about 250 – 500 mL of the eluted fraction was loaded onto a 2-mL SP-Sepharose column (GE Healthcare, Piscataway, NJ) which had been pr eviously equilibrated with SP-Buffer A. The flow rate was typically 4.0 mL/min. The protein was eluted with 0.5 M NaCl in SPBuffer A and the fractions containing Pj Δ 6D protein were pooled and stored at -80 ° C. A total of eight batches were prepared.

Final purification by Hydroxyapatite chromatography – All eight batches of the concentrated Pj Δ 6D preparations were combined and applied onto a 5-mL hydroxyapatite column (Type I ceramic hydroxyapatite, BioRad, Hercules, CA). While the majority of the contaminant proteins bound to the colum n, the flow through fraction was collected and saved as the final Pj Δ 6D protein preparation. Af ter preliminary purity and concentration determination, the final pr eparation was submitted to Monsanto APS program under the Orion lot # 10001532.

C.1.2. Determination of Protein Concentration

The concentration of the MON 87769-produced Pj Δ 6D protein sample was determined by amino acid analysis (AAA) using AccQ-T ag derivatization (Waters Corporation, Milford, MA), a pre-column derivatization method which allows for high sensitivity

fluorescent detection of amino acids. In or der to avoid the interference from buffer components during protein hydrolys is, protein samples were pr ecipitated using ethanol. The sample preparation was as follows: in a hydrolysis tube (~300 μ l), approximately 1 μg of a protein sample was mixed with 200 μ L of chilled 95% ethanol. After incubation overnight at -20 oC, each sample was centrifuged for 45 min at 2-4 °C atMonsanto Company 09-SY-200U Page 208 of 395 12,000 rpm in a microcentrifuge to precipitate proteins. The supernatant was removed and discarded. Each sample was then washed twice sequentially with 100 μ L of both

chilled acetone and water. Repli cates of MON 87769-produced Pj Δ 6D protein, a hydrolysis blank, four dilutions of a calibrati on standard (NIST), and a BSA control were analyzed. An internal calibrant, α -aminobutyric acid, was included in all non-blank samples. All samples were evaporated to dryness in hydrolysis t ubes using a Speed-Vac concentrator. A 500 μL volume of hydrolysis solution (6 N HCl, 1% phenol) was added

and the tubes were transferred to a vacuum chamber. Samp les were hydrolyzed for 90 min at 150±2 °C under vacuum. After cooling, the vacuum was released and the contents of the tube were evaporated to dryness usi ng a Speed-Vac concentrator. A 20 μL volume of reconstitution solution (20 mM HCl) was a dded and tubes were vortexed to resuspend the sample. A 60 μl volume of AccQ-Fluor Borate Buffer and a 20 μl volume of AccQFluor reagent were added sequentially to each vial with vortexing after each addition. The samples were transferred individually to autosampler vials, capped, and heated at 55 °C for 10 min. Samples were analyzed using a 2695 Separation Module (Waters Corp.) in conjunction with reverse-phase C-18 column for separation of AccQ-Tag derivatized amino acids. Chromatographic da ta were collected using Atlas software (Thermo Electron Corp.).

C.1.3. Protein Identity

Immunoblot Analysis

Immunoblot analysis was performed to confirm the identity of Pj Δ 6D protein purified

from MON 87769. Based on the concentratio n and purity, aliquots of the purified Pj Δ 6D protein preparation were diluted in 50 mM sodium acetate, pH 5.6, 10% glycerol and 0.1% Fos-choline 12 to give final amounts of 4, 6, 8, 10 and 12 ng, respectively. Samples were mixed with 5X Loading Bu ffer (LB) (312.5 mM Tris-HCl, 25% (v/v) 2-mercaptoethanol, 10% (w/v) sodium dodecy l sulfate, 0.025% (w/v) bromophenol blue, 50% (v/v) glycerol, pH 6.8 and loaded dire ctly onto gels without boiling. Membrane proteins in (a portion) the presence of de tergents tends to self -associate via hydrophobic contact surfaces, forming irreversible nonspecific aggregates (McGregor et al

. , 2003; Sagne et al. 1996). This results in a smeared appearance on SDS-PAGE. Heating of the samples causes an increase in this behavior. As a result, none of th e protein of interest (POI) samples was heated before analysis using SDS-PAGE. The samples were then separated on SDS-PAGE using 4-20% precast polyacrylamide gels (Invitrogen, Carlsbad, CA) at a constant voltage of 50 V for 25 min followed by 160 V for 70 min. Proteins in the gel were electrotransferred to PVDF membrane (Invitrogen, Carlsbad, CA) at a constant voltage of 25 V for 90 min. Pre-st ained molecular weight markers (Precision Plus protein standard, BioRad, Hercules, CA) were used to c onfirm the electrotransfer of proteins and estimate the size of the immunoreactive bands.

Following electrotransfer, the membrane was blocked for 30 min with 4% (w/v) NFDM in PBST. The membrane was then probe d with a 1:5000 dilution of goat anti-Pj Δ 6D antibody (APS Lot No. 7580957 ̧ Orion number 10000821) in PBST cont aining 2% (w/v) NFDM for 1 hr. The IgG was purified fr om crude antiserum using immobilized Pj Δ 6D protein and was used at dilu tion of 1:5000. Rabbit anti-goat IgG conjugated with horseMonsanto Company 09-SY-200U Page 209 of 395 radish peroxidase (Pierce, Rockford, IL) was used as secondary anti body at a d ilution of 1:10,000. Excess antibody was removed using fi ve 10 min washes with PBST. Finally, the membrane was probed with horseradish peroxidase-conjugated rabbit anti-goat IgG (Pierce, Rockford, IL) at a dilution of 1: 10,000 in PBST containing 2% (w/v) NFDM for 60 min. Following five 10 min washes with PBST, immunoreactive bands were visualized using the ECL detection system (GE Healthcare, Piscat away, NJ) and exposed (30 and 60 sec) to Hyperfilm ECL (G. E. Healthcare, Piscataway, NJ). Films were developed using a Konica SRX-101A automate d film processor (Tokyo, Japan). The

MW of the immunoreactive bands was estimat ed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA).

C.1.4. N-Terminal Sequencing

Edman degradation was used to confirm the N-terminal identity of the MON 87769- produced Pj Δ 6D.

Protein Blot for N-Terminal Analysis

An aliquot of M ON 87769-produced Pj Δ 6D protein was removed from storage and

mixed with 5X LB to a final concentration of 0.42 μg/μl and loaded in five lanes at 10.5 μg per lane. Samples were loaded direc tly onto gels without boiling. SDS-PAGE was conducted using 4-20% precas t polyacrylamide gels (Invi trogen, Carlsbad, CA). Electrophoresis was carried out at constant voltage of 50 V for 25 min followed by 160 V for 70 min. Proteins in the gel were electr otransferred to a PVDF (Invitrogen, Carlsbad, CA) membrane in the electrotransfer buffe r containing 10 mM CAPS, pH 11, and 10% methanol at a constant voltage of 26 V fo r 120 min. Pre-staine d molecular weight markers (Precision Plus protei n standard, BioRad, Hercules, CA) were loaded in parallel to confirm electrotransfer of protein to the membrane and estimate the size of the stained

bands observed. The blot was stained with Coomassie Blue R-250 (BioRad, Hercules, CA) to visualize the protein of interest.

N-Terminal Sequencing

Following electroblotting and staining, the band corresponding to MON 87769-produced Pj Δ 6D protein was excised from the blot and N-terminal sequence analysis was performed for 15 cycles using automated Ed man degradation chemistry (Hunkapiller and Hood, 1983). An Applied Biosystems 494 Procise Sequencing System with 140C Microgradient system and 785 Programmable Ab sorbance Detector and Procise Control Software (version 1.1a) were used. Chromat ographic data were collected using Atlas software (version 3.59a, LabS ystems, Altrincham, Cheshire, England). A PTH-amino acid standard mixture (Applied Biosystems, Foster City, CA) was used to chromatographically calibrate the instrument for each analysis. This mixture served to verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times. A control protein (10 pmole β -lactoglobulin, Applied Biosystems) was analyzed before and after the analysis of the Pj Δ 6D protein, to verify that the sequencer met performance criteria for repetitive yield and sequence identity.Monsanto Company 09-SY-200U Page 210 of 395 C.1.5. MALDI-TOF Tryptic Mass Map Analysis

MALDI-TOF mass spectrometry was used to confirm the identity of the MON 87769- produced Pj Δ 6D protein.

Protein Gel for Tryptic Mass Map Analysis

A 100 μ L aliquot of MON 87769-produced Pj Δ 6D protein was removed from storage,

mixed with 5X LB to a final concentration of 0.42 μg/μL and loaded in five lanes at 10.5 μg per lane. Samples were loaded direc tly onto gels without boiling. SDS-PAGE was conducted using 4-20% precas t polyacrylamide gels (Invi trogen, Carlsbad, CA). Electrophoresis was carried out at constant voltage of 50 V for 25 min followed by 160 V for 70 min. The gel was stained with Coom assie blue dye R-250 (B ioRad, Hercules, CA) to visualize the protein of interest. The band correspond ing to the MON 87769-produced Pj Δ 6D protein was excised from fi ve lanes of the gel, destai ned, reduced, and alkylated. Briefly, each gel band was destaine d for 30 min by incubation in 100 μ L of destaining solution (40% methanol, 10% acetic acid in wa ter) in a microfuge tube. This step was repeated four times with the final destaini ng step proceeding for 60 minutes. Following destaining, the gel band was incubated in 100 μ L of 100 mM ammonium bicarbonate buffer for 30 min at room temperature. The protein was reduced in 100 μ L of 10 mM

DTT solution for 2 h at 37 ° C. The protein was alkylat ed by the addition of 100 μ L of 20 mM iodoacetic acid. The alkyl ation reaction was allowed to proceed at room temperature

for 20 min in the dark. Gel band was incubated in 200 μ L of 25 mM ammonium bicarbonate buffer for 15-25 min at room temp erature. This step was repeated two additional times, following which the gel band was dried using a Speed Vac concentrator (Ramsey, MN). A single ge l band was rehydrated with 20 μ L of 0.02 μg/μL trypsin in 25 mM ammonium bicarbonate, 10% acetonitrile, and was incubated for about 1 h at room temperature, after whic h excess liquid was removed and the sample was incubated overnight at 37 ° C in 40 μ L of 25 mM ammonium bicarbonate, 10% acetonitrile. The following day, the sample was sonicated for 5 min, and the supernatant was transferred to a new tube and dried using a Speed Vac concen trator (Extract 1). The gel band was resuspended in 30 μ L 60% acetonitrile, 0.1% trifl uoroacetic acid, 0.1% octyl- β -Dglucopyranoside solution, and sonicated for 5-10 min. After transfer of the supernatant to a new tube, this step was repeated one time, and the combined supernatants were dried using a Speed Vac concentrator (Extract 2). Extracts 1 and 2 were each resuspended in

20 μ L 0.1% trifluoroacetic acid and then dried using a Speed Vac con centrator. Extract 1 was resuspended in 5 μ L of 50% acetonitrile, 0.1% trifluoroacetic acid, while Extract 2 was re-suspended in 10 μ L of the same solution. Each extract was sonicated for 5 min.

C.1.6. MALDI-TOF Mass Analysis

Mass spectral analyses were performed as follows. Mass calibration of the instrument was performed using an external peptide mi xture (CalMix 2; Applied Biosystems). Extract 1 and Extract 2 samples (0.3 μ L) were co-crystallized with 0.70 μ L each of the following matrix solutions: dihydroxybenzoic acid (DHB), α -cyano-4-hydroxy cinnamic acid ( α -cyano), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic aci d) at separateMonsanto Company 09-SY-200U Page 211 of 395 locations on the analysis plate. The samples in DHB matrix were analyzed in the 300 to 7500 Da range using 200 shots at a laser intensity setting of 3001. Th e laser intensity setting is a unit-less MALDI-TOF instrume nt-specific value. The samples in α -cyano matrix were analyzed in the 300 to 7500 Da range using 200 shots at a laser intensity setting of 2601. The samples in sinapinic aci d matrix were analyz ed in the 850 to 7500 Da range using 200 shots at a laser intensit y setting of 3200. Protonated (MH+) peptide masses were observed monoisotopically in refl ector mode (Aebersold, 1993; Billeci and Stults, 1993), except above 5000 Da, where mass-averaged values were used. GPMAW32 software (Applied Biosystems) was used to generate a theoretical trypsin digest of the expected Pj Δ 6D protein sequence based up on the nucleotide sequence. Masses were calculated for each theoretical peptide and compared to the raw mass data. Experimental masses (MH+) were assigned to peaks in the 500 to 1000 Da range if there were two or more isotopically resolved peak s, and in the 1000 to 8000 Da range if there were three or more isotopically resolved peaks in the spectra. Peaks were not assessed if the peak heights were less th an approximately twice the ba seline noise, or when a mass could not be assigned due to ove rlap with a stronger signal ± 2 Da from the mass analyzed.

C.1.7. Molecular Weight and Puri ty Estimation – SDS-PAGE

Aliquots of the MON 87769-produced Pj Δ 6D protein were dilute d with Milli-Q water

and mixed with 5 X LB to a final protein concentration of 0.2 μ g/ μ L. The MON 87769- produced Pj Δ 6D protein was analyzed in duplicate at 2, 3, and 4 μ g of total protein per lane. Molecular weight standards were heat ed, though the test samples were not, and all samples were applied to a pre-cast 4-20 % polyacrylamide gradient 10-well gel (Invitrogen, Carlsbad, CA). Pre-stained mo lecular weight markers (BioRad, Hercules, CA) were loaded in parallel. Electrophoresis was performed at a constant voltage of 120 V for 60 min followed by 180 V for 30 mi nutes. Proteins we re stained using

Colloidal Coomassie Brilliant Blue stain (Sigma-Aldrich, St. Louis, MO).

Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). The molecular weight markers were used to estimate the appa rent molecular wei ght of the MON 87769- produced Pj Δ 6D protein. For the purity evaluati on, all visible bands within each lane were quantified. The purity and estimat ed molecular weight of the MON 87769- produced Pj Δ 6D protein were reported as the av erage of the six values obtained by densitometric analysis.

C.1.8. Glycosylation Analysis

The GE Healthcare Glycosylation Detection Mo dule (Piscataway, NJ) was used to detect carbohydrate covalently bound to the MON 87769-produced Pj Δ 6D proteins after it was resolved by SDS-PAGE and electrotransfer red onto PVDF membrane. The kit utilizes the specific labeling of biotin to the bound carbohydrate moiety followed by probing with streptavidin-conjugated horse ra dish peroxidase (Strep-HRP) and detection by ECL. The biotin labeling consists of two steps: pretreatment of the glycoprotein on the membrane and covalent conjugation of biotin to the carbohydrate moiety of the glycoprotein. The pre-treatment oxidizes hydroxyl groups of the carbohydrate mo ieties in the glycoproteinsMonsanto Company 09-SY-200U Page 212 of 395 to aldehydes which then react with biotin, re sulting in covalent conjugation of biotin. This biotin-labeling procedure is specific for the detection of conjugation with carbohydrates and sensitive due to the use of biotin’s high affinity binding partner, avidin, in an enzyme linked assay.

Aliquots of 0.5 μg and 1.0 μg of the purity-corrected Pj Δ 6D protein in SP-buffer A were

mixed with 5 X LB and loaded onto a 4-20% precast polyacrylamide gel (Invitrogen, Carlsbad, CA). Along with the Pj Δ 6D samples, 25, 50 100 and 200 ng of the glycosylated control protei n, transferrin, were load ed in parallel. The Pj Δ 6D samples were not heated, but the transferrin cont rols were boiled prior to the loading. Electrophoresis was carried ou t at constant voltage of 90 V for 30 min followed by 159 V for 60 min. Proteins in the gel were electr otransferred to a PVDF membrane (Invitrogen, Carlsbad, CA) at a constant voltage of 25 V for 90 min. Pre-stai ned molecular weight markers (Precision Plus Dual Color protein st andard, BioRad, Hercules, CA) were loaded in parallel to confirm electrotr ansfer of protein to the memb rane and estimate the size of the stained bands observed. Transferrin (~76 – 81 kDa, Sigma-Aldrich, St. Louis, MO) was loaded in parallel as a pos itive control of glycosylated pr otein in series of dilution.

The labeling and detection of carbohydrat e was carried out according to the manufacturer’s instructions and all the reag ents except PBS were provided by the kit. Following electrotransfer to 0.45 μ m PVDF membrane, the blot was incubated first in 30 mL of PBS for 10 min, then in 10 mM NaIO 4 for 20 min in darkness. The membrane

blot was then rinsed twice with 15 mL PBS followed by three washes for 10 min each. To label the carbohydrate moiety on the protein with biotin, the membrane was incubated with biotin-hydrazide for 45 min followed by tw o rinses and three 10 min PBS washes as

described above. The membrane was further blocked for 60 minutes with 5% blocking reagent in PBS followed by two rinses and three 10 min PBS washes. Strep-HRP at

1:6000 dilution was overlaid onto the membrane and incubated at room temperature for

30 min. After two rinses and three 10 mi n PBS washes, the membrane was then developed with ECL detection reagents by mixing 2 mL of Reagent 1 and 2 mL of Reagent 2. After 1 min in cubation, the excess detection solution was removed by blotting with paper towel and the blot was e xposed to Hyperfilm EC L (G. E. Healthcare, Piscataway, NJ). The film was develope d using a Konica SRX-101A automated film processor (Tokyo, Japan). The blot images were captured usin g a Bio-Rad GS-800 densitometer with the supplied Quantity On e software (version 4.4.0, Hercules, CA).

C.1.9. Storage Stability

The short-term stability of the MON 87769-produced Pj Δ 6D protein was evaluated by comparing the purity and molecular weight valu es before and after storage for 29 days in a 4 oC refrigerator, and in - 20 oC, and -80 oC freezers. At th e end of storage, aliquots of the Pj Δ 6D protein samples were removed an d subjected to SDS-PAGE followed by staining, and subsequent purity and MW estimation. The MON 87769-produced Pj Δ 6D protein was analyzed in duplicate at 2, 3, and 4 μ g of total protein per lane. MW standards were heated, though the test samples were not, and all samples were applied to a pre-cast 4-20% polyacrylamide gradient 10- well gel (Invitrogen, Carlsbad, CA). Prestained molecular weight markers (BioRad, He rcules, CA) were loaded in parallel.Monsanto Company 09-SY-200U Page 213 of 395 Electrophoresis was performed at a constant voltage of 120 V for 20 min followed by

180V for 60 minutes. Proteins were staine d using colloidal Coomassie Brilliant Blue stain (Sigma-Aldrich, St. Louis, MO).

Analysis of each gel was performed usi ng a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). The molecular weight markers were used to estimate the appa rent molecular wei ght of the MON 87769- produced Pj Δ 6D protein. The protein samples we re considered to have undergone degradation if a >10% decrease in purity and/or molecular weight was observed relative to the value determined on Day 0.

C.2. Materials and Methods Used for the Characterization of Nc Δ15 D Protein

C.2.1. Protein Purification

The Nc Δ 15 protein was purified from immature MON 87769 seed prior to initiation of this characterization plan. The purification was not pe rformed under a GLP study or plan; however, all procedures were documen ted on workstheets and, where applicable, SOPs were followed. All operations were carried out at 4 °C unless otherwise indicated.

A total of 13.5 kg of immature MON 87769 se ed was used for th e isolation of Nc Δ 15D protein in two separate batc hes. Approximately 2.5 kg of seed (Seed Lot No. GLP-0509- 16624-S) were homogenized in four batches (5 00-800 g seed per batch) for the isolation

of membrane fractions. The membrane was then solubilized in 2% Fos-choline 12 and separated on a 500-mL cation exchange chromatography column followed by immunoaffinity purification. The solubilization and cation exchange chromatography were performed in three different runs. The remaining 11 kg of seed (Seed Lot No. G828102A) was homogenized for membrane isol ation at a larger scale with 1,000-2,500 g of seed per extraction (a to tal of six runs). The solubilized membranes were then separated on a 3,000-mL cation ex change chromatography column (a total of three runs) followed by immuno-affinity purificati on (a total of 30 runs). The Nc Δ 15D protein preparations by both small-scale and large-s cale operations were combined. Although at different scales, conditions (e.g., buffer to sample ratio) used to purify Nc Δ 15D protein at both small and large scales were proportionally comparable and therefore practically identical. After preliminary purity and concentration determination of the purified

Nc Δ 15D preparation, the final preparation was submitted to Monsanto APS program

under Orion lot # 10001516.

Each run included the following series of purification steps:

Homogenization and membrane isolation

In a 5-L Viking Blender (Waring Commercial , Torrington, CT), appr oximately 400 g of immature MON 87769 seed was homogenize d for 3 min in 4,000 mL of ice-cold homogenization buffer consisting of 100 mM Tris-HCl, pH 8.0, 350 mM NaCl, 5.0 mM DTT, 0.5 mM PMSF, 1 μ M leupeptin, and 1.0 mM sodium benzamidine hydrochloride. The process was repeated to homogenize the required amount seed (t ypically 1,000 g to 2,000 g seed per extraction). After filtration of the homogenate through four layers ofMonsanto Company 09-SY-200U Page 214 of 395 cheese cloth, the filtrate was centrifuged at 2,000 x g to remove cell debris. The supernatant was then centrifuged at 37,000 x g for 1 hr. After the centrifugation, the supernatant was discarded and the pe llet re-suspended in ice-cold DI H 2 O at a membrane concentration of approximately 40 mg/mL (f w/v). The membrane suspension was then mixed with an equal volume of 2X Carbonate Wash Buffe r (200 mM sodium carbonate, pH 11.5, 2 mM MgCl 2, 0.5 mM PMSF, 1 μ M leupeptin, and 1.0 mM benzamidine hydrochloride). Immediately after mixi ng, the mixture was centrifuged at 37,000 x g for 30 min. The supernatant was di scarded and the membrane pelle ts were stored at -80 °C

until use.

Solubilization and cation exchange chromatography

The frozen membrane pellets (~100 g FW) were thawed and re-suspended in 5 liters of the solubilization buffer [50 mM sodium acetate, pH 5.6, 100 mM NaCl, 1.0 mM DTT

, 10% Glycerol (v/v), 0.5 mM PMSF, and 1 μ M leupeptin]. Solid Fos-choline 12 was added to a final concentration of 2% (w/v ) and the membrane/detergent solution was incubated for 2 hr with stirring. The mixture was centrifuged at 37,000 x g for 30 min to

remove the insoluble fractions. The sol ubilized supernatant was applied onto a SPSepharose column (bed volume ~3.0 L, GE Healthcare, Piscataway, NJ) which had been previously equilibrated with SP-Buffer A c onsisting of 50 mM sodium acetate, pH 5.6, 10% glycerol (v/v), and 0.1% Fos-choline 12 (w /v). After washing the column with SPBuffer A, the column was further washed with 0.1 M NaCl in SP-Buffer A. The bound proteins were then eluted stepwise with 0.5 M NaCl in SP-Buffer A. Fractions containing protein were pool ed, and stored at -80 ° C.

Immuno-affinity chromatography

Immuno-affinity resin used in the purifi cation was prepared by immobilizing antiNc Δ 15D IgG to agarose resin using an IgG- orientation kit (Pie rce Cat. No. 44990, Rockford, IL) according to the manufact ure’s instructions. The anti-Nc Δ 15D IgG (Lot Numbers: 7580958, 7580959, and 7580965) were used for immuno-affinity resin preparation. The immuno-affinity column (bed vol. ~28 mL) was equilibrated with IAWB consisting of 10 mM sodium phos phate, pH 7.2, 150 mM NaCl, 1 mM MgCl 2, 10% glycerol (v/v) and 0.1% Fos-choline 12 (w/v). Before applying the SP-purified protein preparation to the immuno-affinity column, the pH of the sample was adjusted to pH 7.4 with 2.0 M potassium phosphate, pH 8.5. The flow through fraction was reapplied to the column and the flow through fraction re-application was repeated three

times. The column was washed exte nsively with the IAWB and the Nc Δ 15D protein was eluted with 0.1 M glycine, pH 2.8 containing 1mM MgCl 2, 10% glycerol (v/v) and 0.1% Fos-choline 12 (w/v). The pooled protein fraction (~140 mL) was ne utralized to pH 5.5 with 2.0 M MES, pH 10 , and stored at -80 ° C.

Concentration of Protein solution

To concentrate the immuno-affinity-purified Nc Δ 15D preparations, about 250 – 500 mL

of the eluted fractions were loaded onto a 2-mL SP-Sepharose column (GE Healthcare, Piscataway, NJ) which had been previously e quilibrated with the SP-Buffer A. The flow rate was typically at 4.0 mL/min. The prot ein was eluted with 0.5 M NaCl in the SPBuffer A. Fractions containing the Nc Δ 15D protein were pooled and stored at -80 ° C.Monsanto Company 09-SY-200U Page 215 of 395 C.2.2. Protein Concentration

The total protein concentrati on of the MON 87769-produced Nc Δ 15D protein preparation was determined by amino acid analysis (AAA) using AccQ-Tag derivatization (Waters Corporation, Milford, MA), which allows fo r high sensitivity fluorescent detection of

amino acids. In order to avoid the interf erence from buffer co mponents during protein hydrolysis, protein was precipitated using et hanol. The sample preparation was as follows: in a hydrolysis tube (~300 μ l), approximately 1 μg of protein samples (~10 μ L)

were mixed with 200 μ L of chilled 95% ethanol. After incubation overni ght at -20 oC, samples were centrifuged at 12,000 rpm in a mi crocentrifuge for 30 min at 2-8 °C. The supernatant was removed and discarded. Precipi tates were then washed sequentially with

100 μ L of both chilled acetone and water. Along with replicates of the test sample, a hydrolysis blank, four dilutions of a calibrati on standard (NIST), and a BSA control were also analyzed. An internal calibrant, α -aminobutyric acid, was included in all non-blank samples. All samples were evaporated to dryness in hydrolysis t ubes using a Speed-Vac concentrator. 500 μL of hydrolysis solu tion (6N HCl/1% phenol) was added and the

tubes were transferred to a vacuum chambe r. Samples were hydrolyzed for 90 min at 150 ± 2 °C under vacuum. After cooling, the vacuum was released and the hydrolysates were evaporated to dryness using a Speed-Vac concentrator and recons tituted in 20 μL of 20 mM HCl by vortexing. 60 μL of AccQ-Fluor Borate Buffer and 20 μL of AccQ-Fluor reagent were added sequentially to each vial with vortexing after each addition. The samples were transferred individually to auto sampler vials, capped, and heated to 55 °C for 10 min. Samples were analyzed usi ng a 2695 Separation Module (Waters Corp.) equipped with a reverse-phase C-18 column for separation of AccQ-Tag derivatized amino acids. Chromatographic data were collected using Atlas software (Thermo Electron Corp. Waltham, MA).

C.2.3. Protein Identity

Immunoblot Analysis

Immunoblot analysis was performed to confirm the identity of Nc Δ 15D protein purified

from MON 87769. Based on the concentratio n and purity, aliquots of the purified

Nc Δ 15D protein preparation were diluted in 50 mM sodium acetate, pH 5.6, 10% glycerol and 0.1% Fos-choline 12 to give a final purity -corrected protein preparation of 2, 4, 6, 8, and 10 ng/10 μ L, respectively. The samples (10 μ L) were then mixed with 2.5 μ L of 5X Loading Buffer (312 mM Tris-HCl, pH 6.8, 25% 2-mercaptoethanol, 10% sodiumdodecyl sulfate, 0.025% bromophenol blue, 50% glycerol) and separated on the SDSPAGE at constant voltage of 50 V for 25 mi n followed by 160 V for 70 min. Proteins in the gel were electrotransferred to PVDF membrane (Invitrogen, Carlsbad, CA) at a constant voltage of 25 V for 90 min in the electrotransfer buffer containing 12 mM Tris, pH 8.3, 96 mM glycine, and 20% methanol. Pre-stained MW markers were loaded in parallel to confirm the tran sfer of proteins and estimat e the size of the immuno-reactive

bands observed.

Following the electrotransfer, the membrane was blocked for 30 min with 4% (w/v) NFDM in PBST. The membrane was then probed with a 1:5,000 dilution of goat anti-Monsanto Company 09-SY-200U Page 216 of 395 Nc Δ 15D antibody (Lot No. 7580958) in PBST c ontaining 2% (w/v) NFDM for 1 hr. Excess antibody was removed by three 15 min washes with PBST. Finally, the membrane was probed with horseradish pero xidase-conjugated rabbit anti-goat IgG (Pierce, Rockford, IL) at a dilution of 1: 10,000 in PBST containing 2% (w/v) NFDM for 60 min. Following five 15 min washes with PBST, immunoreactive bands were visualized using the ECL Detection Syst em (GE Healthcare, Piscataway, NJ) and exposed (30 and 60 sec) to Hyperfilm ECL (G . E. Healthcare, Piscataway, NJ). Films were developed using a Konica SRX-101A au tomated film proces sor (Tokyo, Japan). The MW of the immunoreactive bands were estimated using a Bio-Rad GS-800 densitometer with the supplied Quantity On e software (version 4.4.0, Hercules, CA).

C.2.4. N-Terminal Sequencing

Edman degradation was used to confirm the N-terminal iden tity of the MON 87769- produced Nc Δ 15D and to determine if the N-terminal methionine was present in the protein.

Protein Blot for N-Terminal Analysis

Five 20 μL aliquots of MON 87769-produced Nc Δ 15D protein preparation were removed from storage, mixed with 5 μL of 5X LB a nd loaded in five lanes (25μL/lane) onto a 4- 20% gradient polyacrylamide (10-well) gel. Pre-stained MW markers (Precision Plus protein standard, BioRad, Hercules, CA) were loaded in parallel to confirm electrotransfer of proteins to the membrane and estimate the size of the stained bands observed. Electrophoresis was carried out at constant voltage of 50 V for 25 min followed by 160 V for 70 min. Proteins in th e gel were electrotransferred to PVDF membrane (Invitrogen, Carlsbad, CA) at a cons tant voltage of 26 V for 2.0 hr in the electrotransfer buffer containing 10 mM CAPS, pH 11 and 10% methanol. The blot was stained with Coomassie Blue R-250 (BioRad, He rcules, CA) to visualize the protein of

interest.

N-Terminal Sequencing

Following electroblotting and staining, the band corresponding to the Nc Δ 15D protein

was excised from the blot and N-terminal se quence analysis was performed for 15 cycles using automated Edman degradation chem istry (Hunkapiller and Hood, 1983). An Applied Biosystems 494 Procise Sequencing System with 140C Mi crogradient system and 785 Programmable Absorbance Detector an d Procise Control Software (version 1.1a) were used. Chromatographic da ta were collected using Atlas 99 software (version 3.59a, LabSystems, Altrincham, Cheshire, Engla nd). A PTH-amino aci d standard mixture (Applied Biosystems, Foster City, CA) was us ed to chromatographically calibrate the instrument for each analysis. This mixture se rved to verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times. A control protein, 10 pmole β -lactoglobulin, (Applied Biosystems, Foster City, CA) was analyzed before and after the analysis to verify that the sequencer met performance criteria for repetitive yield and sequence identity.Monsanto Company 09-SY-200U Page 217 of 395 C.2.5. MALDI-TOF Tryptic Mass Map Coverage Analysis

Protein Gel for Tryptic Mass Map Analysis

Five 20 μL aliquots of MON 87769-produced Nc Δ 15D protein preparation were removed from storage, mixed with 5 μL of 5X LB and loaded in five lanes (10.25 μ g/lane) onto a 4-20% gradient polyacrylamide (10-well) gel. Pre-stained MW markers (Precision Plus Protein Standard, BioRad, Hercul es, CA) were loaded in parallel to estimate the size of the stained bands observed. Electrophoresis wa s carried out at constant voltage of 50 V for 25 min followed by 160 V for 70 min. Pr e-stained MW marker s were loaded to estimate the size of the protein bands. Fo llowing electrophoresis , the gel was stained with Coomassie blue dye R-250 (BioRad, Herc ules, CA). The bands corresponding to the MON 87769-produced Nc Δ 15D protein were excised from five lanes of the gel, destained, reduced, and alkylated. Briefly, each gel band was destained for 30 min by

incubation in 100 μ L of destaining solution in a microf uge tube. This step was repeated three times with the final destaining st ep proceeding for 60 minutes. Following destaining, the gel bands were incubated in 100 μ L per band of 100 mM ammonium bicarbonate buffer for 30 min at room temper ature. The protein was reduced in 100 μ L

of 10 mM dithiothreitol solution for 2 h at 37 ° C. After removing th e reducing solution, the protein in the gel was alkylated by incubating in 100 μ L of 20 mM iodoacetic acid. The alkylation reaction was allowed to proceed at room temperature for 20 min in the dark. The gel containing the pr otein band was incubated in 200 μ L of 25 mM ammonium bicarbonate buffer for 15-45 min at room temp erature. This step was repeated two additional times, following which, gel band was dried using a Savant Speed Vac concentrator (Ramsey, MN). Each gel band was rehydrated with 20 μ L of 0.02 μg/ μ L trypsin in 25 mM ammonium bicarbonate an d 10% acetonitrile, an d was incubated for about 1 h at room temperature. Follo wing the incubation, the excess solution was removed and the gel/trypsin reaction mixture was incubated overnight at 37 ° C in 40 μ L

of 25 mM ammonium bicarbonate and 10% acet onitrile. The following day, the sample was sonicated for 5 min, and the supernatant transferred to a new tube and dried using Speed Vac concentrator (Extract 1). The gel band was resuspended in 30 μ L of a solution consisting of 60% acetonitrile, 0.1% trifluoroacetic acid and 0.1% octyl- β -Dglucopyranoside, and sonicated for 5-10 min. Af ter transfer of the supernatant to a new tube, this step was repeated one more time, and the combined supernatants were dried using Speed Vac concen trator (Extract 2). Extracts 1 and 2 were separately dissolved in

20 μ L 0.1% trifluoroacetic acid and then dried using a Speed Vac concentrator. Finally,

Extract 1 was dissolved in 5 μ L of 50% acetonitrile/0.1% trifluoroacetic acid, while

Extract 2 was dissolved in 10 μ L of the same solution. To maximize the solubilization, each sample was sonicated for 5 min. The extracts were ready for loading onto the MALDI-TOF sample plate.

C.2.6. MALDI-TOF Mass Analysis

Mass spectral analyses were performed as follows. Mass calibration of the instrument was performed using an external peptide mixt ure (CalMix 2; Applie d Biosystems, Foster City, CA). The samples Extract 1 and Extract 2 (0.1-0.25 μ L) were co-crystallized with

0.75 μ L each of the following matrix solu tions: dihydroxybenzoic acid (DHB), α -cyano-Monsanto Company 09-SY-200U Page 218 of 395 4-hydroxy cinnamic acid ( α -cyano), and 3,5-dimethoxy- 4-hydroxycinnamic acid (sinapinic acid) at separate locations on the analysis plate. The samples in DHB matrix were analyzed in the 550 to 6,000 Da range us ing 200 shots at a laser intensity setting of 3,001. The laser intensity setting is a unit-le ss MALDI-TOF instrument-specific value. The samples in α -cyano matrix were analyzed in the 500 to 6,000 Da range using 200 shots at a laser intensity se tting of 2,601. The samples in sinapinic acid matrix were analyzed in the 900 to 8,000 Da range using 200 shots at a lase r intensity sett ing of 3,100. Protonated (MH+) peptide masses were obser ved monoisotopically in reflector mode (Aebersold, 1993; Billeci and Stults, 1993), except above 3,000 Da, where mass-averaged values were used. GPMAW32 software (App lied Biosystems) was used to generate a theoretical trypsin digest of the deduced Nc Δ 15D amino acid sequence. Masses were calculated for each theoretical peptide and comp ared to the raw mass data. Experimental masses (MH+) were assigned to peaks in the 500 to 1,000 Da range if there were two or more isotopically reso lved peaks, and in th e 1,000 to 8,000 Da range if there were three or more isotopically resolved peaks in the sp ectra. Peaks were not assessed if the peak heights were less than approximately twice th e baseline noise, or when a mass could not be assigned due to overlap with a stronger signal ± 2 Da from the mass analyzed.

C.2.7. Molecular Weight and Puri ty Analysis by SDS-PAGE

Aliquots of the MON 87769-produced Nc Δ 15D protein were diluted with the solubilization buffer (50 mM sodium acetate, pH 5.6, 0.1% Fos-choline 12, and 10% glycerol) and mixed with 5X LB to a final protein concentration of 0.2 μ g/ μ L. The

Nc Δ 15D protein was analyzed in duplicate at 1, 2, and 3 μ g of total protein per lane. MW standards were heated, though the test samples were not, and all samples were applied to a pre-cast 4-20% polyacrylamide gr adient 10-well gel (Invitrogen, Carlsbad, CA). MW markers (Broad Range MW Marker , BioRad, Hercules, CA ) were loaded in parallel. Electrophoresis was performed at a constant voltage of 120 V for 60 min followed by 180 V for 30 minutes. The gel was stained using Colloidal Brilliant Blue G stain (Sigma-Aldrich, St. Louis, MO).

Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). The MW markers were used to estimate the apparent MW of the Nc Δ 15D protein. For the purity evaluation, all visible bands within each lane were quan tified. The purity and estimated MW of the MON 87769-produced Nc Δ 15D protein were reported as the average of the six values obtained by densitometric analysis.

C.2.8. Glycosylation Analysis

Glycosylation Detection M odule (Cat. No. RPN 2190, GE H ealthcare, Piscataway, NJ) was used to detect carbohydrate co valently bound to MON 87769-produced Nc Δ 15D after the proteins were resolved by SD S-PAGE and electrot ransferred onto PVDF membrane. The kit utilizes the specific labeling of biotin to the bound carbohydrate moiety followed by probing with streptavidin-c onjugated horse radish peroxidase (StrepHRP) and detection by ECL. Th e biotin labeling consists of two steps: pretreatment of the glycoprotein on the membrane and covale nt conjugation of bio tin to the carbohydrateMonsanto Company 09-SY-200U Page 219 of 395 moiety of the glycoprotein. This biotin-labe ling procedure is specifi c for the conjugation with carbohydrates and is sensitive due to the use of biotin’s high affinity binding partner, avidin, in an enzyme linked assay. This method of det ecting glycoprotein on membrane blots is widely used for th e determination of glycoproteins.

Aliquots of 0.5 μ g and 1.0 μ g of Nc Δ 15D protein were mixed with 5X LB and loaded onto a 4-20% precast polyacrylamide gel (Invitrogen, Carlsbad, CA). Along with the

Nc Δ 15D samples, 25, 50, 100, and 200 ng of the glyc osylated control protein, transferrin, were loaded in parallel. The Nc Δ 15D samples were not heat ed, but the transferrin controls were boiled prior to the loading. Electrophoresis was carried out at constant voltage of 90 V for 30 min followed by 159 V for 60 min. Following electrophoresis, proteins in the gel were electrotransferred to 0.45 μ M PVDF membrane (Invitrogen, Carlsbad, CA) at a constant voltage of 25 V for 90 min in the electrotransfer buffer containing 12 mM Tris, pH 8.3, 96 mM glyc ine, and 20% methanol. Pre-stained MW markers were loaded to verify electrotransfe r of proteins and estimate the size of the

bands on the blot.

The labeling and detection of carbohydrat e was carried out according to the manufacturer’s instruction and all the reagen ts except PBS were provided in the kit. Following electrotransfer to PVDF membrane, the blot was incubated first in 30 mL of PBS for 10 min, then in 10 mM NaIO 4 for 20 min in darkness. Following the treatment,

the membrane was then rinsed twice with 15 mL PBS and washed three times with 15

mL PBS for 10 min each. The membrane was incubated with biotin-hydrazide for 60 min followed by two PBS rinses and three 10 min washes with 15 mL PBS as described

above. The membrane was blocked for 60 min with 5% blocki ng reagent in PBS followed by two PBS rinses and three 10 min washes with 15 mL PBS. Strep-HRP at

1:6000 dilution was overlaid onto the membrane and incubated at room temperature for

30 min. After two PBS rinses and three 10 mi n washes with 15 mL PBS, the membrane was then developed with ECL detection reag ents by mixing 2 mL of the Reagent 1 and 2 mL of Reagent 2. After 1 min incubation, th e excess detection solu tion was removed by blotting with paper towel and the blot was e xposed to Hyperfilm EC L (G. E. Healthcare, Piscataway, NJ). The film was develope d using a Konica SRX-101A automated film processor (Tokyo, Japan). The blot images were captured usin g a Bio-Rad GS-800 densitometer with the supplied Quantity On e software (version 4.4.0, Hercules, CA).

C.2.9 Storage Stability

The short-term stability of the MON 87769-produced Nc Δ 15D protein was evaluated by comparing the purity and MW values before and after the storage for 29 days in a 4 oC refrigerator and, in-20 oC and -80 o C freezers. At the end of storage, aliquots of the

Nc Δ 15D protein samples equivalent to 1, 2, and 3 μ g of the purity-corrected Nc Δ 15D at Day 0 were removed and analysed by SDS-PA GE followed by staining, and subsequent purity and MW estimation. The MON 87769-produced Nc Δ 15D protein was analyzed in duplicate at 1, 2, and 3 μ g of total protein per lane. MW standards were heated, though the test samples were not, and all samples were applied to a pre-cast 4-20% polyacrylamide gradient 10-well gel (Invitrog en, Carlsbad, CA). Electrophoresis was performed at a constant vo ltage of 120 V for 20 min followed by 180 V for 60 minutes.Monsanto Company 09-SY-200U Page 220 of 395 The gels were stained using Colloidal Brilli ant Blue G stain (Sigma-Aldrich, St. Louis, MO).

Analysis of each gel was performed usi ng a Bio-Rad GS-800 densitometer with the supplied ‘Quantity One’ software (version 4.4. 0, Hercules, CA). The molecular weight markers were used to estimate the appa rent molecular wei ght of the MON 87769- produced Nc Δ 15D protein on the gel. The protein samples were considered to have undergone degradation if a >10% decrease in purity and/or molecular weight was observed relative to the value determined on Day 0.

C.3. Characterization of the MON 87769-Produced Pj Δ 6D Protein

To characterize the purified Pj Δ 6D protein produced in MON 87769, a number of analyses were performed as described in Section C.1. The anal yses employed for the characterization of MON 87769-produced Pj Δ 6D protein included:

1. N-terminal sequence analysis.

2. MALDI-TOF to generate a tryptic peptide map.

3. Immunoblot analysis to es tablish protein identity th rough immunoreactivity with Pj Δ 6D–specific antibody.

4. SDS-PAGE to assess the apparent molecular weight of the protein.

5. Glycosylation analysis to evaluate potenti al post-translational modification of the protein.

C.3.1. N-terminal Sequence Analysis

N-terminal sequence analysis of the first 15 amino acids performed on the major protein band with a molecular weight of ~46 kDa observed on stained SDS-PAGE (Figure C-1) and in immunoblot analysis with Pj Δ 6D antibody (Figure C-2) resulted in the expected sequence for the Pj Δ 6D protein (Table C-1). The N-terminal methionine was not observed, indicating that it was removed dur ing post-translational processing of the protein. This result is expected as removal of the N-terminal methionine, catalyzed by methionine aminopeptidase, is a common mo dification that occurs co-translationally before completion of the nascent protein ch ain in many organisms and its removal has no effect on protein structure or activity (Arf in and Bradshaw, 1988; Polevoda and Sherman, 2000; Schmidt et al., 1992) and is common in many organisms. There was a second band of approximate molecular weight 7 kDa wh ich comprised 10.6% of the total protein present on the gel. The sequence analysis of this band indicated the presence of multiple sequences that are not related to Pj Δ 6D protein. The protein preparation was not highly purified; therefore the presence of additional peptide fragments is not unexpected. The N-terminal sequence information, therefor e, confirms the identity of the Pj Δ 6D protein

isolated from MON 87769 and that its N-terminus is intact.Monsanto Company 09-SY-200U Page 221 of 395 C.3.2. MALDI-TOF Mass Spectrometry Analysis

The identity of the MON 87769-produced Pj Δ 6D protein was further confirmed by tryptic peptide mass mapping analysis usi ng MALDI-TOF MS. In general, protein identification made by peptide mapping is cons idered to be reliable if the measured coverage of the sequence is 15% or higher with a minimum of five matched peptides (Jensen et al., 1997). Observed tryptic peptides were considered a matc h to the expected tryptic mass when differences in molecula r weight of less than one Da were found between the observed and predicted fragment masses. Such matches were made without consideration for potential natural amino acid modifications such as glycosylation. The MON 87769-produced Pj Δ 6D protein sample was heat-d enatured, chemically reduced, alkylated, digested with tryps in, guanidinated, and the mass of the tryptic peptides were

measured.

There were 30 unique protein fr agments identified that matched the expected masses of the Pj Δ 6D trypsin-digested peptides. The iden tified protein fragments were used to assemble a coverage map of the entire Pj Δ 6D protein (Figure C-3), resulting in ~42% (188 out of 446 amino acids) coverage of the to tal protein. This analysis confirmed the identity of the MON 87769-produced Pj Δ 6D protein.Monsanto Company 09-SY-200U Page 222 of 395 Figure C-1. SDS-PAGE of the MON 87769-Produced Pj Δ 6D Protein Aliquots of the MON 87769-produced Pj Δ 6D protein were separated by SDS-PAGE, followed by Coomassie blue staining. Appr oximate molecular weights (kDa) are shown on the left and correspond to the marker lo aded in lanes 2 and 9. Arrow indicates Pj Δ 6D protein.

Lane Sample Amount loaded (μg)

1 Blank 0
2 BioRad Broad Range Marker 4.5

3 MON 87769-produced Pj Δ 6D protein 2.0

4 MON 87769-produced Pj Δ 6D protein 2.0

5 MON 87769-produced Pj Δ 6D protein 3.0

6 MON 87769-produced Pj Δ 6D protein 3.0

7 MON 87769-produced Pj Δ 6D protein 4.0

8 MON 87769-produced Pj Δ 6D protein 4.0
9 BioRad Broad Range Marker 4.5

10 Blank 0Monsanto Company 09-SY-200U Page 223 of 395 Figure C-2. Western Blot Analysis of MON 87769-Produced Pj Δ 6D Protein Aliquots of the purifie d, MON 87769-produced Pj Δ 6D protein were separated by SDSPAGE, and electro-transferred to a PVDF membrane. The membrane was probed with goat anti-Pj Δ 6D antibody and developed using an ECL system (GE Healthcare, Piscataway, NJ). Approximate molecular wei ghts (kDa) of markers loaded in Lane 2 are

shown on the left side of the blot.

Lane Sample Amount Loaded (ng) 1 Empty -

2 Precision Plus All Blue protein Standards 7.5

3 MON 87769-produced Pj Δ 6D protein 4

4 MON 87769-produced Pj Δ 6D protein 6

5 MON 87769-produced Pj Δ 6D protein 8

6 MON 87769-produced Pj Δ 6D protein 10

7 MON 87769-produced Pj Δ 6D protein 12 8 Empty - 9 Empty - 10 Empty -Monsanto Company 09-SY-200U Page 224 of 395 Table C-1. N-terminal Amino Acid Seque nce Analysis of the MON 87769-Produced Pj Δ 6D Protein

Amino acid

residue # from

the Nterminus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Predicted Pj Δ 6D Sequence 1,2 M T K T I Y I T S S E L E K H N │ │ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Observed Sequence 3 T K T I Y I T S S E L E K H N

1 The single letter IUPAC-IUB amino acid code is; E, glutamic acid; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; R, arginine; S, serine; T, threonine; Y, tyrosine.

2 The predicted amino acid sequence of the Pj Δ 6D protein was deduced from the coding region of the full length Pj.D6D gene present in MON 87769 (Figure VI-1).

3 The N-terminal methionine was removed by the action of methionine aminopeptidase during co-translation, and therefore, was not observed.

1 MTK TIYITSS ELEK HNKPGD LWISIHGQVY DVSSWAALHP GGIAPLLALA

51 GHDVTDAFLA YHPPSTSR LL PPFSTNLLLE KHSVSETSSD YRKLLDSFHK

101 MGMFR ARGHT AYATFVIMIL MLVSSVTGVL CSENPWVHLV CGAAMGFAWI

151 QCGWIGHDSG HYRIMTDR KW NRFAQILSSN CLQGISIGWW K WNHNAHHIA
201 CNSLEYDPDL QYIPLLVVSP K FFNSLTSRF YDKKLNFDGV SRFLVQYQHW

251 SFYPVMCVAR LNMLAQSFIL LFSRREVANR VQEILGLAVF WLWFPLLLSC
301 LPNWGERIMF LLASYSVTGI QHVQFSLNHF SSDVYVGPPV GNDWFKKQTA

351 GTLNISCPAW MDWFHGGLQF QVEHHLFPR M PRGQFRKISP FVRDLCKKHN

401 LTYNIASFTK ANVLTLETLR NTAIEAR DLS NPIPK NMVWE AVK NVG

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87769-Produced Pj Δ 6D

Protein The amino acid sequence of the MON 87769 Pj Δ 6D protein was deduced from the coding region of the full-length Pj.D6D gene present in MON 87769 (see Figure VI-1). Shaded regions correspond to tryptic peptide masses that were identified from the protein sample using MALDITOF MS. In total, ~42% (188 of 446 total ami no acids) of the expected protein sequence was

identified.Monsanto Company 09-SY-200U Page 225 of 395 C.3.3. Pj Δ 6D Protein Immuno-reactivity

A western blot analysis using goat anti-Pj Δ 6D antibody was conducted to further confirm the identity of th e MON 87769-produced Pj Δ 6D protein. As demonstrated in Figure C-2,

an immuno-reactive band was observed at th e molecular weight of approximately 46 kDa. As expected, the immuno-reactive si gnal increased with increasing amount of protein loaded.

The immuno-reactive profiles shown in Figur e C-2 reveal a slightly diffuse signal (streak) in the high molecular weight area between 100 kDa and 250 kDa. When membrane proteins are separated on SDS-PAGE , a portion tends to migrate as aggregates (McGregor et al., 2003; Von Jagow et al., 1994). While all possible precautions were taken during electrophor esis (i.e., the samples were not heated to prevent further aggregation), it is reasonable to assume th at the diffuse signal observed in these blots consisted mainly of Pj Δ 6D protein as it is immu no-reactive with anti-Pj Δ 6D antibody. It is also possible that the diffuse signal obs erved in high molecular area originates from endogenous proteins cross-reacting with Pj Δ 6D antibody proteins. The Pj Δ 6D-specific antibody also recognized a band of approximately 15 kDa. This fragment is likely the result of proteolytic degradation of the Pj Δ 6D protein generated during the purification procedure or originates from endogenous cr oss-reacting proteins co-purified with the Pj Δ 6D protein.

Based on the above analysis, the id entity of the MON 87769-produced Pj Δ 6D protein

was confirmed.

C.3.4. Pj Δ 6D Protein Molecular Weight

The molecular weight of the MON 87769-produced Pj Δ 6D protein was determined using SDS-PAGE (Figure C-1). The MON 87769-produced Pj Δ 6D protein migrated with an apparent molecular weight of 46 kDa. The average purity of the full length Pj Δ 6D protein was estimated to be 47%.

C.3.5. Pj Δ 6D Protein Glycosy lation Analysis

Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate moieties (Rademacher et al., 1988), while many prokaryotic organisms such

as non-virulent E. coli strains used for cloning and expression purposes lack the necessary biochemical synthetic capacity requi red for protein glycosylation. There are two forms of glycosylation. The first occurs on asparagine side ch ains, and is known as N-glycosylation. The other form is O-glycosylation, which is the addition of N-acetylglucosamine (N-GlcNAc) to the β -hydroxyl of either a serine or threonine residue. O-glycosylation sites are less well defined (Christlet and Veluraja, 2001) and may occur at any serine or threonine residue. Pj Δ 6D is an integral membrane protein of higher plant origin ( Primula juliae ) which contains two putat ive N-glycosylation sites Asn-Xxx-Ser/Thr (Marshall, 1972).

To test whether the MON 87769-produced Pj Δ 6D protein was glycos ylated, the purified protein was analyzed for the presence of covalently bound carbohydrate moieties using aMonsanto Company 09-SY-200U Page 226 of 395 Glycoprotein Detection Module (GE Healthcare, Piscataway, NJ) which detects N- and O-linked carbohydrates. A naturally glycosylat ed protein, transferrin, was utilized as a positive control for the assay. The transferrin protein and purified Pj Δ 6D protein isolated from MON 87769 were separated on SDS-PAGE , transferred to a PVDF membrane, and glycosylation analysis was performed to detect carbohydrate moieties on the proteins (Figure C-4). The positive control, transferrin, was detected at the expected molecular weights of ~75 kDa in a con centration-dependent manner (Fi gure C-4, Lanes 2-5). No detectable signal was observed at the expect ed molecular weight of approximately 46 kDa for the Pj Δ 6D protein (Figure C-4, lanes 7-8).

Three faint and slightly diffuse signals were detected by this analysis in the lane containing the highest protein concentration (Lane 8, Figure C-4) in the molecular weight range of 60-200 kDa. Taking into considerati on that the purity of Pj Δ 6D protein is approximately 47% and that no signal wa s observed at the expected MW for Pj Δ 6D protein (~46 kDa), it was concluded that the faint signals observed on the blot are not derived from the Pj Δ 6D protein. Most likely, the obser ved faint bands or iginated from plant proteins that co-purified during the preparation of the Pj Δ 6D protein. Therefore,

the above data demonstrate that the MON 87769-produced Pj Δ 6D is not glycosylated.Monsanto Company 09-SY-200U Page 227 of 395 Figure C-4. Glycosylation Analysis of the MON 87769-Produced Pj Δ 6D Protein Aliquots of transferrin (positive control) and MON 87769-produced Pj Δ 6D protein were separated by SDS-PAGE and transferred to a PVDF membrane. Carbohydrate moieties were labeled with biotin-hydrazide and de tected using a Streptavidin-horseradish peroxidase (Streptavidin-HR P) antibody. Chemiluminescent activity of the HRP tag was detected using Hyperfilm ECL. Approxima te molecular weights indicated in kDa correspond to protein standard markers (lane 6 and 9). The arrow i ndicates the expected position of Pj Δ 6D protein.

Lane Sample Amount Loaded (μg)

1 Blank -

2 Transferrin 0.025

3 Transferrin 0.050

4 Transferrin 0.100

5 Transferrin 0.200

6 Precision Plus Dual Color MW marker na

7 MON 87769-produced Pj Δ 6D 0.500

8 MON 87769-produced Pj Δ 6D 1.0

9 Precision Plus Dual Color MW marker na 10 Empty -Monsanto Company 09-SY-200U Page 228 of 395 C.3.6. Conclusions on the Characterization of the MON87769-Produced Pj Δ 6D

Protein

A panel of analytical tec hniques was used to char acterize the purified MON 87769- produced Pj Δ 6D protein. The identit y of the MON 87769-produced Pj Δ 6D protein was confirmed by its recognition with anti-Pj Δ 6D antibodies, identification of the first 15 amino acids of the expected N-terminus by amino acid sequencing, and identification of tryptic peptide masses that yielded ~42% ov erall coverage of the expected protein sequence. The apparent molecula r weight of the MON 87769-produced Pj Δ 6D protein was estimated to be 46 kDa. The MON 87769-produced Pj Δ 6D protein was confirmed to be a non-glycosylated protein. Taken together, these data confirm the identity of the Pj Δ 6D protein isolated from MON 87769.

C.4. Characterization of the MON 87769-Produced Nc Δ 15D Protein

To characterize the purified Nc Δ 15D protein produced in MON 87769 a number of analyses were performed as described in Section C.2. The anal yses employed for the characterization of MON 87769-produced Nc Δ 15D protein were the same as the analyses utilized to characterize Pj Δ 6D protein and included:

1. N-terminal sequence analysis.

2. MALDI-TOF mass spectrometry to ge nerate a tryptic peptide map.

3. Immunoblot analysis to es tablish protein identity through immunoreactivity with

Nc Δ 15D –specific antibody.

4. SDS-PAGE to assess the apparent molecular weight of the protein.

5. Glycosylation analysis to evaluate potenti al post-translational modification of the protein.

C.4.1. N-terminal Sequence Analysis

N-terminal sequence analysis performed on the major protein band with an apparent molecular weight of ~46 kDa observed on st ained SDS-polyacrylamide gel (Figure C-5) resulted in the sequence expected for the Nc Δ 15D protein (Table C-2). The N-terminal methionine was not observed, indicating that it was removed during post-translational processing of the protein. This result is expected as removal of the N-terminal methionine, catalyzed by methionine aminope ptidase, is a common modification that occurs co-translationally before completion of the nascen t protein chain and has no effect on protein structure or activity (Arfin a nd Bradshaw, 1988; Polevoda and Sherman, 2000; Schmidt et al., 1992) and is common in many organisms.

The N-terminal sequence information, theref ore, confirms the identity of the Nc Δ 15D protein isolated from MON 87769 and that its N-terminus is intact.Monsanto Company 09-SY-200U Page 229 of 395 Figure C-5. SDS-PAGE of the MON 87769-Produced Nc Δ 15D Protein Aliquots of the MON 87769-produced Nc Δ 15D protein were separated by SDS-PAGE, followed by Coomassie blue staining. Appr oximate molecular weights (kDa) are shown on the left and correspond to the marker loaded in lanes 2 and 9. Arrow indicates

Nc Δ 15D protein.

Lane Sample Amount loaded (μg) 1 Empty 2 BioRad Broad Range Marker 0.2 3 MON 87769-produced Nc Δ 15D protein 1 4 MON 87769-produced Nc Δ 15D protein 1 5 MON 87769-produced Nc Δ 15D protein 2 6 MON 87769-produced Nc Δ 15D protein 2 7 MON 87769-produced Nc Δ 15D protein 3 8 MON 87769-produced Nc Δ 15D protein 3 9 BioRad Broad Range Marker 0.2 10 EmptyMonsanto Company 09-SY-200U Page 230 of 395 Table C-2. N-terminal Amino Acid Seque nce Analysis of the MON 87769-Produced

Nc Δ 15D Protein

Amino acid

residue # from

the Nterminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Predicted

Nc Δ 15D
Sequence 1,2 → M A V T T R S H K A A A A T E P

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Observed Sequence 3 → A V T T R S H K A A A A T E P

1 The single letter IUPAC-IUB amino acid code is; A, alanine; E, glutamate; H, histidine; M, methionine; P, proline; R, arginine; S, serine; T, threonine.

2 The predicted amino acid sequence of the Nc Δ 15D protein was deduced from the coding region

of the full length Nc.Fad3 gene present in MON 87769 (see Figure VI-3).

3 The N-terminal methionine was removed by the action of methionine aminopeptidase during

co-translation, and therefore, was not observed.

C.4.2. MALDI-TOF Mass Spectrometry Analysis

The identity of the MON 87769-produced Nc Δ 15D protein was further confirmed by tryptic peptide mass mapping analysis usi ng MALDI-TOF MS. In general, protein identification made by peptide mapping is cons idered to be reliable if the measured coverage of the sequence is 15% or higher with a minimum of five matched peptides (Jensen et al., 1997). Observed tryptic peptides were considered a matc h to the expected tryptic mass when differences in molecular weight of less than one Da were found between the observed and predicted fragment masses. Such matches were made without consideration for potential natural amino acid modifications such as glycosylation. The protein sample was heat-denatured, chemically reduced, alkylated, di gested with trypsin, guanidinated, and the masses of the tryptic peptides were measured.

There were 15 unique protein fr agments identified that matched the expected masses of

the Nc Δ 15D trypsin-digested peptides. The identified masses were used to assemble a coverage map indicating the matched peptide sequences for the entire Nc Δ 15D protein (Figure C-6), resulting in ~45% (193 out of 429 amino acids) coverage of the total protein. This analysis confirmed the identity of the MON 87769-produced Nc Δ 15D protein.Monsanto Company 09-SY-200U Page 231 of 395 Figure C-6. MALDI-TOF MS Coverage Map of the MON 87769-Produced Nc Δ 15D

Protein The amino acid sequence of the MON 87769 Nc Δ 15D protein was deduced from the coding region of the full-length Nc.Fad3 gene present in MON 87769 (see Figure VI-3). Shaded regions correspond to tryptic peptide masses that were identified from the protein sample using MALDITOF MS. In total, ~45% (193 of 429 total ami no acids) of the expected protein sequence was

identified.

C.4.3. Nc Δ 15D Protein Immuno-reactivity

A western blot analys is using goat anti-Nc Δ 15D antibody was conducted to further confirm the identity of the MON 87769-produced Nc Δ 15D protein. As demonstrated on Figure C-7, a predominant immuno-reactiv e band was observed at the expected molecular weight of approximately 46 kDa (F igure C-7, lanes 2-6). As expected, the immuno-reactive signal increased with increasing amount of protein loaded.

The immuno-reactive profiles shown in Figu re C-7 reveal slightly diffuse immunostaining in the high MW portion of the gel. When membrane proteins are separated on SDS-PAGE, a small portion tends to aggregate (McGregor et al ., 2003; Von Jagow et al., 1994). While all possible precautions were ta ken during electrophoresis (i.e., the samples were not heated to prevent furt her aggregation), it is reasonabl e to assume that the diffuse signal observed in these blots consisted mainly of the Nc Δ 15D protein as it is immunoreactive with anti-Nc Δ 15D antibody.

In addition, two minor bands with molecula r weights between 25 kDa and 37 kDa were recognized by the antibody (Figure C-7, lanes 2-6). It is likely that these signals are the results of proteolytic degradation of the Nc Δ 15D protein that occurs during the purification procedure or originate from e ndogenous cross-reacting proteins co-purified

with the Nc Δ 15D protein.

Based on the above analysis, the id entity of the MON 87769-produced Nc Δ 15D protein

was confirmed.Monsanto Company 09-SY-200U Page 232 of 395 Figure C-7. Western Blot Analysis of MON 87769-Produced Nc Δ 15D Protein Aliquots of the purified, MON 87769-produced Nc Δ 15D protein were separated by SDSPAGE, and electro-transferred to a PVDF membrane. The membrane was probed with goat anti-Nc Δ 15D antibody and developed using an ECL system (GE Healthcare, Piscataway, NJ ). Approximate molecular wei ghts (kDa) of markers loaded in Lane 1 are

shown on the left side of the blot.

Lane Sample Amount loaded (ng)

1 Precision Plus MWT Marker 198

2 MON 87769-produced Nc Δ 15D protein 2.0

3 MON 87769-produced Nc Δ 15D protein 4.0

4 MON 87769-produced Nc Δ 15D protein 6.0

5 MON 87769-produced Nc Δ 15D protein 8.0

6 MON 87769-produced Nc Δ 15D protein 10.0 7 Empty 8 Empty 9 Empty 10 EmptyMonsanto Company 09-SY-200U Page 233 of 395 C.4.4. Nc Δ 15D Protein Molecular Weight

The molecular weight of the MON 87769-produced Nc Δ 15D protein was determined using SDS-PAGE (Figure C- 5). The MON 87769-produced Nc Δ 15D protein migrated with an apparent molecular weight of 46 kDa. The average purity of the full length

Nc Δ 15D protein was estimated to be 74%.

C.4.5. Nc Δ 15D Protein Glycosylation Analysis

Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate moieties (Rademacher et al., 1988), while many prokaryotic organisms such

as non-virulent E. coli strains used for cloning and expression purposes, lack the necessary biochemical synthetic capacity requi red for protein glycosylation. There are two forms of glycosylation. The first occurs on asparagine side ch ains, and is known as N-glycosylation. The other form is O-glycosylation, which is the addition of N-acetylglucosamine (N-GlcNAc) to the β -hydroxyl of either serine or threonine residue. O-glycosylation sites are less well defined (Christlet and Veluraja, 2001) and may occur at largely any serine or threonine residue. Nc Δ 15D is an integral membrane protein of microbial origin, which cont ains two putative N-glycosyl ation sites Asn-Xxx-Ser/Thr (Marshall, 1972).

To test whether th e MON 87769-produced Nc Δ 15D protein was gl ycosylated, the purified protein was analyzed for the presen ce of covalently bound carbohydrate moieties using a Glycoprotein Detection Module (GE H ealthcare, Piscataway, NJ) which detects N- and O-linked carbohydrates. A naturally glycosylated protein, transferrin, was utilized as a positive control for the assay. The transferrin protein and purified Nc Δ 15D protein isolated from MON 87769 were separated on SDS-PAGE , transferred to a PVDF membrane, and glycosylation analysis was perf ormed to detect carbohydrate moieties on the proteins (Figure C-8). The positive control, transferrin, was detected at the expected molecular weights of ~75 kDa in a concentr ation-dependent manner (Figure C-8, lanes 2- 5). No detectable signal was observed at th e expected MW of approximately 46 kDa for

the Nc Δ 15D protein (Figure C-8, lanes 7-8).

Two faint bands were detected at ~70 kDa in lanes 7-8 containing purified Nc Δ 15D protein (Figure C-8, lanes 7-8). An additional very weak signal was observed in the same lanes between 150 kDa and 250 kDa. Taking into consideration that the purity of

Nc Δ 15D protein is only 74% and that no signal was observed at the expected MW for

Nc Δ 15D protein ~46 kDa, it was c oncluded that the faint signa ls observed on the blot are

not derived from the Nc Δ 15D protein. Most likely, the observed faint bands originate from plant proteins that co-purif ied during the prep aration of the Nc Δ 15D protein. Therefore, the above data demons trate that the MON 87769-produced Nc Δ 15D is not glycosylated.Monsanto Company 09-SY-200U Page 234 of 395 Figure C-8. Glycosylation Analysis of the MON 87769-Produced Nc Δ 15D Protein Aliquots of transferrin (positiv e control) and MON 87769-produced Nc Δ 15D protein were separated by SDS-PAGE and transferred to a PVDF membrane. Carbohydrate

moieties were labeled with biotin-hydrazide and detected using a Streptavidin-HRP antibody. Chemiluminescent activity of the HR P tag was detected us ing Hyperfilm ECL. Approximate molecular weights indicated in kDa correspond to protein standard markers (lane 6 and 9). The arrow indi cates the expected position of Nc Δ 15D protein.

Lane Sample Amount loaded ( μ g) 1 Empty

2 Transferrin 0.025

3 Transferrin 0.05

4 Transferrin 0.10

5 Transferrin 0.20

6 BioRad Precision Plus MWT Marker 0.20

7 MON 87769-produced Nc Δ 15D protein 0.50

8 MON 87769-produced Nc Δ 15D protein 1.00

9 BioRad Precision Plus MWT Marker 0.20 10 EmptyMonsanto Company 09-SY-200U Page 235 of 395 C.4.6. Conclusions on the Characterization of the MON 87769-Produced Nc Δ 15D

Protein

A panel of analytical tec hniques was used to char acterize the purified MON 87769- produced Nc Δ 15D protein. The identity of the purified Nc Δ 15D protein was confirmed by its recognition with anti-Nc Δ 15D antibodies, identification of the first 15 amino acids of the expected N-terminus by amino acid sequencing, and identification of tryptic peptide masses that yielded 45% overall covera ge of the expected protein sequence. The apparent molecular weight of the Nc Δ 15D protein was estimated to be 46 kDa. In addition, the MON 87769-produced Nc Δ 15D was confirmed to be a non-glycosylated protein. Taken together, these data confirm the identity of the Nc Δ 15D protein isolated

from MON 87769.

References Aebersold, R. 1993. Mass spectrometry of prot eins and peptides in biotechnology. Curr. Opin. Biotechnol. 4:412-419.

Arfin, S.M. and R.A. Bradshaw. 1988. Cotranslat ional processing and protein-turnover in eukaryotic cells. Biochemistry 27:7979-7984.

Billeci, T. M. and Stults, J. T. 1993. Tryptic mapping of recombinant proteins by matrixassisted laser desorption/ionization mass spectrometry. Anal. Chem. 65:1709-1716.

Christlet, T.H.T. and K. Veluraja. 2001. Data base analysis of O-gl ycosylation sites in proteins. Biophys J. 80:952-960.

Hunkapillar, M.W. and L.E. Hood. 1983. Protein sequence analysis: automated microsequencing. Science 219:650-659.

Jensen, O.N., A.V. Podtelejnikov, and M. Mann. 1997. Identification of the components of simple protein mixtures by high accu racy peptide mass mapping and database searching. Analytical Chemistry 69:4741-4750.

Marshall, R.D. 1972. Glycoproteins. Annua l Review of Biochemistry 41:673-702.

McGregor, C., L. Chen, N.C. Pomroy, P. Hwang, S. Go, A. Chakrabarrty, and G.G. Prive. 2003. Lipopeptide deterg ents designed for the stru ctural study of membrane proteins. Nature Biotech. 21:171-176.

Polevoda, B. and Sherman, F. 2000. N-terminal acetylation of eukaryotic proteins. J.

Biol. Chem. 275:36479-36482.

Rademacher, T.W., R.B. Parekh, and R.A. Dwek. 1988. Glycobiology. Annu Rev

Biochem. 57:785-838.

Sagne, C., M.F. Isambert, J.P. Henry and B. Gasnier. 1996. SDS-resi stant aggregation of membrane proteins: Aapplication to the purification of the vesicular monoamine transporter. Biochem. J. 316:825-831.Monsanto Company 09-SY-200U Page 236 of 395 Schmidt, J., E. Herfurth, and A.R. Subr amanian. 1992. Purification and characterization of 7 chloroplast ribosomal-prote ins: Evidence that organell e ribosomal-protein genes are functional and that NH2-terminal processing occurs via multiple pathways in chloroplasts. Plant Molecular Biology 20:459-465.

Von Jagow, G., T. Link, and H. Schagger. 1994. Purication strategies for membrane proteins. Pages 3-21 in A pr actical guide to membrane protein purirification. Von Jagow, G. and H. Schagger (eds.). Academic Press, San Diego, CA.Monsanto Company 09-SY-200U Page 237 of 395 Appendix D. Materials a nd Methods Used for the Determination of Protein Expression in MON 87769 Tissue

D.1. Tissue Processing and Protein Extraction

All tissue samples produced at the field site s were shipped to the Monsanto Sample Processing facility. Processed tiss ue samples were stored in a -80 ° C freezer until shipped on dry ice to Monsanto’s analytical facility. All processe d tissue samples were

stored in a -80 ° C freezer during the study.

The Pj Δ 6D and Nc Δ 15D proteins were extracted from soybean tissue by shaking tubes mounted in a Harbil mixer for two 3.5 minute cy cles. Each extraction tube contained two 1⁄4” diameter Chrome-steel beads, buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 2% Triton X-100, 1X protease inhibito rs [Roche Diagnostics, Indianapolis, IN])

and a tissue-to-buffer ratio of 1:10 for root, leaf, and fora ge or 1:20 for immature and mature seed. Extracts were clarified by centrifugation (14,000 rpm x 15 minutes at 4 o C)

and stored in a -80 ° C freezer until western blot analyses.

D.2. Pj Δ 6D Antibodies

Goat polyclonal antibodies (lot # 7580971) were raised against a peptide fragment of the Pj Δ 6D protein (EARDLSNPIPKN), which is located close to the C terminus of the Pj Δ 6D protein. The antibodies were affinity purified by I nvitrogen Life Sciences (Carlsbad, CA). The concentration of the purified IgG was determined to be 2.2 mg/mL by spectrophotometric methods. The purifie d antibody was stored in 25 mM sodium borate, 100 mM boric acid, 75 mM NaCl, 5 mM EDTA, pH 8.2 to 8.4 (Borate Buffered Saline).

D.3. Nc Δ 15D Antibodies

Goat polyclonal antibodies (lot # 7580965) were raised against a peptide fragment of the

Nc Δ 15D protein (TEADRNQRKLANLYMDKET) lo cated in the middle region of the

Nc Δ 15D protein. The antibodies were affinity purified by Invitrogen Life Sciences. The concentration of the purified IgG was determ ined to be 2.6 mg/mL by spectrophotometric methods. The purified antibody was stor ed in Borate Buffered Saline.

D.4. Pj Δ 6D Western Blot Method

Extracts were analyzed by SDS-PAGE on a 4-20% Tris-HCl gradient gel. Prior to loading, the samples were diluted in 2X L aemmli Buffer (Bio-Rad, Hercules, CA). Sample extracts were loaded on the gels with the appropriate reference standards. The reference standards were prepared in tissue- specific nontransgenic extracts for each tissue type and diluted in 2X Laemmli buffer. Th e reference standards ranged from 20 ng to 1 ng. The nontransgenic extracts used for the reference standa rds for each tissue type were pooled from all sites. Samples were loaded in triplicate and at l east two were used for densitometry and quantitation. Additionally, th e Precision Plus molecular weight markerMonsanto Company 09-SY-200U Page 238 of 395 (Bio-Rad) was loaded on the gel to demonstrat e the transfer of protein to membrane and for the approximate molecular weight determination.

Electrophoresis was conducted according to the current version of SOP BR-ME-0388 at 200 V for approximately 5 minutes and at 120 V for approximately 80 minutes in 1X Tris-Glycine-SDS running buffer (Bio-Rad). Electrotransfer and we stern blot analysis were conducted according to the current versions ofSOPs BR-ME-0924 and BR-ME0392, respectively. Proteins separated by SDS-PAGE were electrophoretically transferred to 0.45 μm Criteri on Nitrocellulose membrane (Bio-Rad) using 1X Trisglycine transfer buffer (Bio-R ad) containing 20% methanol. After transfer, nonspecific sites on the membrane were blocked using 5% (w/v) NFDM (Bio-Rad, Hercules, CA) in 1X Phosphate-Buffered Saline with 0.05% (v/v) Tween-20 (1X PBST).

The membrane was probed for the presence of the Pj Δ 6D protein with a 1:2500 dilution of purified goat antibodies, anti-Pj Δ 6D peptide antibody (Lot 7580971) in 1X PBST with 1% (w/v) NFDM. Unbound antibodies were removed by rinsing the membrane briefly and then washing three times of 10 minutes each in 1X PBST. Bound antibodies were probed with a 1:5000 dilution of anti-goat IgG antibody conjugated to horseradish peroxidase (Pierce, Rockford, IL) in 1X PBST with 1% (w/v) NFDM. Unbound antigoat IgG-HRP antibodies were removed by rinsing the membrane briefly and then washing four times of 10 minutes each in 1X PBST. The ECL substrate (Amersham, Piscataway, NJ) was added to the membrane acco rding to the manufacturers’ instructions. The membrane was exposed to Hyperfilm ECL (Amersham, Piscataway, NJ) to record an image of the immunoreactive bands.

D.5. Nc Δ 15D Western Blot Method

Extracts were analyzed by SDS-PAGE on a Novex 4-20% gradient gel (Invitrogen). Prior to loading, the samples were diluted in 2X Laemmli Buffer (Bio-Rad). Sample extracts were loaded on the gels with the a ppropriate reference standards. The reference standards were prepared in tissue-specific non transgenic extracts for each tissue type and

diluted in 2X Laemmli buffer. The referen ce standards ranged from 75 ng to 1 ng. The nontransgenic extracts used for the reference standards for each tis sue type were pooled from all sites. Samples were loaded in triplicate and at least two were used for densitometry and quantitation. Additionally, th e Precision Plus molecular weight marker (Bio-Rad) was loaded on the gel to demonstrat e the transfer of protein to membrane and for the approximate molecular weight determination.

Electrophoresis was conducted according to the current version of SOP BR-ME-0388 at 120 V for approximately 120 minutes in 1X Novex Tris-Glycine SDS running buffer (Invitrogen, Carlsbad, CA). Electrotransfer and western bl ot analysis were conducted according to the current version of SOPs BR-ME-0924 and BR-ME-0392, respectively. Proteins separated by SDS-PA GE were electrophoretically transferred to Invitrolon PVDF membrane (Invitrogen) using 1X Novex Tris-Glycine transfer buffer (Invitrogen) containing 20% methanol. After transfer, nonspecific sites on the membrane were blocked using 5% (w/v) NFDM in 1X Tris-Buffered Salin e with 0.1% (v/v) Tween-20 (1X TBST).Monsanto Company 09-SY-200U Page 239 of 395 The membrane was probed for the presence of the Nc Δ 15D protein with a 1:2500 dilution of purified goat antibodies, anti-Nc Δ 15D peptide antibody (Lot 7580965) in 1X TBST with 5% (w/v) NFDM. Unbound antibodies were removed by rinsing the membrane briefly and then washing three times of 10 minutes each in 1X TBST. Bound antibodies were probed with a 1:5000 dilution of anti-goa t IgG antibody conjugate d to HRP (Pierce, Rockford, IL) in 1X TBST with 5% (w/v) NFDM. Unbound anti-goat IgG-HRP antibodies were removed by rinsing the membra ne briefly and then washing four times

of 10 minutes each in 1X TBST. The SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was a dded to the membrane according to the

manufacturers’ instructions. The membrane was exposed to Hyperfilm ECL (Amersham, Piscataway, NJ) to record an image of the immunoreactive bands.

D.6. Moisture Analysis

Forage and immature and mature seed were analyzed for moisture content using an IR-200 Moisture Analyzer (Denver Instrume nt Company, Arvada, CO) according to SOP AG-EQ-1023-01. A homogeneous tissue-specific site pool (TSSP) was prepared using MON 87769 and conventional soybean control sa mples of a given tissu e type grown at a given site. These pools were prepared for all tissue types in this study. The average percent moisture for each T SSP was calculated from triplicate analyses. A TSSP Dry Weight Conversion Factor (DWC F) was calculated as follows:

DWCF = 1 – [Mean % TSSP Moisture / 100]

The DWCF was used to convert protein levels assessed on a μg/g fresh weight (FW) basis into levels reported on a μg/g dr y weight (DW) basis using the following

calculation:

The protein levels that were reported to be le ss than or equal to the LOD or less than the LOQ on a fresh weight basis were no t reported on a dry weight basis.

D.7. Data Analyses

Quantitation of the bands was performed acco rding to the current version of BR-ME0932. At least three standards we re present on the film and us ed to generate the standard curve for sample quantitation. For the tissues that protei n expression was not detected, only the standard curve was generated. For th e tissue types where protein expression was detected, at least two sample replicates were used for densitometry and quantitation. The coefficient of variation (CV) met the pre-set criteria of ≤ 33%. 

### ( ) ( )

DWCF Weight Fresh Level otein Pr Weight Dry in Level otein Pr =Monsanto Company 09-SY-200U Page 240 of 395 Appendix E. Materials and Methods Used for the Compositional Analysis of MON 87769 Soybean Seed, Forage and Processed Fractions from the Five Replicated Field Sites in the U.S. in 2006 Growing Season

E.1. Materials

MON 87769, a conventional soybean contro l (A3525) and conventional reference soybean varieties were grown at five U.S. locations in 2006. MON 87769 and the conventional soybean control were grow n from seed lots GLP-0604-17267-S and GLP0604-17278-S, respectively. The conventional soybean control has background genetics representative of MON 87769 but does not contain neither the Nc.Fad3 nor the Pj.D15D coding sequence. In additi on, 10 conventional soybean va rieties produced alongside of MON 87769 were included for the generation of a 99% tolerance interval. The varieties, locations, and seed lot nu mbers are listed below:

Vendor/Variety Starting Seed Lot No. Field Site

Stine/ST3300 GLP-0605-17335-S IA-1

Asgrow/A3244 GLP-0604-17273-S IA-1

Asgrow/A2869 GLP-0604-17264-S IA-1

Stine/ST2788 GLP-0605-17334-S IA-2

Lewis 372 GLP-0604-17261-S IA-2

Stine/ST3300 GLP-0605-17335-S IA-2

Stine/ST3600 GLP-0605-17336-S IL

P-93B82 GLP-0604-17260-S IL

Lewis 392 GLP-0604-17262-S IL

Asgrow/A2553 GLP-0604-17263-S MI

Asgrow/A2804 REF-0506-16373-S MI

Lewis 372 GLP-0604-17261-S MI

Lewis 372 GLP-0604-17261-S OH

Asgrow/A3244 GLP-0604-17273-S OH

Stine/ST3300 GLP-0605-17335-S OH

E.2. Characterization of the Materials

The identities of MON 87769, the conventional soybean control, and reference soybean varieties were verified prio r to their use in the study by confirming the chain-of-custody documentation of the samples from the field cooperators. The seed samples from MON 87769, the conventional soybean control, and reference soybean varieties wereMonsanto Company 09-SY-200U Page 241 of 395 further characterized by an event-specific PCR an alysis of the DNA extracted from the seed to confirm the presence or absence of MON 87769.

E.3. Field Production of the Samples

Soybean forage and seed of MON 87769 and conventional soybean control collected from each of three replicate plots and conven tional reference soybean varieties collected from one replicate plot at each of five U.S. field sites were analyzed. Seed was planted in a randomized complete block design with three replicates per block of MON 87769, a conventional soybean control, and conventional reference s oybean varieties. All the samples at the field sites were grown under normal agronomic field conditions for their respective geographic regions. The five U.S. sites were: Site IA -1, Richland, IA; Site IA-2, Bagley, IA; Site IL, Carlyle, IL; Site MI, Conklin, MI; and Site OH, New Holland, OH. Forage and seed samples were harves ted from all plots and shipped on dry ice (forage) or ambient temperature (seed) to Monsanto Company, St. Louis, MO. A subsample for use in compositional analyses was prepared from each of the bulk forage and seed samples generated in the field. E ach sub-sample was ground, stored in a -20 °C freezer located at Monsanto Company (St. L ouis, MO), and then shipped overnight, on dry ice to Covance Laboratories Inc. (Mad ison, WI) for compositional analyses. The labels on the samples shipped to Covance La boratories Inc. listed the composition study number, tissue type, material name, storage conditions, and a unique sample ID number.

E.4. Analytical Method Summaries and Ref erence Standards for Seed and Forage Analysis

E.4.1. Acid Detergent Fiber

The sample was placed in a fritted vessel and washed with an acidic boiling detergent solution that dissolved the protein, carbohydrat e, and ash. An acetone wash removed the fats and pigments. The lignocellulose fracti on was collected on the frit and determined gravimetrically. The limit of qua ntitation for this study was 0.100%.

Reference USDA. 1970. Forage and Fiber Analyses. Agriculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

E.4.2. Amino Acid Composition

Total aspartic acid (i ncluding asparagine)

Total threonine

Total serine Total glutamic acid (including glutamine) Total proline Total glycine

Total alanine

Total valine

Total isoleucine

Total leucineMonsanto Company 09-SY-200U Page 242 of 395 Total tyrosine Total phenylalanine

Total histidine Total lysine Total arginine Total tryptophan Sulfur-containing amino acids: Total methionine Total cystine (including cysteine)

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. The limit of quantitation was 0.100 mg/g.

Reference Standards

• Beckman K18, 2.5 μ mol/mL per constituent except cystine (1.25 μ mol/mL), Lot

Number S702114

• Sigma, L-Tryptophan, 100%, Lot Number 076K50075

• Fluka, L-Cysteic Acid Monohydr ate, 100%, Lot Number 1157629

• Sigma, L-Methionine Sulfone, 100%, Lot Number 012H3349

Reference

AOAC. 2005. Method 982.30 in Official Methods of Analysis of AO AC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.3. Ash

The sample was placed in an electric furnace at 550 ° C and ignited to drive off all volatile organic matter. The nonvolatile matter re maining was quantitated gravimetrically and

calculated to determine percent ash. The limit of quantitation was 0.100%.

Reference

AOAC. 2005. Method 923 in Official Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.4. Carbohydrates

The total carbohydrate level was calculated by difference usin g the fresh weight-derived data and the following equation:

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The limit of quantitation was 0.100%.Monsanto Company 09-SY-200U Page 243 of 395 Reference USDA. 1973. Energy value of foods. Agri culture Handbook No. 74. United States Department of Agriculture, Washington, D.C.

E.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 steambath unde r nitrogen, dried, and weighed. The limit of quantitation was 0.100%.

Reference

AOAC. 2005. Methods 922.06 and 954.02 in Official Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.6. Fat by Soxhlet Extraction

The sample was weighed into a cellulose 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 weighed. The limit of quantitation was 0.100%.

Reference

AOAC. 2005. Method 960.39 in Official Methods of Analysis of AO AC International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.7. Fatty Acid Profile with Trans Fat by GC

The lipid was extracted, saponified with 0.5N methanolic sodium hydroxide, and methylated with 14% BF 3 -methanol. The resulting methyl es ters of the fatty acids were extracted with heptane. An internal standard was added prior to th e lipid extraction. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation. Th e limit of quantitation was 0.0200%.

Reference Standards

• Nu Chek Prep GLC Reference Standa rd Hazelton No. 1, Lot Number AU22-P

• Nu Chek Prep GLC Reference Standard Hazelton No. 2, Lot Number M13-O

• Nu Chek Prep GLC Reference Standa rd Hazelton No. 3, Lot Number N28-Q

• Nu Chek Prep GLC Reference Standa rd Hazelton No. 4, Lot Number N28-Q

• Nu Chek Prep Methyl Gamma Linolenate, used as 100% Lot Number U-63M-N30-Q

• Sigma Methyl Tridecanoate, Lot Numb er 036K2636, used as 99%, Lot Numbers

035K1392 and 046K1065, used as 100%

• Nu Chek Prep Methyl Butyrate, used as 100%, Lot Number N-4M-A4-R

• Nu Chek Prep Methyl Hexanoate, us ed as 100%, Lot Number N-6M-S1-Q

• Nu Chek Prep Methyl Erucate, us ed as 100%, Lot Number U-79M-AU3-Q

• Nu Chek Prep Methyl Lignocerate, us ed as 100%, Lot Number N-24M-A5-RMonsanto Company 09-SY-200U Page 244 of 395 • Nu Chek Prep Methyl Docosapentaenoate , used as 100%, Lot Number U-101M-MA27-

R

• Nu Chek Prep Methyl Docosahexaenoate , used as 100%, Lot Number U-84M-D18-Q

• Nu Chek Prep Methyl Eicosapentaenoate , used as 100%, Lot Number U-99M-F13-R

• Cayman Chemicals Steariodonic Acid Met hyl Ester, used as 100%, Lot Number

171721-172473

• Nu Chek Prep Methyl Elaidate, us ed as 100%, Lot Number U-47M-JA18-R

• Nu Chek Prep Methyl Linoelaidate, us ed as 100%, Lot Number U-60M-MA27-R

References

AOAC. 2005. Method 996.06 in Official Methods of Analysis of AO AC International, 18th ed. AOAC International, Gaithersburg, MD.

AOCS. 1997. Method Ce 2c-66 and Ce 1C-89 in Official Methods and Recommended Practices
of the AOCS, 5th ed. American Oil Chemists' Society, Champaign, IL.

AOAC. 2005. Method 983.23 in Official Methods of Analysis of AO AC International, 18th ed. AOAC International, Gaithersburg, MD

E.4.8. Isoflavones Analysis

The sample was extracted using a solution of hydrochloric acid and reagent alcohol heated on steam baths or hot plates. The ex tract 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 ultraviole t detection and was compared to an external standard curve of known standards for qua ntitation. The limit of quantitation for each component was 10.0 ppm.

Reference Standards Indofine, Daidzein, 99%, Lot Number 020508146 Indofine, Genistein, > 99%, (used as 100%), Lot Number 0604043 Indofine, Glycitein, 99%, Lot Number 0704034

References Seo, A. and C.V. Morr. 1984. Improved high-performance liquid chromatographic analysis of phenolic acids and isoflavonoids from soybean protein products. Journal of Agricultural and Food Chem istry, 32(3):530-533, (1984).

Pettersson, H., and K.H. Kie ssling. 1984. Liquid Chromatogra phic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official Analytical Chemists Journal, 67(3):503-506.

E.4.9. Lectin

The sample was suspended in phosphate buffered saline (PBS), shaken, and filtered. AnMonsanto Company 09-SY-200U Page 245 of 395 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. The limit of quantitation was 0.10 H.U./mg.

Reference Standard Sigma-Aldrich, Red Blood Cells, Rabb it, Product #R1629, Lot Number 105K6042

References Klurfeld, D. M. and Kritchevsky, D., “I solation and Quantitation of Lectins from Vegetable Oils,” Lipids, 22:667-668, (1987).

Klurfeld, D. M., Pe rsonal communication.

Liener, I. E., “The Photometric Determinati on of the Hemagglutina ting Activity of Soyin and Crude Soybean Extracts,” Archives of Bioche mistry and Biophysics, 54:223-231, (1955).

E.4.10. Moisture

The sample was dried in a vacuum oven at approximately 100 ° C to a constant weight. The moisture weight loss was determined a nd converted to percent moisture. The limit of quantitation for this study was 0.100%.

Reference Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 926.08 and 925.09, AOAC INTERNATIONAL: Ga ithersburg, Maryland, (2005).

E.4.11. Neutral Detergent Fiber, Enzyme Method

The sample was placed in a fritted vessel and washed with a neutral boiling detergent solution that dissolved the protein, 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 grav imetrically. The limit of quantitation was

0.100%.

References Approved Methods of the American Association of Cereal Chemists , 9th Ed., Method 32.20, (1998).

USDA. 1970. Forage and Fiber Analyses. Agriculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.Monsanto Company 09-SY-200U Page 246 of 395 E.4.12. Phytic Acid

The sample was extracted using 0.5 M HCl with ultrasonication. Purification and concentration were accomplished on a silica- based anion-exchange column. The sample was analyzed on a polymer high-performan ce liquid chromatography column PRP-1,

5 μ m (150 x 4.1mm) with a refractive index de tector. The limit of quantitation was

0.100%.

Reference Standard Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 97%, Lot Number 035K0590

References Lehrfeld, Jacob, “HPLC Separation and Quantita tion of Phytic Acid and Some Inositol Phosphates in Foods: Problem and Solutions,” Journal of Agricultural and Food Chemistry , 42:2726-2731, (1994).

Lehrfeld, Jacob, “High-Performance Liquid Chro matography Analysis of Phytic Acid on a pH-Stable, Macroporous Polymer Column,” Cereal Chemistry , 66(6):510-515, (1989).

E.4.13. Protein

Nitrogenous compounds in the sample were redu ced in the presence of boiling sulfuric acid and a mercury catalyst mixture to form am monia. The acid digest was made alkaline.

The ammonia was distilled and then titrated with a previously standardized acid. The percent nitrogen was calculated and converted to equivalent protein using the factor 6.25. The limit of quantitation was 0.100%.

References Official Methods of Analysis of AOAC INTERNATIONAL, 18 th Ed., Methods 955.04 and 979.09, AOAC INTERNATIONAL, Ga ithersburg, Maryland, (2005).

Bradstreet, R. B., The Kjeldahl Method for Organic Nitrogen , Academic Press: New York, New York, (1965).

Kalthoff, I. M., and Sandell, E. B., Quantitative Inorganic Analysis , MacMillan: New York, (1948).Monsanto Company 09-SY-200U Page 247 of 395 E.4.14. Raffinose and Stachyose

The sample was extracted with deionize d water and the extract treated with a hydroxylamine hydrochloride solution in pyridine, containing phenyl- β -D-glucoside as an internal standard. The resulting oxime s were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization de tector. The quantitation limit was 0.0500%.

Reference Standards

• Sigma, Raffinose Pentahydrate, 99% (84% af ter correction for degree of hydration),

Lot Number 035K1371

• Sigma, Stachyose, 98% (96.4% after correcti on for degree of hydration), Lot Number

065K3775

References Brobst, K. M. 1972. Gas-Liquid Chromatogr aphy of Trimethylsilyl Derivatives in Methods in Carbohydrate Chemistry, Vol. 6. Academic Press, New York.

Mason, B.S., and H.T. Slover. 1971. A Gas Chromatographic Method for the Determination of Sugars in Foods. Jour nal of Agricultural and Food Chemistry, 19(3):551-554

E.4.15. 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. The lim it of quantitation was 1.00 Trypsin Inhibitor Units (TIU)/mg.

Reference

AOCS. 1997. Method Ba 12-75 in Official Me thods and Recommended Practices of the American Oil Chemists’ Society. AOCS Press, Champaign, IL.

E.4.16. Vitamin E

The sample was saponified to break down any fat and release vitamin E. The saponified mixture was extracted with ethyl ether and then quantitated by hi gh-performance liquid chromatography using a silica column. The limit of quantitation was 0.500 mg/100 g.

Reference Standard USP, Alpha Tocopherol, 100%, Lot Number MMonsanto Company 09-SY-200U Page 248 of 395 References Cort, W.M., T.S. Vincente, E.H. Waysek, and B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high-performance liquid chromatographic Fluorescence. Journal of Agricultural Food Chemistry, 31:1330-1333.

Speek, A.J., J. Schijver, and W.H.P. Schreu rs. 1985. Vitamin E composition of some seed oils as determined by high-performance liquid chromatography with fluorometric quantitation. Journal of Food Science, 50(1):121-124.

McMurray, C.H., W.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction techniques on determination of α -tocopherol in animal feedstuffs. Journal of the

Association of Official Analytical Chemists

, 63(6):1258-1261.

E.5. Data Processing

After compositional analyses were performed at Covance Laboratories Inc., data spreadsheets were forwarded to Monsanto Co mpany. The data were reviewed, formatted, and sent to Certus International, Inc. for st atistical analysis. A statistical sub-report was generated by Certus and sent to Monsanto Company.

In all, 75 different analytical compone nts were measured. Of these evaluated components, 26 had more than 50% of observa tions below the assay limit of quantitation (LOQ) and, as a result, were excluded from the statistical analysis. The following analytes were excluded from statistical anal ysis: 8:0 caprylic acid, 10:0 capric acid, 12:0 lauric acid, 14:0 myristic acid, 14:1 my ristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic aci d, 17:1 heptadecenoic acid, 18:1 total trans-octadecenoic acid, 18:2 iso linoleic acid, 18:2 total trans-linoleic acid, 18:3 9c,12c,15t trans-ALA, 18:3 gamma linolen ic acid, 18:4 6c,9c,12c,15t trans-SDA, 18:4 stearidonic acid, 20:2 eicosadienoic ac id, 20:3 eicosatrienoic acid, 20:4 arachidonic acid, 20:5 eicosapentaenoic acid, 22:1 eruc ic acid, 22:5 docosapentaenoic acid, 22:6 docosehexaenoic acid, and 24:0 lignoceric acid. The LOQ for the fatty acid method was 0.0200% fresh weight.

For four of the seed fatty acids excluded from statistical analysis (GLA, trans-ALA, SDA, and trans-SDA), the majority of values were below the LOQ for the conventional soybean control and conventional reference s oybean varieties, but were measured in MON 87769. Because of their presence in quantities higher than the LOQ in MON 87769, data for the above four fatty acid s were excluded from the calculation of total fatty acids for use in the fatty acid composition data re-expression formula.

The following 21 observations for seed tissue samples were below the LOQ: 18:4 6c,9c,12c,15t trans-SDA (three values), 20:1 ei cosenoic acid (17 values), and vitamin E (one value). To include a complete data set for these analytes in the statistical analysis, observations below the limit of quantitation were assigned a value equal to half the limit of quantitation (LOQ) for these 21 data points.

The data were assessed for potential outliers using a st udentized PRESS residuals

calculation. No outliers were identified in the data set.Monsanto Company 09-SY-200U Page 249 of 395 Statistical Methodology

At the field sites, MON 87769, the conventiona l soybean control, and reference soybean varieties were grown in single plots randomly assigned within each of three replication blocks. The compositional components for MON 87769 and the conventional soybean control were statistically anal yzed using a mixed-model analysis of variance. The data from the five replicated sites were analyzed separately and as a combined data set. Individual replicated site analyses used the model:

Y ij = U + T i + B j + e ij ,

where Y ij = unique individual observation, U = overall mean, T i = variety effect, B j = random block effect, and e ij = residual error.

Combined-site analyses used the model:

Y ijk = U + T i + L j + B(L) jk

+ LT ij + e ijk ,

where Y ijk = unique individual observation, U = overall mean, T i = variety effect, L j = random location effect, B(L) jk

= random block within location effect, LT ij = random location by variety inte raction effect, and e ijk = residual error. For each of the forage and seed compositional components, MON 87769 was compared to the conventional soybean

control.

A range of observed values from the referen ce soybean varieties was determined for each analytical component. Additionally, the refere nce soybean varieties’ data were used to develop population tolerance intervals. A tole rance interval is an interval that one can claim, with a specified degree of confidence, contains at least a sp ecified proportion, p, of an entire sampled population for the para meter measured. For each compositional component, 99% tolerance intervals were calcu lated that are expect ed to contain, with 95% confidence, 99% of the quantities expresse d in the population of conventional reference soybean varieties (G eorge et al., 2004; Ridley et al., 2002). Each tolerance interval estimate was based upon one observa tion per unique reference variety. Since individual variety with multiple observations would first be summarized across replicates

within each site and then summarized across si tes to obtain a single value for inclusion in tolerance interval calculations, a single repl icate from each unique reference variety was analyzed for inclusion in tolerance interval calculations. Because negative quantities are not possible, calculated negative lower tolerance bounds were set to zero. SAS programming was used to generate all summar y statistics and perform all analyses (SAS Software Release 9.1, 2002-2003). Report tables present p-values from SAS as either

<0.001 or the actual value trunc ated to three decimals.Monsanto Company 09-SY-200U Page 250 of 395Individual-Site Compositional Comparison of MON 87769 vs.the Conventional Soybean Control for Forage and SeedTable E-1. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Forage from IA-1Site for Fiber and ProximatesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber (% DW)Acid Detergent Fiber 32.11 (1.36) 31.48 (1.36) 0.63 (1.35) -5.20, 6.46 0.688 (24.31 - 31.73)[31.12 - 32.90] [28.48 - 34.85] [-1.95 - 2.64] [19.24, 38.36]Neutral Detergent Fiber33.00 (2.18) 29.47 (2.18) 3.53 (0.83)-0.052, 7.11 0.051 (24.37 - 38.09)[30.50 - 36.34] [25.48 - 34.20] [2.14 - 5.02] [19.01, 46.73]Proximate (% DW, unless noted)Ash 6.26 (0.13) 6.03 (0.13) 0.23 (0.19) -0.57, 1.03 0.342 (5.33 - 7.77)[6.06 - 6.46] [5.74 - 6.18] [-0.12 - 0.53] [3.73, 9.32]Carbohydrates 64.75 (0.58) 66.01 (0.58) -1.26(0.82) -4.79, 2.28 0.265 (64.94 - 72.18)[64.27 - 65.07] [65.13 - 67.57] [-3.30 - -0.055] [60.10, 76.68]Moisture (% FW) 74.67 (0.57) 73.07 (0.57) 1.60 (0.80) -1.85, 5.05 0.183 (71.10 - 74.90)[73.20 - 75.90] [72.80 - 73.30] [0.10 - 3.10] [67.41, 78.15]Protein 23.17 (0.50) 22.22 (0.50) 0.95 (0.70) -2.06, 3.97 0.306 (16.96 - 21.65)[22.13 - 23.82] [21.36 - 22.96] [-0.22 - 2.21] [13.69, 25.14]Total Fat 5.82 (0.32) 5.74 (0.32) 0.075 (0.26) -1.04,1.19 0.800 (4.15 - 7.02)[5.22 - 6.34] [5.33 - 6.36] [-0.32 - 0.56] [1.46, 9.88]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 251 of 395Table E-2. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and IsoflavonesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Alanine 1.78 (0.015) 1.72 (0.015) 0.057 (0.021) -0.033, 0.15 0.112 (1.63 - 1.86)[1.77 - 1.78] [1.68 - 1.75] [0.033 - 0.10] [1.45, 2.02]Arginine 3.20 (0.056) 2.94 (0.056) 0.26 (0.079) -0.083, 0.60 0.082 (2.61 - 3.15)[3.12 - 3.25] [2.81 - 3.04] [0.14 - 0.44] [2.13, 3.62]Aspartic Acid 4.57 (0.055) 4.39 (0.055) 0.18 (0.065) -0.097, 0.46 0.106 (4.01 - 4.71)[4.51 - 4.63] [4.27 - 4.51] [0.12 - 0.31] [3.45, 5.29]Cystine 0.64 (0.0098) 0.61 (0.0098) 0.026 (0.0081) -0.0086, 0.061 0.083 (0.55 - 0.62)[0.63 - 0.65] [0.60 - 0.64] [0.010 - 0.036] [0.49, 0.68]Glutamic Acid 7.66 (0.079) 7.29 (0.079) 0.36 (0.11) -0.11, 0.84 0.081 (6.67 - 7.99)[7.59 - 7.70] [7.10 - 7.46] [0.22 - 0.60] [5.51, 9.04]Glycine 1.80 (0.012) 1.72 (0.012) 0.075 (0.017) 0.0027, 0.15 0.046 (1.61 - 1.86)[1.79 - 1.80] [1.69 - 1.75] [0.047 - 0.11] [1.39, 2.05]Histidine 1.09 (0.010) 1.05 (0.010) 0.040 (0.014) -0.023, 0.10 0.111 (0.98 - 1.13)[1.09 - 1.10] [1.03 - 1.08] [0.013 - 0.068] [0.86, 1.27]Isoleucine 1.88 (0.026) 1.79 (0.026) 0.090(0.036) -0.067, 0.25 0.132 (1.62 - 2.00)[1.88 - 1.88] [1.72 - 1.84] [0.037 - 0.16] [1.34, 2.28]Monsanto Company 09-SY-200U Page 252 of 395Table E-2. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Leucine 3.19 (0.022) 3.08 (0.022) 0.11 (0.027) -0.00097, 0.23 0.050 (2.86 - 3.37)[3.19 - 3.20] [3.04 - 3.14] [0.061 - 0.15] [2.45, 3.76]Lysine 2.70 (0.020) 2.62 (0.020) 0.077 (0.024) -0.027, 0.18 0.085 (2.42 - 2.78)[2.68 - 2.71] [2.59 - 2.67] [0.039 - 0.12] [2.13, 3.06]Methionine 0.60 (0.0064) 0.58 (0.0064) 0.026 (0.0085) -0.010,0.063 0.089 (0.52 - 0.61)[0.60 - 0.61] [0.57 - 0.59] [0.0098 - 0.038] [0.48, 0.66]Phenylalanine 2.13 (0.025) 2.03 (0.025) 0.10 (0.017) 0.034, 0.18 0.024 (1.92 - 2.29)[2.08 - 2.16] [1.99 - 2.07] [0.086 - 0.14] [1.61, 2.55]Proline 2.12 (0.020) 2.00 (0.020) 0.12 (0.024) 0.014, 0.22 0.039 (1.81 - 2.16)[2.11 - 2.13] [1.97 - 2.06] [0.070 - 0.15] [1.53, 2.45]Serine 2.21 (0.035) 2.13 (0.035) 0.073 (0.049) -0.14,0.28 0.278 (1.97 - 2.27)[2.16 - 2.24] [2.05 - 2.19] [-0.023 - 0.19] [1.75, 2.51]Threonine 1.60 (0.026) 1.57 (0.026) 0.039 (0.029) -0.084, 0.16 0.304 (1.45 - 1.65)[1.57 - 1.64] [1.51 - 1.61] [-0.0089 - 0.090] [1.30, 1.82]Tryptophan 0.47 (0.011) 0.45 (0.011) 0.019 (0.016) -0.051, 0.088 0.363 (0.43 - 0.52)[0.45 - 0.50] [0.44 - 0.46] [-0.0067 - 0.059] [0.35, 0.59]Monsanto Company 09-SY-200U Page 253 of 395Table E-2. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromSite IA for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Tyrosine 1.40 (0.056) 1.34 (0.056) 0.064 (0.079) -0.28, 0.41 0.504 (1.21 - 1.49)[1.27 - 1.47] [1.26 - 1.41] [-0.072 - 0.21] [1.03, 1.67]Valine 1.99 (0.027) 1.89 (0.027) 0.097 (0.038) -0.066, 0.26 0.124 (1.70 - 2.11)[1.98 - 2.00] [1.82 - 1.94] [0.037 - 0.18] [1.42, 2.41]Fatty Acid (% Total FA)16:0 Palmitic 12.39 (0.056) 12.06 (0.056) 0.34(0.078) -0.0012, 0.67 0.050 (9.88 - 12.33)[12.27 - 12.54] [12.05 - 12.06] [0.21 - 0.48] [7.28, 14.20]18:0 Stearic 4.00 (0.027) 4.04(0.027) -0.043 (0.015) -0.11,0.019 0.096 (3.68 - 4.89)[3.96 - 4.06] [4.01 - 4.08] [-0.064 - -0.015] [2.87, 5.85]18:1 Total 18:1 13.42 (0.14) 18.45 (0.14) -5.04 (0.20) -5.92, -4.15 0.001 (16.70 - 23.16)[13.14 - 13.80] [18.35 - 18.52] [-5.38 - -4.55] [12.56, 27.98]18:2 9c,12c Linoleic 18.46 (0.11)54.90 (0.11) -36.45 (0.078) -36.78, -36.11 <0.001 (53.36 - 57.39)[18.24 - 18.68] [54.80 - 55.07] [-36.60 - -36.35] [50.46, 59.96]18:3 Linolenic 11.11 (0.019) 9.85 (0.019) 1.27 (0.0091) 1.23, 1.30 <0.001 (6.95 - 10.58)[11.08 - 11.13] [9.80 - 9.88] [1.25 - 1.28] [3.72, 13.46]20:0 Arachidic 0.33 (0.0038) 0.30 (0.0038) 0.032 (0.0024) 0.021, 0.042 0.005 (0.27 - 0.36)[0.32 - 0.34] [0.29 - 0.30] [0.029 - 0.037] [0.20, 0.45]Monsanto Company 09-SY-200U Page 254 of 395Table E-2. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromSite IA-1 for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Fatty Acid (% Total FA)20:1 Eicosenoic 0.10 (0.017) 0.086 (0.017) 0.017(0.023) -0.084, 0.12 0.540 (0.071 - 0.19)[0.075 - 0.15] [0.082 - 0.089] [-0.013 - 0.063] [0, 0.31]22:0 Behenic 0.29 (0.014) 0.31(0.014) -0.028 (0.0039) -0.045,-0.011 0.018 (0.30 - 0.41)[0.26 - 0.31] [0.30 - 0.34] [-0.035 - -0.022] [0.22, 0.49]Fiber (% DW)Acid Detergent Fiber 15.21 (0.50) 16.19 (0.50) -0.98 (0.70) -4.01, 2.05 0.298 (14.57 - 18.85)[14.38 - 16.48] [15.70 - 16.70] [-1.91 - 0.77] [10.36, 22.77]Neutral Detergent Fiber15.79 (0.80) 15.80 (0.80) -0.0054 (1.13)-4.87, 4.86 0.996 (15.03 - 18.92)[15.46 - 16.05] [14.43 - 18.01] [-2.55 - 1.44] [10.91, 22.59]Proximate (% DW, unless noted)Ash 5.74 (0.079) 5.72 (0.079) 0.025 (0.11) -0.46, 0.51 0.843 (5.59 - 6.20)[5.66 - 5.82] [5.60 - 5.92] [-0.17 - 0.21] [5.16, 6.64]Carbohydrates 37.57 (0.89) 40.99 (0.89) -3.41(1.25) -8.81, 1.98 0.112 (33.50 - 40.22)[35.70 - 39.54] [39.93 - 41.94] [-6.24 - -0.39] [26.76, 45.99]Moisture (% FW) 7.55 (0.21) 7.37 (0.21) 0.18 (0.29) -1.08, 1.44 0.607 (6.68 - 8.16)[7.14 - 8.01] [7.17 - 7.66] [-0.52 - 0.84] [5.23, 9.56]Protein 41.93 (0.32) 40.09 (0.32) 1.84 (0.45) -0.080, 3.77 0.054 (37.52 - 42.37)[41.35 - 42.27] [39.43 - 40.55] [1.07 - 2.75] [33.37, 46.00]Monsanto Company 09-SY-200U Page 255 of 395Table E-2. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-1Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]ProximateTotal Fat 14.75 (0.65) 13.21(0.65) 1.54 (0.92) -2.42,5.50 0.235 (13.99 - 20.56)[13.35 - 16.31] [12.73 - 13.86] [-0.51 - 3.27] [11.04, 25.03]VitaminVitamin E (mg/100g DW) 1.02 (0.11)1.10 (0.11) -0.082 (0.11) -0.57, 0.40 0.543 (0.27 - 2.93)[0.86 - 1.30] [0.97 - 1.19] [-0.28 - 0.11] [0, 4.65]Antinutrient (% DW, unless noted)Lectin (H.U./mg DW) 4.28 (1.49) 2.31 (1.49)1.97 (1.59) -4.88, 8.82 0.341 (0.81 - 9.73)[1.52 - 8.07] [0.75 - 3.23] [0.014 - 5.13] [0, 16.00]Phytic Acid 1.28 (0.048) 1.17 (0.048) 0.11(0.023) 0.011, 0.21 0.041 (0.81 - 1.27)[1.22 - 1.34] [1.06 - 1.26] [0.079 - 0.16] [0.51, 1.59]Raffinose 0.35 (0.019) 0.31 (0.019) 0.039 (0.022) -0.055, 0.13 0.215 (0.31 - 0.42)[0.32 - 0.40] [0.29 - 0.32] [0.0029 - 0.079] [0.19, 0.52]Stachyose 3.05 (0.087) 2.78 (0.087) 0.27 (0.076) -0.055, 0.60 0.070 (2.23 - 3.29)[2.93 - 3.27] [2.69 - 2.86] [0.16 - 0.41] [1.61, 4.05]Trypsin Inhibitor (TIU/mg DW) 45.27 (5.77) 35.70 (5.77) 9.57 (8.16) -25.56, 44.70 0.361 (24.29 - 46.29)[33.06 - 54.80] [25.67 - 41.69] [-6.68 - 29.14] [8.09, 57.27]Isoflavone (μg/g DW)Daidzein 995.39 (13.83) 1550.96 (13.83) -555.58 (19.56) -639.75, -471.40 0.001 (783.49 - 1691.97)[978.37 - 1026.28] [1531.49 - 1572.77] [-594.40 - -522.35] [0, 2594.50]Monsanto Company 09-SY-200U Page 256 of 395Table E-2. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Isoflavone (μg/g DW)Genistein 594.53 (7.52) 973.04 (7.52) -378.51 (4.09) -396.09,-360.93 <0.001 (741.53 - 1580.48)[584.75 - 612.91] [966.28 - 983.61] [-384.49 - -370.70] [254.31, 1976.30]Glycitein 87.40 (9.32) 103.25 (9.32) -15.86(6.20) -42.55, 10.83 0.124 (74.87 - 189.64)[73.40 - 106.72] [86.17 - 113.71] [-27.80 - -6.99] [0, 243.40]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 257 of 395Table E-3. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Forage from IA-2Site for Fiber and ProximatesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber (% DW)Acid Detergent Fiber 26.08 (0.67) 25.76 (0.67) 0.33 (0.23) -0.66, 1.32 0.290 (24.31 - 31.73)[24.91 - 26.79] [24.39 - 26.92] [-0.13 - 0.59] [19.24, 38.36]Neutral Detergent Fiber 30.83 (1.42) 32.11 (1.42) -1.28 (2.00) -9.90, 7.35 0.589 (24.37 - 38.09)[28.06 - 34.06] [30.71 - 33.99] [-5.94 - 2.44] [19.01, 46.73]Proximate (% DW, unless noted)Ash 7.19 (0.13) 7.45 (0.13) -0.26 (0.16) -0.95, 0.43 0.244 (5.33 - 7.77)[7.00 - 7.30] [7.25 - 7.75] [-0.49 - 0.048] [3.73, 9.32]Carbohydrates 64.72 (0.85) 62.33 (0.85) 2.39(1.07) -2.19, 6.98 0.154 (64.94 - 72.18)[62.88 - 65.63] [61.18 - 63.79] [0.88 - 4.45] [60.10, 76.68]Moisture (% FW) 72.93 (0.94) 74.03 (0.94) -1.10 (0.67) -3.96, 1.76 0.240 (71.10 - 74.90)[70.70 - 74.80] [72.90 - 74.70] [-2.20 - 0.10] [67.41, 78.15]Protein 23.23 (0.76) 23.95 (0.76) -0.73 (0.64) -3.50, 2.05 0.376 (16.96 - 21.65)[22.18 - 24.53] [22.33 - 24.98] [-2.01 - -0.017] [13.69, 25.14]Total Fat 4.87 (0.20) 6.27 (0.20) -1.40 (0.28) -2.60, -0.20 0.037 (4.15 - 7.02)[4.40 - 5.28] [6.13 - 6.49] [-2.09 - -0.92] [1.46, 9.88]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 258 of 395Table E-4. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-2 Site for Amino Acids, Fatty Acids, Fiber,Proximates, Vitamin E, Antinutrients and IsoflavonesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Alanine 1.79 (0.018) 1.78 (0.018) 0.0095 (0.022) -0.083, 0.10 0.703 (1.63 - 1.86)[1.76 - 1.81] [1.74 - 1.81] [-0.018 - 0.052] [1.45, 2.02]Arginine 3.42 (0.037) 3.11 (0.037) 0.31(0.020) 0.23, 0.40 0.003 (2.61 - 3.15)[3.36 - 3.50] [3.05 - 3.15] [0.28 - 0.35] [2.13, 3.62]Aspartic Acid 4.56 (0.053) 4.50 (0.053) 0.060 (0.072) -0.25, 0.37 0.490 (4.01 - 4.71)[4.51 - 4.61] [4.36 - 4.60] [-0.034 - 0.20] [3.45, 5.29]Cystine 0.62 (0.0028) 0.60 (0.0028) 0.024 (0.0019) 0.016, 0.032 0.005 (0.55 - 0.62)[0.62 - 0.63] [0.59 - 0.61] [0.021 - 0.027] [0.49, 0.68]Glutamic Acid 7.73 (0.088) 7.56 (0.088) 0.17 (0.11) -0.32, 0.66 0.283 (6.67 - 7.99)[7.62 - 7.83] [7.36 - 7.71] [-0.0052 - 0.38] [5.51, 9.04]Glycine 1.81 (0.018) 1.78 (0.018) 0.031 (0.022) -0.062, 0.12 0.284 (1.61 - 1.86)[1.78 - 1.83] [1.74 - 1.81] [0.0039 - 0.074] [1.39, 2.05]Histidine 1.10 (0.012) 1.08 (0.012) 0.015 (0.016) -0.056, 0.086 0.455 (0.98 - 1.13)[1.08 - 1.11] [1.05 - 1.10] [-0.013 - 0.044] [0.86, 1.27]Isoleucine 1.89 (0.027) 1.81 (0.027) 0.082(0.038) -0.081, 0.25 0.162 (1.62 - 2.00)[1.84 - 1.94] [1.78 - 1.86] [-0.018 - 0.16] [1.34, 2.28]Monsanto Company 09-SY-200U Page 259 of 395Table E-4. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Leucine 3.21 (0.028) 3.16 (0.028) 0.047 (0.032) -0.092, 0.19 0.279 (2.86 - 3.37)[3.17 - 3.26] [3.10 - 3.19] [-0.015 - 0.094] [2.45, 3.76]Lysine 2.70 (0.029) 2.69 (0.029) 0.014 (0.041) -0.16, 0.19 0.760 (2.42 - 2.78)[2.65 - 2.73] [2.62 - 2.73] [-0.059 - 0.094] [2.13, 3.06]Methionine 0.60 (0.0088) 0.58 (0.0088) 0.025 (0.0099) -0.017,0.067 0.126 (0.52 - 0.61)[0.59 - 0.61] [0.56 - 0.60] [0.0057 - 0.038] [0.48, 0.66]Phenylalanine 2.14 (0.018) 2.11 (0.018) 0.032(0.025) -0.075, 0.14 0.330 (1.92 - 2.29)[2.13 - 2.15] [2.07 - 2.15] [-0.016 - 0.084] [1.61, 2.55]Proline 2.12 (0.021) 2.06 (0.021) 0.061 (0.0073) 0.029, 0.092 0.014 (1.81 - 2.16)[2.09 - 2.15] [2.01 - 2.09] [0.048 - 0.073] [1.53, 2.45]Serine 2.19 (0.028) 2.22 (0.028) -0.030 (0.040) -0.20,0.14 0.529 (1.97 - 2.27)[2.18 - 2.21] [2.17 - 2.29] [-0.11 - 0.041] [1.75, 2.51]Threonine 1.58 (0.015) 1.60 (0.015) -0.013(0.017) -0.086, 0.061 0.542 (1.45 - 1.65)[1.58 - 1.60] [1.56 - 1.62] [-0.039 - 0.020] [1.30, 1.82]Tryptophan 0.46 (0.0039) 0.47 (0.0039) -0.0056(0.0055) -0.029, 0.018 0.417 (0.43 - 0.52)[0.46 - 0.46] [0.46 - 0.48] [-0.019 - 0.0016] [0.35, 0.59]Monsanto Company 09-SY-200U Page 260 of 395Table E-4. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Tyrosine 1.41 (0.022) 1.32 (0.022) 0.089 (0.031) -0.043, 0.22 0.100 (1.21 - 1.49)[1.35 - 1.45] [1.30 - 1.34] [0.015 - 0.14] [1.03, 1.67]Valine 2.01 (0.031) 1.92 (0.031) 0.097 (0.043) -0.089, 0.28 0.154 (1.70 - 2.11)[1.95 - 2.07] [1.89 - 1.96] [-0.017 - 0.18] [1.42, 2.41]Fatty Acid (% Total FA)16:0 Palmitic 11.77 (0.12) 11.36 (0.12) 0.41(0.16) -0.29, 1.12 0.128 (9.88 - 12.33)[11.53 - 11.90] [11.14 - 11.52] [0.0098 - 0.75] [7.28, 14.20]18:0 Stearic 4.21 (0.058) 4.11 (0.058) 0.097 (0.056) -0.14,0.34 0.224 (3.68 - 4.89)[4.15 - 4.27] [3.96 - 4.21] [-0.0058 - 0.19] [2.87, 5.85]18:1 Total 18:1 13.56 (0.31) 18.53 (0.31) -4.97 (0.31) -6.32, -3.63 0.003 (16.70 - 23.16)[12.93 - 14.36] [18.29 - 18.72] [-5.36 - -4.36] [12.56, 27.98]18:2 9c,12c Linoleic 21.19 (0.57)55.33 (0.57) -34.14 (0.80) -37.60, -30.68 <0.001 (53.36 - 57.39)[20.36 - 22.78] [55.21 - 55.58] [-35.15 - -32.43] [50.46, 59.96]18:3 Linolenic 11.14 (0.12) 9.96 (0.12) 1.18 (0.16) 0.47, 1.89 0.018 (6.95 - 10.58)[11.10 - 11.18] [9.73 - 10.28] [0.82 - 1.45] [3.72, 13.46]20:0 Arachidic 0.34 (0.0072) 0.30 (0.0072) 0.036(0.010) -0.0077, 0.080 0.071 (0.27 - 0.36)[0.33 - 0.34] [0.28 - 0.32] [0.017 - 0.058] [0.20, 0.45]Monsanto Company 09-SY-200U Page 261 of 395Table E-4. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Fatty Acid (% Total FA)20:1 Eicosenoic 0.13 (0.015) 0.084 (0.015) 0.043(0.021) -0.048, 0.13 0.180 (0.071 - 0.19)[0.086 - 0.15] [0.075 - 0.092] [-0.0061 - 0.072] [0, 0.31]22:0 Behenic 0.27 (0.019) 0.32(0.019) -0.051 (0.027) -0.17,0.067 0.206 (0.30 - 0.41)[0.27 - 0.27] [0.28 - 0.37] [-0.11 - -0.019] [0.22, 0.49]Fiber (% DW)Acid Detergent Fiber 17.23 (0.90) 16.16 (0.90) 1.07 (0.85) -2.57, 4.71 0.333 (14.57 - 18.85)[16.45 - 17.62] [13.80 - 17.85] [-0.24 - 2.65] [10.36, 22.77]Neutral Detergent Fiber 17.34 (0.97) 17.64 (0.97) -0.31 (1.33) -6.03, 5.42 0.839 (15.03 - 18.92)[15.47 - 19.15] [16.73 - 19.37] [-1.98 - 2.32] [10.91, 22.59]Proximate (% DW, unless noted)Ash 5.52 (0.091) 5.41 (0.091) 0.11 (0.065) -0.17, 0.39 0.229 (5.59 - 6.20)[5.46 - 5.63] [5.24 - 5.64] [-0.0074 - 0.22] [5.16, 6.64]Carbohydrates 36.95 (0.79) 40.25 (0.79) -3.30(1.12) -8.12, 1.52 0.098 (33.50 - 40.22)[35.58 - 39.06] [39.89 - 40.93] [-4.71 - -0.88] [26.76, 45.99]Moisture (% FW) 8.09 (0.066) 7.98 (0.066) 0.11 (0.093) -0.29, 0.51 0.348 (6.68 - 8.16)[7.99 - 8.21] [7.88 - 8.11] [-0.12 - 0.26] [5.23, 9.56]Protein 42.80 (0.78) 40.42 (0.78) 2.37 (0.95) -1.73, 6.48 0.130 (37.52 - 42.37)[42.50 - 43.36] [38.31 - 41.58] [0.96 - 4.19] [33.37, 46.00]Monsanto Company 09-SY-200U Page 262 of 395Table E-4. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]ProximateTotal Fat 14.73 (0.76) 13.91 (0.76) 0.81 (0.74) -2.37,4.00 0.386 (13.99 - 20.56)[12.95 - 15.65] [13.24 - 15.13] [-0.30 - 2.22] [11.04, 25.03]VitaminVitamin E (mg/100g DW) 1.05 (0.11)1.18 (0.11) -0.13 (0.15) -0.79, 0.53 0.489 (0.27 - 2.93)[0.92 - 1.22] [0.94 - 1.37] [-0.35 - 0.27] [0, 4.65]Antinutrient (% DW, unless noted)Lectin (H.U./mg DW) 2.84 (0.65) 1.69 (0.65)1.15 (0.36) -0.41, 2.70 0.086 (0.81 - 9.73)[2.07 - 4.28] [0.71 - 2.71] [0.43 - 1.57] [0, 16.00]Phytic Acid 0.97 (0.059) 1.01 (0.059) -0.047(0.057) -0.29, 0.20 0.495 (0.81 - 1.27)[0.86 - 1.11] [0.94 - 1.06] [-0.13 - 0.064] [0.51, 1.59]Raffinose 0.38 (0.024) 0.34 (0.024) 0.042 (0.034) -0.10, 0.19 0.344 (0.31 - 0.42)[0.35 - 0.45] [0.31 - 0.37] [-0.019 - 0.099] [0.19, 0.52]Stachyose 2.87 (0.19) 2.74 (0.19) 0.13 (0.26) -0.99, 1.26 0.659 (2.23 - 3.29)[2.53 - 3.24] [2.43 - 3.00] [-0.46 - 0.44] [1.61, 4.05]Trypsin Inhibitor (TIU/mg DW) 28.51 (1.63) 29.19 (1.63)-0.69 (1.71) -8.02, 6.65 0.726 (24.29 - 46.29)[27.39 - 29.81] [24.81 - 31.50] [-3.18 - 2.58] [8.09, 57.27]Isoflavone (μg/g DW)Daidzein 1076.04 (29.87) 1583.00 (29.87) -506.96 (25.06) -614.77, -399.15 0.002 (783.49 - 1691.97)[999.02 - 1130.31] [1553.50 - 1599.74] [-554.48 - -469.43] [0, 2594.50]Monsanto Company 09-SY-200U Page 263 of 395Table E-4. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Isoflavone (μg/g DW)Genistein 656.80 (21.40) 1044.68 (21.40) -387.88 (16.63) -459.42, -316.33 0.001 (741.53 - 1580.48)[612.27 - 687.55] [1023.36 - 1084.46] [-411.09 - -355.65] [254.31, 1976.30]Glycitein 75.18 (6.78) 76.19 (6.78) -1.01 (1.36) -6.84, 4.82 0.533 (74.87 - 189.64)[67.00 - 87.16] [65.51 - 90.33] [-3.16 - 1.49] [0, 243.40]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 264 of 395Table E-5. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Forage from IL Sitefor Fiber and ProximatesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber (% DW)Acid Detergent Fiber 31.77 (1.82) 31.12 (1.82) 0.65 (1.56) -6.06, 7.37 0.715 (24.31 - 31.73)[30.24 - 34.02] [27.30 - 35.23] [-1.21 - 3.76] [19.24, 38.36]Neutral Detergent Fiber 36.52 (3.76) 37.15 (3.76) -0.64 (4.89) -21.69, 20.42 0.908 (24.37 - 38.09)[29.02 - 42.44] [30.49 - 42.62] [-9.33 - 7.60] [19.01, 46.73]Proximate (% DW, unless noted)Ash 6.79 (0.18) 6.72 (0.18) 0.068 (0.18) -0.70, 0.84 0.741 (5.33 - 7.77)[6.70 - 6.92] [6.44 - 7.20] [-0.28 - 0.31] [3.73, 9.32]Carbohydrates 69.55 (0.72) 69.94 (0.72) -0.39 (0.36) -1.95, 1.17 0.396 (64.94 - 72.18)[68.50 - 70.34] [68.21 - 70.84] [-0.95 - 0.29] [60.10, 76.68]Moisture (% FW) 71.63 (0.62) 71.27 (0.62) 0.37 (0.88) -3.41, 4.14 0.716 (71.10 - 74.90)[70.60 - 72.90] [70.20 - 72.10] [-0.90 - 2.70] [67.41, 78.15]Protein 18.60 (0.40) 18.57 (0.40) 0.028 (0.39) -1.67, 1.72 0.949 (16.96 - 21.65)[17.76 - 19.23] [18.09 - 19.25] [-0.63 - 0.73] [13.69, 25.14]Total Fat 5.06 (0.28) 4.76(0.28) 0.29 (0.33) -1.14,1.73 0.470 (4.15 - 7.02)[4.61 - 5.35] [4.25 - 5.34] [-0.085 - 0.96] [1.46, 9.88]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 265 of 395Table E-6. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and IsoflavonesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Alanine 1.80 (0.020) 1.74 (0.020) 0.052 (0.024) -0.049, 0.15 0.158 (1.63 - 1.86)[1.76 - 1.84] [1.71 - 1.76] [0.0060 - 0.084] [1.45, 2.02]Arginine 3.07 (0.049) 2.90 (0.049) 0.17 (0.070) -0.13, 0.47 0.129 (2.61 - 3.15)[3.00 - 3.14] [2.80 - 3.00] [0.0027 - 0.27] [2.13, 3.62]Aspartic Acid 4.55 (0.063) 4.35 (0.063) 0.20 (0.085) -0.17, 0.56 0.146 (4.01 - 4.71)[4.45 - 4.70] [4.26 - 4.42] [0.031 - 0.31] [3.45, 5.29]Cystine 0.63 (0.0056) 0.61 (0.0056) 0.020 (0.0020) 0.011, 0.028 0.010 (0.55 - 0.62)[0.62 - 0.64] [0.60 - 0.62] [0.016 - 0.022] [0.49, 0.68]Glutamic Acid 7.65 (0.11) 7.26 (0.11) 0.39 (0.14) -0.24, 1.01 0.116 (6.67 - 7.99)[7.46 - 7.90] [7.08 - 7.36] [0.098 - 0.56] [5.51, 9.04]Glycine 1.82 (0.019) 1.75 (0.019) 0.070 (0.023) -0.028, 0.17 0.091 (1.61 - 1.86)[1.78 - 1.87] [1.72 - 1.76] [0.028 - 0.11] [1.39, 2.05]Histidine 1.10 (0.015) 1.06 (0.015) 0.044 (0.020) -0.042, 0.13 0.158 (0.98 - 1.13)[1.08 - 1.14] [1.04 - 1.07] [0.0068 - 0.075] [0.86, 1.27]Isoleucine 1.91 (0.025) 1.82 (0.025) 0.087(0.036) -0.066, 0.24 0.134 (1.62 - 2.00)[1.87 - 1.97] [1.79 - 1.84] [0.022 - 0.18] [1.34, 2.28]Monsanto Company 09-SY-200U Page 266 of 395Table E-6. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Leucine 3.21 (0.048) 3.10 (0.048) 0.11 (0.059) -0.14, 0.36 0.196 (2.86 - 3.37)[3.14 - 3.32] [3.03 - 3.14] [0.0023 - 0.20] [2.45, 3.76]Lysine 2.68 (0.031) 2.59 (0.031) 0.085 (0.040) -0.087, 0.26 0.168 (2.42 - 2.78)[2.63 - 2.75] [2.55 - 2.62] [0.014 - 0.15] [2.13, 3.06]Methionine 0.61 (0.0070) 0.59 (0.0070) 0.028 (0.0068) -0.0014,0.057 0.054 (0.52 - 0.61)[0.61 - 0.62] [0.57 - 0.60] [0.015 - 0.038] [0.48, 0.66]Phenylalanine 2.12 (0.026) 2.07 (0.026) 0.051(0.025) -0.058, 0.16 0.182 (1.92 - 2.29)[2.08 - 2.18] [2.03 - 2.10] [0.0051 - 0.092] [1.61, 2.55]Proline 2.09 (0.030) 1.99 (0.030) 0.094 (0.043) -0.090, 0.28 0.159 (1.81 - 2.16)[2.05 - 2.15] [1.95 - 2.04] [0.016 - 0.17] [1.53, 2.45]Serine 2.20 (0.044) 2.11 (0.044) 0.094 (0.059) -0.16,0.35 0.253 (1.97 - 2.27)[2.14 - 2.25] [2.00 - 2.18] [0.0050 - 0.21] [1.75, 2.51]Threonine 1.58 (0.028) 1.56 (0.028) 0.027 (0.040) -0.14, 0.20 0.564 (1.45 - 1.65)[1.54 - 1.63] [1.50 - 1.60] [-0.058 - 0.087] [1.30, 1.82]Tryptophan 0.50 (0.0096) 0.48 (0.0096) 0.013 (0.010) -0.030, 0.056 0.322 (0.43 - 0.52)[0.49 - 0.50] [0.46 - 0.50] [-0.0014 - 0.032] [0.35, 0.59]Monsanto Company 09-SY-200U Page 267 of 395Table E-6. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Tyrosine 1.38 (0.032) 1.37 (0.032) 0.014 (0.042) -0.17, 0.19 0.773 (1.21 - 1.49)[1.34 - 1.41] [1.33 - 1.44] [-0.058 - 0.087] [1.03, 1.67]Valine 2.01 (0.029) 1.92 (0.029) 0.098 (0.040) -0.076, 0.27 0.136 (1.70 - 2.11)[1.96 - 2.08] [1.88 - 1.95] [0.011 - 0.20] [1.42, 2.41]Fatty Acid (% Total FA)16:0 Palmitic 12.31 (0.052) 12.00 (0.052) 0.30(0.048) 0.095, 0.51 0.024 (9.88 - 12.33)[12.24 - 12.39] [11.89 - 12.08] [0.21 - 0.35] [7.28, 14.20]18:0 Stearic 4.42 (0.027) 4.33 (0.027) 0.090 (0.036) -0.064,0.24 0.128 (3.68 - 4.89)[4.37 - 4.45] [4.28 - 4.37] [0.035 - 0.16] [2.87, 5.85]18:1 Total 18:1 17.89 (0.22) 20.89 (0.22) -3.00 (0.31) -4.31, -1.68 0.010 (16.70 - 23.16)[17.52 - 18.18] [20.42 - 21.17] [-3.65 - -2.46] [12.56, 27.98]18:2 9c,12c Linoleic 30.48 (0.20)54.33 (0.20) -23.86 (0.087) -24.23, -23.48 <0.001 (53.36 - 57.39)[30.26 - 30.81] [54.05 - 54.77] [-23.96 - -23.68] [50.46, 59.96]18:3 Linolenic 10.27 (0.090) 7.59 (0.090) 2.68 (0.13) 2.13, 3.22 0.002 (6.95 - 10.58)[10.20 - 10.38] [7.42 - 7.81] [2.42 - 2.82] [3.72, 13.46]20:0 Arachidic 0.36 (0.0038) 0.34 (0.0038) 0.027 (0.0045) 0.0083, 0.047 0.025 (0.27 - 0.36)[0.36 - 0.37] [0.33 - 0.34] [0.020 - 0.036] [0.20, 0.45]Monsanto Company 09-SY-200U Page 268 of 395Table E-6. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Fatty Acid (% Total FA)20:1 Eicosenoic 0.20 (0.0013) 0.19 (0.0013) 0.0062(0.0019) -0.0019, 0.014 0.081 (0.071 - 0.19)[0.20 - 0.20] [0.19 - 0.19] [0.0027 - 0.0098] [0, 0.31]22:0 Behenic 0.31 (0.0039) 0.33(0.0039) -0.022 (0.0049) -0.043,-0.00089 0.046 (0.30 - 0.41)[0.30 - 0.31] [0.32 - 0.34] [-0.029 - -0.013] [0.22, 0.49]Fiber (% DW)Acid Detergent Fiber 16.16 (0.20) 17.76 (0.20) -1.60 (0.12) -2.12, -1.08 0.005 (14.57 - 18.85)[15.91 - 16.61] [17.47 - 18.03] [-1.83 - -1.41] [10.36, 22.77]Neutral Detergent Fiber 17.06 (0.12) 17.87 (0.12) -0.81 (0.17) -1.56, -0.060 0.043 (15.03 - 18.92)[16.72 - 17.25] [17.78 - 17.92] [-1.20 - -0.53] [10.91, 22.59]Proximate (% DW, unless noted)Ash 6.06 (0.13) 5.78 (0.13) 0.28 (0.17) -0.44, 1.01 0.234 (5.59 - 6.20)[5.90 - 6.17] [5.51 - 6.07] [0.099 - 0.62] [5.16, 6.64]Carbohydrates 34.20 (0.77) 36.20 (0.77) -2.00 (0.55) -4.38, 0.37 0.068 (33.50 - 40.22)[33.23 - 36.11] [35.30 - 37.13] [-2.92 - -1.01] [26.76, 45.99]Moisture (% FW) 6.98 (0.11) 7.28 (0.11) -0.29 (0.16) -0.98, 0.40 0.209 (6.68 - 8.16)[6.71 - 7.24] [7.21 - 7.37] [-0.66 - 0.030] [5.23, 9.56]Protein 41.64 (0.45) 39.58 (0.45) 2.06 (0.63) -0.64, 4.76 0.081 (37.52 - 42.37)[41.05 - 42.26] [38.97 - 40.65] [0.97 - 3.14] [33.37, 46.00]Monsanto Company 09-SY-200U Page 269 of 395Table E-6. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]ProximateTotal Fat 18.10 (0.45) 18.44(0.45) -0.34 (0.49) -2.44,1.75 0.554 (13.99 - 20.56)[16.94 - 19.03] [18.14 - 18.64] [-1.20 - 0.49] [11.04, 25.03]VitaminVitamin E (mg/100g DW) 2.42 (0.070)2.10 (0.070) 0.31 (0.099) -0.11, 0.74 0.087 (0.27 - 2.93)[2.27 - 2.54] [2.01 - 2.21] [0.059 - 0.45] [0, 4.65]Antinutrient (% DW, unless noted)Lectin (H.U./mg DW) 5.00 (1.67) 7.07 (1.67) -2.07 (2.36) -12.21, 8.07 0.472 (0.81 - 9.73)[3.69 - 7.02] [4.88 - 11.32] [-7.01 - 2.00] [0, 16.00]Phytic Acid 1.07 (0.030) 1.04 (0.030) 0.023(0.043) -0.16, 0.21 0.641 (0.81 - 1.27)[1.02 - 1.10] [1.00 - 1.11] [-0.094 - 0.10] [0.51, 1.59]Raffinose 0.43 (0.015) 0.43 (0.015) 0.00042 (0.021) -0.091, 0.092 0.985 (0.31 - 0.42)[0.40 - 0.44] [0.40 - 0.45] [-0.049 - 0.044] [0.19, 0.52]Stachyose 3.06 (0.088) 3.07 (0.088) -0.0061(0.12) -0.54, 0.53 0.965 (2.23 - 3.29)[2.91 - 3.17] [2.88 - 3.21] [-0.30 - 0.29] [1.61, 4.05]Trypsin Inhibitor (TIU/mg DW) 38.06 (1.03) 35.27 (1.03) 2.80 (1.46) -3.49, 9.09 0.195 (24.29 - 46.29)[36.23 - 40.86] [34.82 - 35.95] [0.28 - 5.83] [8.09, 57.27]Isoflavone (μg/g DW)Daidzein 1079.87 (87.82) 1484.63 (87.82) -404.76 (120.69) -924.03, 114.51 0.078 (783.49 - 1691.97)[957.23 - 1193.55] [1380.05 - 1691.99] [-603.16 - -186.51] [0, 2594.50]Monsanto Company 09-SY-200U Page 270 of 395Table E-6. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Isoflavone (μg/g DW)Genistein 655.90 (47.11) 837.93 (47.11) -182.04(52.30) -407.05, 42.97 0.073 (741.53 - 1580.48)[576.70 - 716.13] [770.81 - 940.83] [-265.97 - -86.03] [254.31, 1976.30]Glycitein 84.86 (5.58) 120.49 (5.58) -35.63 (7.78) -69.10, -2.17 0.044 (74.87 - 189.64)[83.23 - 87.74] [106.01 - 132.56] [-49.33 - -22.40] [0, 243.40]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 271 of 395Table E-7. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Forage from MISite for Fiber and ProximatesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber (% DW)Acid Detergent Fiber 33.02 (2.24) 33.97 (2.24) -0.95 (3.17) -14.61, 12.71 0.793 (24.31 - 31.73)[27.92 - 36.39] [30.72 - 37.06] [-9.14 - 5.67] [19.24, 38.36]Neutral Detergent Fiber36.16 (2.10) 36.13 (2.10) 0.029 (2.96)-12.72, 12.78 0.993 (24.37 - 38.09)[31.87 - 40.66] [33.13 - 38.13] [-5.27 - 7.53] [19.01, 46.73]Proximate (% DW, unless noted)Ash 5.99 (0.33) 6.00 (0.33) -0.014 (0.077) -0.35, 0.32 0.875 (5.33 - 7.77)[5.29 - 6.36] [5.38 - 6.39] [-0.091 - 0.14] [3.73, 9.32]Carbohydrates 70.54 (1.12) 70.07 (1.12) 0.47(1.26) -4.94, 5.87 0.745 (64.94 - 72.18)[68.27 - 72.79] [68.44 - 71.51] [-2.00 - 2.12] [60.10, 76.68]Moisture (% FW) 71.30 (0.93) 71.37 (0.93) -0.067 (1.32) -5.73, 5.60 0.964 (71.10 - 74.90)[69.50 - 72.80] [70.10 - 73.10] [-1.50 - 2.70] [67.41, 78.15]Protein 18.80 (0.73) 18.54 (0.73) 0.26 (1.02) -4.11, 4.63 0.822 (16.96 - 21.65)[17.05 - 20.44] [18.23 - 19.14] [-1.20 - 2.21] [13.69, 25.14]Total Fat 4.67 (0.68) 5.39(0.68) -0.71 (0.54) -3.05,1.62 0.318 (4.15 - 7.02)[3.80 - 5.99] [4.02 - 6.12] [-1.80 - -0.13] [1.46, 9.88]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 272 of 395Table E-8. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and IsoflavonesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Alanine 1.79 (0.021) 1.72 (0.021) 0.068 (0.030) -0.062, 0.20 0.152 (1.63 - 1.86)[1.76 - 1.83] [1.69 - 1.76] [0.0015 - 0.11] [1.45, 2.02]Arginine 3.32 (0.10) 2.94 (0.10) 0.39 (0.14) -0.23, 1.01 0.114 (2.61 - 3.15)[3.16 - 3.61] [2.91 - 2.98] [0.19 - 0.68] [2.13, 3.62]Aspartic Acid 4.57 (0.064) 4.29 (0.064) 0.28 (0.090) -0.10, 0.67 0.087 (4.01 - 4.71)[4.48 - 4.73] [4.21 - 4.37] [0.11 - 0.43] [3.45, 5.29]Cystine 0.58 (0.011) 0.57 (0.011) 0.012 (0.015) -0.054, 0.077 0.528 (0.55 - 0.62)[0.56 - 0.60] [0.56 - 0.58] [-0.024 - 0.037] [0.49, 0.68]Glutamic Acid 7.62 (0.11) 7.16 (0.11) 0.46 (0.15) -0.20, 1.12 0.094 (6.67 - 7.99)[7.47 - 7.87] [7.03 - 7.32] [0.15 - 0.76] [5.51, 9.04]Glycine 1.78 (0.019) 1.70 (0.019) 0.086 (0.027) -0.032, 0.20 0.088 (1.61 - 1.86)[1.76 - 1.83] [1.67 - 1.72] [0.034 - 0.14] [1.39, 2.05]Histidine 1.09 (0.017) 1.04 (0.017) 0.048 (0.024) -0.053, 0.15 0.176 (0.98 - 1.13)[1.07 - 1.13] [1.02 - 1.07] [0.0020 - 0.090] [0.86, 1.27]Isoleucine 1.82 (0.048) 1.75 (0.048) 0.068(0.068) -0.23, 0.36 0.422 (1.62 - 2.00)[1.75 - 1.94] [1.70 - 1.80] [-0.052 - 0.24] [1.34, 2.28]Monsanto Company 09-SY-200U Page 273 of 395Table E-8. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Leucine 3.19 (0.047) 3.06 (0.047) 0.13 (0.066) -0.15, 0.41 0.189 (2.86 - 3.37)[3.13 - 3.28] [3.01 - 3.15] [-0.019 - 0.26] [2.45, 3.76]Lysine 2.67 (0.033) 2.56 (0.033) 0.11 (0.047) -0.092, 0.31 0.142 (2.42 - 2.78)[2.62 - 2.75] [2.51 - 2.61] [0.013 - 0.19] [2.13, 3.06]Methionine 0.56 (0.011) 0.56 (0.011) 0.00074 (0.016) -0.068,0.069 0.967 (0.52 - 0.61)[0.54 - 0.59] [0.56 - 0.57] [-0.030 - 0.033] [0.48, 0.66]Phenylalanine 2.16 (0.032) 2.05 (0.032) 0.11(0.046) -0.086, 0.31 0.135 (1.92 - 2.29)[2.11 - 2.24] [2.02 - 2.10] [0.013 - 0.20] [1.61, 2.55]Proline 2.09 (0.039) 1.94 (0.039) 0.14 (0.055) -0.091, 0.38 0.119 (1.81 - 2.16)[2.03 - 2.19] [1.91 - 1.98] [0.056 - 0.25] [1.53, 2.45]Serine 2.23 (0.022) 2.12 (0.022) 0.11 (0.030) -0.020,0.23 0.067 (1.97 - 2.27)[2.23 - 2.23] [2.06 - 2.17] [0.067 - 0.17] [1.75, 2.51]Threonine 1.63 (0.023) 1.55 (0.023) 0.082 (0.032) -0.057, 0.22 0.125 (1.45 - 1.65)[1.60 - 1.65] [1.49 - 1.58] [0.023 - 0.15] [1.30, 1.82]Tryptophan 0.48 (0.014) 0.46 (0.014) 0.018 (0.011) -0.030, 0.065 0.253 (0.43 - 0.52)[0.45 - 0.51] [0.44 - 0.47] [-0.00067 - 0.037] [0.35, 0.59]Monsanto Company 09-SY-200U Page 274 of 395Table E-8. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Tyrosine 1.42 (0.049) 1.35 (0.049) 0.072 (0.048) -0.14, 0.28 0.275 (1.21 - 1.49)[1.29 - 1.50] [1.32 - 1.40] [-0.020 - 0.14] [1.03, 1.67]Valine 1.92 (0.054) 1.86 (0.054) 0.065 (0.076) -0.26, 0.39 0.485 (1.70 - 2.11)[1.84 - 2.06] [1.79 - 1.91] [-0.063 - 0.26] [1.42, 2.41]Fatty Acid (% Total FA)16:0 Palmitic 12.11 (0.057) 11.79 (0.057) 0.31(0.044) 0.12, 0.50 0.019 (9.88 - 12.33)[12.03 - 12.19] [11.70 - 11.92] [0.26 - 0.40] [7.28, 14.20]18:0 Stearic 3.87 (0.054) 3.90(0.054) -0.022 (0.070) -0.32,0.28 0.779 (3.68 - 4.89)[3.73 - 3.98] [3.85 - 3.94] [-0.16 - 0.059] [2.87, 5.85]18:1 Total 18:1 12.92 (0.13) 17.44 (0.13) -4.53 (0.097) -4.94, -4.11 <0.001 (16.70 - 23.16)[12.66 - 13.16] [17.24 - 17.59] [-4.66 - -4.34] [12.56, 27.98]18:2 9c,12c Linoleic 18.40 (0.71)55.60 (0.71) -37.21 (0.90) -41.08, -33.33 <0.001 (53.36 - 57.39)[16.46 - 19.58] [55.36 - 56.04] [-38.90 - -35.83] [50.46, 59.96]18:3 Linolenic 11.76 (0.029) 10.59 (0.029) 1.16 (0.038) 1.00, 1.33 0.001 (6.95 - 10.58)[11.72 - 11.80] [10.54 - 10.66] [1.09 - 1.22] [3.72, 13.46]20:0 Arachidic 0.32 (0.0037) 0.29 (0.0037) 0.030 (0.0052) 0.0074, 0.052 0.029 (0.27 - 0.36)[0.31 - 0.32] [0.28 - 0.29] [0.019 - 0.042] [0.20, 0.45]Monsanto Company 09-SY-200U Page 275 of 395Table E-8. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Fatty Acid (% Total FA)20:1 Eicosenoic 0.081 (0.014) 0.096 (0.014) -0.015 (0.020) -0.10, 0.072 0.535 (0.071 - 0.19)[0.077 - 0.084] [0.069 - 0.14] [-0.058 - 0.013] [0, 0.31]22:0 Behenic 0.30 (0.0096) 0.29 (0.0096) 0.0060 (0.012) -0.044,0.056 0.656 (0.30 - 0.41)[0.27 - 0.31] [0.29 - 0.30] [-0.016 - 0.022] [0.22, 0.49]Fiber (% DW)Acid Detergent Fiber 17.27 (0.32) 16.77 (0.32) 0.50 (0.21) -0.42, 1.43 0.143 (14.57 - 18.85)[16.91 - 17.59] [15.98 - 17.35] [0.23 - 0.93] [10.36, 22.77]Neutral Detergent Fiber17.30 (0.55) 17.34 (0.55) -0.038 (0.78)-3.38, 3.30 0.965 (15.03 - 18.92)[16.15 - 18.17] [16.42 - 18.11] [-1.34 - 1.75] [10.91, 22.59]Proximate (% DW, unless noted)Ash 5.46 (0.10) 5.51 (0.10) -0.051 (0.15) -0.68, 0.58 0.763 (5.59 - 6.20)[5.23 - 5.66] [5.35 - 5.61] [-0.34 - 0.31] [5.16, 6.64]Carbohydrates 39.11 (1.09) 39.75 (1.09) -0.63 (1.54) -7.27, 6.00 0.720 (33.50 - 40.22)[38.34 - 39.93] [37.70 - 42.60] [-4.26 - 2.23] [26.76, 45.99]Moisture (% FW) 7.15 (0.13) 7.15 (0.13) 0(0.13) -0.55, 0.55 1.000 (6.68 - 8.16)[7.01 - 7.31] [6.84 - 7.39] [-0.25 - 0.17] [5.23, 9.56]Protein 41.86 (0.67) 39.10 (0.67) 2.76 (0.95) -1.33, 6.86 0.100 (37.52 - 42.37)[41.08 - 43.29] [38.22 - 40.31] [0.90 - 5.07] [33.37, 46.00]Monsanto Company 09-SY-200U Page 276 of 395Table E-8. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromMI Site for Amino Acids, Fatty Acids,Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]ProximateTotal Fat 13.57 (0.77) 15.65(0.77) -2.08 (0.81) -5.56,1.40 0.123 (13.99 - 20.56)[13.14 - 14.20] [13.61 - 16.96] [-3.01 - -0.47] [11.04, 25.03]VitaminVitamin E (mg/100g DW) 1.26 (0.087)0.86 (0.087) 0.40 (0.12) -0.13, 0.93 0.083 (0.27 - 2.93)[1.22 - 1.34] [0.70 - 1.09] [0.14 - 0.55] [0, 4.65]AntinutrientLectin (H.U./mg DW) 1.49 (0.63) 3.44 (0.63) -1.95 (0.28) -3.14, -0.77 0.019 (0.81 - 9.73)[0.55 - 2.14] [1.95 - 4.39] [-2.25 - -1.40] [0, 16.00]Phytic Acid (% DW) 0.86 (0.063) 0.88 (0.063) -0.017 (0.061) -0.28, 0.25 0.811 (0.81 - 1.27)[0.81 - 0.92] [0.75 - 1.03] [-0.10 - 0.10] [0.51, 1.59]Raffinose (% DW) 0.34 (0.0085) 0.32 (0.0085) 0.021(0.012) -0.031, 0.073 0.225 (0.31 - 0.42)[0.32 - 0.36] [0.32 - 0.33] [-0.0059 - 0.044] [0.19, 0.52]Stachyose (% DW) 2.43 (0.066) 2.52 (0.066) -0.093 (0.094) -0.50, 0.31 0.425 (2.23 - 3.29)[2.28 - 2.53] [2.45 - 2.63] [-0.35 - 0.080] [1.61, 4.05]Trypsin Inhibitor (TIU/mg DW) 25.92 (2.94) 27.17 (2.94)-1.24 (4.16) -19.15, 16.67 0.793 (24.29 - 46.29)[24.30 - 27.94] [21.34 - 34.89] [-10.58 - 6.60] [8.09, 57.27]Isoflavone (μg/g DW)Daidzein 1662.22 (99.21) 2750.13 (99.21) -1087.92 (140.30) -1691.59, -484.25 0.016 (783.49 - 1691.97)[1389.19 - 1838.91] [2705.02 - 2775.08] [-1385.89 - -866.12] [0, 2594.50]Monsanto Company 09-SY-200U Page 277 of 395Table E-8. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Isoflavone (μg/g DW)Genistein 1000.90 (59.25) 1683.74 (59.25) -682.83 (70.98) -988.25, -377.41 0.010 (741.53 - 1580.48)[841.05 - 1118.40] [1662.89 - 1706.74] [-821.84 - -588.34] [254.31, 1976.30]Glycitein 83.59 (17.61) 114.34 (17.61) -30.75(24.91) -137.92, 76.41 0.342 (74.87 - 189.64)[65.37 - 101.09] [84.16 - 158.73] [-93.36 - 0.95] [0, 243.40]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 278 of 395Table E-9. Summary of the Compositional Comparisonof MON 87769 vs. the Conventional Control Forage from OHSite for Fiber and ProximatesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fiber (% DW)Acid Detergent Fiber 32.29 (0.91) 28.31 (0.91) 3.99 (0.40) 2.28, 5.70 0.009 (24.31 - 31.73)[30.74 - 33.95] [26.53 - 29.41] [3.21 - 4.54] [19.24, 38.36]Neutral Detergent Fiber 32.93 (1.50) 31.22 (1.50) 1.70 (1.80)-6.04, 9.45 0.443 (24.37 - 38.09)[29.20 - 35.58] [29.79 - 32.90] [-1.78 - 4.22] [19.01, 46.73]Proximate (% DW, unless noted)Ash 6.67 (0.24) 6.59 (0.24) 0.078 (0.25) -0.99, 1.14 0.782 (5.33 - 7.77)[6.05 - 7.07] [6.44 - 6.88] [-0.40 - 0.44] [3.73, 9.32]Carbohydrates 67.50 (0.78) 66.87 (0.78) 0.64(0.90) -3.26, 4.53 0.553 (64.94 - 72.18)[66.33 - 69.34] [65.73 - 67.76] [-0.91 - 2.22] [60.10, 76.68]Moisture (% FW) 70.87 (0.56) 71.27 (0.56) -0.40 (0.80) -3.82, 3.02 0.664 (71.10 - 74.90)[69.90 - 72.40] [71.00 - 71.60] [-1.70 - 1.40] [67.41, 78.15]Protein 19.77 (0.58) 20.08 (0.58) -0.32 (0.81) -3.82, 3.19 0.734 (16.96 - 21.65)[18.74 - 20.87] [19.07 - 20.87] [-1.58 - 1.80] [13.69, 25.14]Total Fat 6.06 (0.34) 6.46(0.34) -0.40 (0.48) -2.45,1.65 0.490 (4.15 - 7.02)[5.40 - 6.90] [6.13 - 6.72] [-1.33 - 0.37] [1.46, 9.88]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 279 of 395Table E-10. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and IsoflavonesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Alanine 1.77 (0.0068) 1.74 (0.0068) 0.031 (0.0096) -0.010, 0.073 0.083 (1.63 - 1.86)[1.76 - 1.78] [1.72 - 1.75] [0.0084 - 0.043] [1.45, 2.02]Arginine 3.11 (0.020) 2.86 (0.020) 0.25 (0.028) 0.13, 0.37 0.012 (2.61 - 3.15)[3.07 - 3.15] [2.82 - 2.89] [0.21 - 0.32] [2.13, 3.62]Aspartic Acid 4.43 (0.017) 4.27 (0.017) 0.16 (0.024) 0.056, 0.26 0.021 (4.01 - 4.71)[4.41 - 4.47] [4.25 - 4.29] [0.12 - 0.22] [3.45, 5.29]Cystine 0.60 (0.0030) 0.59 (0.0030) 0.015 (0.0043) -0.0037, 0.033 0.075 (0.55 - 0.62)[0.60 - 0.60] [0.58 - 0.59] [0.0058 - 0.021] [0.49, 0.68]Glutamic Acid 7.51 (0.034) 7.17 (0.034) 0.33 (0.048) 0.13, 0.54 0.020 (6.67 - 7.99)[7.42 - 7.59] [7.16 - 7.19] [0.23 - 0.43] [5.51, 9.04]Glycine 1.76 (0.0055) 1.71 (0.0055) 0.049 (0.0078) 0.015, 0.083 0.024 (1.61 - 1.86)[1.76 - 1.77] [1.70 - 1.73] [0.030 - 0.064] [1.39, 2.05]Histidine 1.07 (0.0021) 1.03 (0.0021) 0.033(0.0021) 0.024, 0.042 0.003 (0.98 - 1.13)[1.06 - 1.07] [1.03 - 1.04] [0.031 - 0.037] [0.86, 1.27]Isoleucine 1.87 (0.024) 1.75 (0.024) 0.11(0.018) 0.035, 0.19 0.025 (1.62 - 2.00)[1.84 - 1.90] [1.70 - 1.80] [0.095 - 0.15] [1.34, 2.28]Monsanto Company 09-SY-200U Page 280 of 395Table E-10. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Leucine 3.16 (0.015) 3.05 (0.015) 0.10 (0.014) 0.046, 0.16 0.016 (2.86 - 3.37)[3.14 - 3.18] [3.02 - 3.07] [0.080 - 0.13] [2.45, 3.76]Lysine 2.62 (0.0092) 2.56 (0.0092) 0.063 (0.0091) 0.024, 0.10 0.020 (2.42 - 2.78)[2.61 - 2.63] [2.54 - 2.58] [0.045 - 0.074] [2.13, 3.06]Methionine 0.59 (0.0038) 0.59 (0.0038) 0.0064 (0.0027) -0.0052,0.018 0.141 (0.52 - 0.61)[0.59 - 0.60] [0.58 - 0.59] [0.0028 - 0.012] [0.48, 0.66]Phenylalanine 2.12 (0.0068) 2.05 (0.0068) 0.076 (0.0096) 0.034, 0.12 0.015 (1.92 - 2.29)[2.12 - 2.13] [2.04 - 2.06] [0.053 - 0.099] [1.61, 2.55]Proline 2.05 (0.0089) 1.95 (0.0089) 0.097 (0.010) 0.052, 0.14 0.011 (1.81 - 2.16)[2.03 - 2.06] [1.94 - 1.96] [0.085 - 0.12] [1.53, 2.45]Serine 2.16 (0.028) 2.13 (0.028) 0.029 (0.028) -0.092, 0.15 0.412 (1.97 - 2.27)[2.08 - 2.21] [2.11 - 2.14] [-0.023 - 0.075] [1.75, 2.51]Threonine 1.57 (0.0094) 1.56 (0.0094) 0.013 (0.013) -0.043, 0.068 0.432 (1.45 - 1.65)[1.56 - 1.58] [1.54 - 1.58] [-0.012 - 0.032] [1.30, 1.82]Tryptophan 0.46 (0.0059) 0.45 (0.0059) 0.0084 (0.0084) -0.028, 0.045 0.421 (0.43 - 0.52)[0.45 - 0.47] [0.45 - 0.46] [-0.011 - 0.026] [0.35, 0.59]Monsanto Company 09-SY-200U Page 281 of 395Table E-10. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Amino Acid (% DW)Tyrosine 1.40 (0.032) 1.34 (0.032) 0.059 (0.044) -0.13, 0.25 0.312 (1.21 - 1.49)[1.36 - 1.44] [1.28 - 1.41] [-0.024 - 0.13] [1.03, 1.67]Valine 1.96 (0.030) 1.84 (0.030) 0.12 (0.021) 0.033, 0.21 0.027 (1.70 - 2.11)[1.94 - 2.01] [1.78 - 1.90] [0.096 - 0.16] [1.42, 2.41]Fatty Acid (% Total FA)16:0 Palmitic 11.74 (0.11) 11.63 (0.11) 0.11(0.15) -0.53, 0.75 0.544 (9.88 - 12.33)[11.56 - 12.02] [11.58 - 11.71] [-0.080 - 0.44] [7.28, 14.20]18:0 Stearic 4.46 (0.056) 4.37 (0.056) 0.088 (0.048) -0.12,0.29 0.208 (3.68 - 4.89)[4.32 - 4.53] [4.32 - 4.44] [0.0019 - 0.17] [2.87, 5.85]18:1 Total 18:1 18.10 (0.49) 20.65 (0.49) -2.54(0.57) -5.00, -0.084 0.046 (16.70 - 23.16)[16.73 - 18.80] [20.41 - 20.79] [-3.68 - -1.95] [12.56, 27.98]Linoleic 25.37 (0.16) 54.50 (0.16) -29.13 (0.22) -30.07, -28.19 <0.001 (53.36 - 57.39)[25.06 - 25.75] [54.38 - 54.67] [-29.61 - -28.70] [50.46, 59.96]18:3 Linolenic 11.63 (0.11) 8.02 (0.11) 3.61 (0.15) 2.95, 4.28 0.001 (6.95 - 10.58)[11.41 - 11.75] [7.85 - 8.21] [3.19 - 3.89] [3.72, 13.46]20:0 Arachidic 0.36 (0.0043) 0.33 (0.0043) 0.028 (0.0031) 0.015, 0.041 0.011 (0.27 - 0.36)[0.35 - 0.36] [0.32 - 0.33] [0.024 - 0.034] [0.20, 0.45]Monsanto Company 09-SY-200U Page 282 of 395Table E-10. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Fatty Acid (% Total FA)20:1 Eicosenoic 0.18 (0.0029) 0.17 (0.0029) 0.0072(0.0032) -0.0064, 0.021 0.150 (0.071 - 0.19)[0.17 - 0.19] [0.17 - 0.18] [0.0019 - 0.013] [0, 0.31]22:0 Behenic 0.29 (0.015) 0.33(0.015) -0.037 (0.018) -0.11,0.039 0.172 (0.30 - 0.41)[0.28 - 0.30] [0.31 - 0.37] [-0.071 - -0.014] [0.22, 0.49]Fiber (% DW)Acid Detergent Fiber 17.96 (0.36)17.65 (0.36) 0.31 (0.51) -1.88, 2.51 0.601 (14.57 - 18.85)[17.78 - 18.31] [16.69 - 18.15] [-0.35 - 1.62] [10.36, 22.77]Neutral Detergent Fiber 16.70 (0.75) 17.25 (0.75) -0.55 (1.06) -5.13, 4.02 0.655 (15.03 - 18.92)[15.06 - 18.13] [16.10 - 17.88] [-2.82 - 2.03] [10.91, 22.59]Proximate (% DW, unless noted)Ash 5.83 (0.040) 5.71 (0.040) 0.12 (0.019) 0.041, 0.20 0.022 (5.59 - 6.20)[5.78 - 5.93] [5.68 - 5.77] [0.099 - 0.16] [5.16, 6.64]Carbohydrates 34.40 (0.53) 36.24 (0.53) -1.83 (0.70) -4.86, 1.20 0.121 (33.50 - 40.22)[33.55 - 35.89] [36.17 - 36.33] [-2.67 - -0.43] [26.76, 45.99]Moisture (% FW) 7.59 (0.15) 7.27 (0.15) 0.32 (0.18) -0.47, 1.11 0.222 (6.68 - 8.16)[7.20 - 7.87] [7.15 - 7.43] [-0.040 - 0.56] [5.23, 9.56]Protein 41.37 (0.24) 39.54 (0.24) 1.83 (0.17) 1.09, 2.57 0.008 (37.52 - 42.37)[40.92 - 41.70] [39.11 - 39.96] [1.54 - 2.14] [33.37, 46.00]Monsanto Company 09-SY-200U Page 283 of 395Table E-10. Summary of the Statistical Comparison ofMON 87769 vs. the Conventional Control Soybean Seed from OHSite for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]ProximateTotal Fat 18.40 (0.42) 18.51(0.42) -0.12 (0.59) -2.67,2.44 0.860 (13.99 - 20.56)[17.26 - 18.97] [18.20 - 18.80] [-1.54 - 0.76] [11.04, 25.03]VitaminVitamin E (mg/100g DW) 2.06 (0.18)1.90 (0.18) 0.17 (0.092) -0.23, 0.56 0.213 (0.27 - 2.93)[1.68 - 2.33] [1.64 - 2.22] [0.040 - 0.34] [0, 4.65]AntinutrientLectin (H.U./mg DW) 4.17 (1.51) 4.15 (1.51)0.016 (2.13) -9.14, 9.18 0.994 (0.81 - 9.73)[2.76 - 6.67] [2.08 - 7.56] [-4.80 - 4.60] [0, 16.00]Phytic Acid (% DW) 1.10 (0.039) 1.01 (0.039)0.085 (0.033) -0.058, 0.23 0.124 (0.81 - 1.27)[1.02 - 1.20] [0.97 - 1.04] [0.049 - 0.15] [0.51, 1.59]Raffinose (% DW) 0.35 (0.020) 0.35 (0.020) 0.0081(0.028) -0.11, 0.13 0.801 (0.31 - 0.42)[0.32 - 0.39] [0.32 - 0.38] [-0.067 - 0.069] [0.19, 0.52]Stachyose (% DW) 2.74 (0.10) 2.64 (0.10) 0.099 (0.14) -0.52, 0.72 0.562 (2.23 - 3.29)[2.61 - 3.01] [2.54 - 2.70] [-0.087 - 0.47] [1.61, 4.05]Trypsin Inhibitor (TIU/mg DW) 31.30 (2.41) 28.18 (2.41) 3.12 (3.41) -11.55, 17.79 0.456 (24.29 - 46.29)[26.11 - 36.42] [25.71 - 31.34] [-5.23 - 8.93] [8.09, 57.27]Isoflavone (μg/g DW)Daidzein 1125.54 (39.70) 1668.07 (39.70) -542.53 (26.89) -658.22, -426.83 0.002 (783.49 - 1691.97)[1094.38 - 1183.11] [1583.20 - 1750.03] [-571.84 - -488.82] [0, 2594.50]Monsanto Company 09-SY-200U Page 284 of 395Table E-10. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Seed fromOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrientsand Isoflavones (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% Tolerance Interval2]Isoflavone (μg/g DW)Genistein 760.07 (20.50) 1143.19 (20.50) -383.12 (21.06) -473.72, -292.52 0.003 (741.53 - 1580.48)[750.00 - 773.91] [1110.39 - 1199.09] [-425.19 - -360.39] [254.31, 1976.30]Glycitein 82.62 (3.92) 96.62 (3.92) -14.00 (5.55) -37.86, 9.86 0.127 (74.87 - 189.64)[74.89 - 90.42] [90.63 - 101.88] [-26.98 - -0.22] [0, 243.40]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 285 of 395Table E-11. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Forage for Fiber and ProximatesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Fiber (% DW)Acid Detergent Fiber 31.06 (1.36) 30.13 (1.36) 0.93 (0.93) -1.06, 2.92 0.332 (24.31 - 31.73)[24.91 - 36.39] [24.39 - 37.06] [-9.14 - 5.67] [19.24, 38.36]Neutral Detergent Fiber 33.89 (1.37) 33.22 (1.37) 0.67 (1.27)-2.05, 3.39 0.605 (24.37 - 38.09)[28.06 - 42.44] [25.48 - 42.62] [-9.33 - 7.60] [19.01, 46.73]Proximate (% DW, unless noted)Ash 6.58 (0.24) 6.56 (0.24) 0.020 (0.081) -0.20, 0.24 0.814 (5.33 - 7.77)[5.29 - 7.30] [5.38 - 7.75] [-0.49 - 0.53] [3.73, 9.32]Carbohydrates 67.41 (1.32) 67.04 (1.32) 0.37(0.61) -1.32, 2.06 0.575 (64.94 - 72.18)[62.88 - 72.79] [61.18 - 71.51] [-3.30 - 4.45] [60.10, 76.68]Moisture (% FW) 72.28 (0.64) 72.20 (0.64) 0.080 (0.46) -0.90, 1.06 0.863 (71.10 - 74.90)[69.50 - 75.90] [70.10 - 74.70] [-2.20 - 3.10] [67.41, 78.15]Protein 20.71 (1.05) 20.67 (1.05) 0.040 (0.34) -0.69, 0.77 0.908 (16.96 - 21.65)[17.05 - 24.53] [18.09 - 24.98] [-2.01 - 2.21] [13.69, 25.14]Total Fat 5.30 (0.29) 5.72(0.29) -0.43 (0.30) -1.26,0.41 0.226 (4.15 - 7.02)[3.80 - 6.90] [4.02 - 6.72] [-2.09 - 0.96] [1.46, 9.88]1DW = dry weight; FW = fresh weight; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 286 of 395Table E-12. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Soybean Seed for Amino Acids, Fatty Acids,Fiber, Proximates, Vitamin E,Antinutrients and IsoflavonesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Alanine 1.78 (0.0084) 1.74 (0.0084) 0.044 (0.011) 0.021, 0.066 0.001 (1.63 - 1.86)[1.76 - 1.84] [1.68 - 1.81] [-0.018 - 0.11] [1.45, 2.02]Arginine 3.23 (0.056) 2.95 (0.056) 0.28(0.037) 0.20, 0.35 <0.001 (2.61 - 3.15)[3.00 - 3.61] [2.80 - 3.15] [0.0027 - 0.68] [2.13, 3.62]Aspartic Acid 4.54 (0.035) 4.36 (0.035) 0.18 (0.036) 0.077, 0.28 0.007 (4.01 - 4.71)[4.41 - 4.73] [4.21 - 4.60] [-0.034 - 0.43] [3.45, 5.29]Cystine 0.62 (0.0098) 0.60 (0.0098) 0.019 (0.0038) 0.011, 0.027 <0.001 (0.55 - 0.62)[0.56 - 0.65] [0.56 - 0.64] [-0.024 - 0.037] [0.49, 0.68]Glutamic Acid 7.63 (0.059) 7.29 (0.059) 0.34 (0.055) 0.22, 0.46 <0.001 (6.67 - 7.99)[7.42 - 7.90] [7.03 - 7.71] [-0.0052 - 0.76] [5.51, 9.04]Glycine 1.79 (0.012) 1.73 (0.012) 0.062 (0.0098) 0.035, 0.090 0.003 (1.61 - 1.86)[1.76 - 1.87] [1.67 - 1.81] [0.0039 - 0.14] [1.39, 2.05]Histidine 1.09 (0.0073) 1.05 (0.0073) 0.036(0.0074) 0.021, 0.051 <0.001 (0.98 - 1.13)[1.06 - 1.14] [1.02 - 1.10] [-0.013 - 0.090] [0.86, 1.27]Isoleucine 1.87 (0.018) 1.78 (0.018) 0.088(0.018) 0.050, 0.13 <0.001 (1.62 - 2.00)[1.75 - 1.97] [1.70 - 1.86] [-0.052 - 0.24] [1.34, 2.28]Monsanto Company 09-SY-200U Page 287 of 395Table E-12. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Soybean Seed for Amino Acids, Fatty Acids,Fiber, Proximates, Vitamin E,Antinutrients and Isoflavones(continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Leucine 3.19 (0.017) 3.09 (0.017) 0.10 (0.020) 0.059, 0.14 <0.001 (2.86 - 3.37)[3.13 - 3.32] [3.01 - 3.19] [-0.019 - 0.26] [2.45, 3.76]Lysine 2.67 (0.020) 2.60 (0.020) 0.070 (0.016) 0.036, 0.10 <0.001 (2.42 - 2.78)[2.61 - 2.75] [2.51 - 2.73] [-0.059 - 0.19] [2.13, 3.06]Methionine 0.60 (0.0067) 0.58 (0.0067) 0.017 (0.0057) 0.0015,0.033 0.038 (0.52 - 0.61)[0.54 - 0.62] [0.56 - 0.60] [-0.030 - 0.038] [0.48, 0.66]Phenylalanine 2.14 (0.011) 2.06 (0.011) 0.075 (0.015) 0.038, 0.11 0.002 (1.92 - 2.29)[2.08 - 2.24] [1.99 - 2.15] [-0.016 - 0.20] [1.61, 2.55]Proline 2.09 (0.018) 1.99 (0.018) 0.10 (0.015) 0.070, 0.13 <0.001 (1.81 - 2.16)[2.03 - 2.19] [1.91 - 2.09] [0.016 - 0.25] [1.53, 2.45]Serine 2.20 (0.016) 2.14(0.016) 0.055 (0.023) 0.0020,0.11 0.043 (1.97 - 2.27)[2.08 - 2.25] [2.00 - 2.29] [-0.11 - 0.21] [1.75, 2.51]Threonine 1.60 (0.0095) 1.57 (0.0095) 0.030(0.013) 0.0022, 0.057 0.035 (1.45 - 1.65)[1.54 - 1.65] [1.49 - 1.62] [-0.058 - 0.15] [1.30, 1.82]Tryptophan 0.47 (0.0064) 0.46 (0.0064) 0.010 (0.0053) -0.00097, 0.022 0.069 (0.43 - 0.52)[0.45 - 0.51] [0.44 - 0.50] [-0.019 - 0.059] [0.35, 0.59]Monsanto Company 09-SY-200U Page 288 of 395Table E-12. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Soybean Seed for Amino Acids, Fatty Acids,Fiber, Proximates, Vitamin E,Antinutrients and Isoflavones(Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Amino Acid (% DW)Tyrosine 1.40 (0.016) 1.34 (0.016) 0.059(0.021) 0.014, 0.10 0.013 (1.21 - 1.49)[1.27 - 1.50] [1.26 - 1.44] [-0.072 - 0.21] [1.03, 1.67]Valine 1.98 (0.019) 1.88 (0.019) 0.096 (0.021) 0.053, 0.14 <0.001 (1.70 - 2.11)[1.84 - 2.08] [1.78 - 1.96] [-0.063 - 0.26] [1.42, 2.41]Fatty Acid (% Total FA)16:0 Palmitic 12.06 (0.13) 11.77 (0.13) 0.29(0.052) 0.19, 0.40 <0.001 (9.88 - 12.33)[11.53 - 12.54] [11.14 - 12.08] [-0.080 - 0.75] [7.28, 14.20]18:0 Stearic 4.19 (0.10) 4.15(0.10) 0.042 (0.031) -0.044,0.13 0.245 (3.68 - 4.89)[3.73 - 4.53] [3.85 - 4.44] [-0.16 - 0.19] [2.87, 5.85]18:1 Oleic 15.18 (0.95) 19.19 (0.95) -4.02 (0.52) -5.46, -2.57 0.001 (16.70 - 23.16)[12.66 - 18.80] [17.24 - 21.17] [-5.38 - -1.95] [12.56, 27.98]18:2 Linoleic 22.78 (1.64) 54.93 (1.64) -32.16 (2.32) -37.50,-26.81 <0.001 (53.36 - 57.39)[16.46 - 30.81] [54.05 - 56.04] [-38.90 - -23.68] [50.46, 59.96]18:3 Linolenic 11.18 (0.46) 9.20 (0.46) 1.98 (0.50) 0.60, 3.36 0.016 (6.95 - 10.58)[10.20 - 11.80] [7.42 - 10.66] [0.82 - 3.89] [3.72, 13.46]20:0 Arachidic 0.34 (0.0090) 0.31 (0.0090) 0.031(0.0027) 0.025, 0.036 <0.001 (0.27 - 0.36)[0.31 - 0.37] [0.28 - 0.34] [0.017 - 0.058] [0.20, 0.45]Monsanto Company 09-SY-200U Page 289 of 395Table E-12. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Soybean Seed for Amino Acids, FattyAcids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones(Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Fatty Acid (% Total FA)20:1 Eicosenoic 0.14 (0.023) 0.13 (0.023) 0.012(0.0094) -0.014, 0.038 0.282 (0.071 - 0.19)[0.075 - 0.20] [0.069 - 0.19] [-0.058 - 0.072] [0, 0.31]22:0 Behenic 0.29 (0.0069) 0.32(0.0069) -0.026 (0.0087) -0.047,-0.0050 0.023 (0.30 - 0.41)[0.26 - 0.31] [0.28 - 0.37] [-0.11 - 0.022] [0.22, 0.49]Fiber (% DW)Acid Detergent Fiber 16.77 (0.42) 16.90 (0.42) -0.14 (0.50) -1.51, 1.24 0.794 (14.57 - 18.85)[14.38 - 18.31] [13.80 - 18.15] [-1.91 - 2.65] [10.36, 22.77]Neutral Detergent Fiber 16.84 (0.38) 17.18 (0.38) -0.34 (0.41) -1.19, 0.50 0.411 (15.03 - 18.92)[15.06 - 19.15] [14.43 - 19.37] [-2.82 - 2.32] [10.91, 22.59]Proximate (% DW, unless noted)Ash 5.72 (0.092) 5.63 (0.092) 0.098 (0.057) -0.024, 0.22 0.106 (5.59 - 6.20)[5.23 - 6.17] [5.24 - 6.07] [-0.34 - 0.62] [5.16, 6.64]Carbohydrates 36.45 (0.99) 38.68 (0.99) -2.24 (0.53) -3.32, -1.15 <0.001 (33.50 - 40.22)[33.23 - 39.93] [35.30 - 42.60] [-6.24 - 2.23] [26.76, 45.99]Moisture (% FW) 7.47 (0.17) 7.41 (0.17) 0.063 (0.10) -0.22, 0.35 0.572 (6.68 - 8.16)[6.71 - 8.21] [6.84 - 8.11] [-0.66 - 0.84] [5.23, 9.56]Protein 41.92 (0.27) 39.75 (0.27) 2.18 (0.31) 1.51, 2.84 <0.001 (37.52 - 42.37)[40.92 - 43.36] [38.22 - 41.58] [0.90 - 5.07] [33.37, 46.00]Monsanto Company 09-SY-200U Page 290 of 395Table E-12. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Soybean Seed for Amino Acids, Fatty Acids,Fiber, Proximates, Vitamin E,Antinutrients and Isoflavones(Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]ProximateTotal Fat (% DW) 15.91 (1.05) 15.94 (1.05) -0.037 (0.61) -1.74, 1.66 0.955 (13.99 - 20.56)[12.95 - 19.03] [12.73 - 18.80] [-3.01 - 3.27] [11.04, 25.03]VitaminVitamin E (mg/100g DW) 1.56 (0.26)1.43 (0.26) 0.13 (0.10) -0.16, 0.42 0.271 (0.27 - 2.93)[0.86 - 2.54] [0.70 - 2.22] [-0.35 - 0.55] [0, 4.65]Antinutrient (% DW, unless noted)Lectin (H.U./mg DW) 3.55 (0.80) 3.73 (0.80) -0.18 (0.81) -2.43, 2.07 0.836 (0.81 - 9.73)[0.55 - 8.07] [0.71 - 11.32] [-7.01 - 5.13] [0, 16.00]Phytic Acid 1.05 (0.059) 1.02 (0.059) 0.031(0.030) -0.052, 0.11 0.357 (0.81 - 1.27)[0.81 - 1.34] [0.75 - 1.26] [-0.13 - 0.16] [0.51, 1.59]Raffinose 0.37 (0.019) 0.35 (0.019) 0.022 (0.011) -0.00076, 0.045 0.057 (0.31 - 0.42)[0.32 - 0.45] [0.29 - 0.45] [-0.067 - 0.099] [0.19, 0.52]Stachyose 2.83 (0.11) 2.75 (0.11) 0.081 (0.070) -0.064, 0.23 0.259 (2.23 - 3.29)[2.28 - 3.27] [2.43 - 3.21] [-0.46 - 0.47] [1.61, 4.05]Trypsin Inhibitor (TIU/mg DW) 33.81 (2.81) 31.10 (2.81) 2.71 (2.01) -1.44, 6.87 0.190 (24.29 - 46.29)[24.30 - 54.80] [21.34 - 41.69] [-10.58 - 29.14] [8.09, 57.27]Monsanto Company 09-SY-200U Page 291 of 395Table E-12. Statistical Summary fromthe Combined-Site Compositional Analysis of MON 87769 vs. the ConventionalControl (A3525) Soybean Seed for Amino Acids, Fatty Acids,Fiber, Proximates, Vitamin E,Antinutrients and Isoflavones(Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)[Range]95% CI(Lower, Upper)p-ValueCommercial(Range)[99% ToleranceInterval2]Isoflavone (μg/g DW)Daidzein 1187.81 (188.32) 1807.36 (188.32) -619.55(120.04) -952.84, -286.26 0.006 (783.49 - 1691.97)[957.23 - 1838.91] [1380.05 - 2775.08] [-1385.89 - -186.51] [0, 2594.50]Genistein 733.64 (114.81) 1136.52 (114.81) -402.88(80.11) -625.30, -180.45 0.007 (741.53 - 1580.48)[576.70 - 1118.40] [770.81 - 1706.74] [-821.84 - -86.03] [254.31, 1976.30]Glycitein 82.73 (5.66) 102.18 (5.66) -19.45 (6.25) -32.35, -6.55 0.004 (74.87 - 189.64)[65.37 - 106.72] [65.51 - 158.73] [-93.36 - 1.49] [0, 243.40]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.Monsanto Company 09-SY-200U Page 292 of 395 Table E-13. Trans-ALA, GLA, tran s-SDA and SDA Levels in MON 87769
Individual-Site Summary

Analytical Component (Units)1 MON 87769 Mean (S.E.) 2 MON 87769 (Range)

Site IA-1 Seed Fatty Acid (% Total FA) 18:3 9c,12c,15t (trans-ALA) 0.47 (0.0087) [0.46 - 0.48]

18:3 Gamma Linolenic (GLA) 7.26 (0.010) [7.25 - 7.28]

18:4 6c,9c,12c,15t (trans-SDA) 0.24 (0.0081) [0.23 - 0.26]

18:4 Stearidonic (SDA) 31.93 (0.23) [31.51 - 32.28]

Site IA-2 Seed Fatty Acid (% Total FA) 18:3 9c,12c,15t (trans-ALA) 0.43 (0.012) [0.41 - 0.45]

18:3 Gamma Linolenic (GLA) 7.99 (0.033) [7.92 - 8.03]

18:4 6c,9c,12c,15t (trans-SDA) 0.20 (0.010) [0.19 - 0.22]

18:4 Stearidonic (SDA) 28.77 (1.09) [26.60 - 30.14]

Site IL Seed Fatty Acid (% Total FA) 18:3 9c,12c,15t (trans-ALA) 0.38 (0.0054) [0.38 - 0.39]

18:3 Gamma Linolenic (GLA ) 6.22 (0.062) [6.15 - 6.34]

18:4 6c,9c,12c,15t (trans-SDA) 0.061 (0.0022) [0.058 - 0.065]

18:4 Stearidonic (SDA) 17.11 (0.26) [16.83 - 17.64]

1 FA = fatty acid; 2 S.E. = standard error.Monsanto Company 09-SY-200U Page 293 of 395 Table E-13. Trans-ALA, GLA, tran s-SDA and SDA Levels in MON 87769 Individual- Site Summary (Continued)

Analytical Component (Units)1 MON 87769 Mean (S.E.) 2 MON 87769 (Range)

Site MI Seed Fatty Acid (% Total FA) 18:3 9c,12c,15t (trans-ALA) 0.47 (0.0081) [0.45 - 0.48]

18:3 Gamma Linolenic (GLA ) 7.64 (0.14) [7.36 - 7.78]

18:4 6c,9c,12c,15t (trans-SDA) 0.24 (0.0090) [0.22 - 0.25]

18:4 Stearidonic (SDA) 31.91 (1.03) [30.49 - 33.92]

Site OH Seed Fatty Acid (% Total FA) 18:3 9c,12c,15t (trans-ALA) 0.43 (0.0025) [0.43 - 0.44]

18:3 Gamma Linolenic (GLA) 6.33 (0.24) [6.07 - 6.82]

18:4 6c,9c,12c,15t (trans-SDA) 0.14 (0.0074) [0.14 - 0.16]

18:4 Stearidonic (SDA) 20.95 (0.65) [20.14 - 22.25]

1FA = fatty acid; 2 S.E. = standard error.Monsanto Company 09-SY-200U Page 294 of 395 Table E-14. Literature and ILSI Database Values for Components in Conventional
Soybean Seed and Forage

Analytical Component (Units) 1 Literature Range ILSI Range 2

SEED
Fiber (% DW)
Acid Detergent Fiber 9.0 – 11.1 i 7.81 – 18.61
Neutral Detergent Fiber 10.0 – 14.9 i 8.53 – 21.25
Proximate (% DW, unless noted)

Ash 4.61 – 5.94 b 3.89 – 6.99
Carbohydrates 29.3 – 41.3 c 29.6 – 50.2
Moisture (% FW) 5.3 – 8.73 c 4.7 – 34.4

Protein 36.0 – 48.4 e 33.19 – 45.48

Total Fat 16.0 – 23.1 e 8.10 – 23.56

19.8 – 26.7 d

Amino Acids (% DW)

Alanine 1.60 – 1.86 c 1.513 – 2.104
Arginine 2.56 – 3.46 c 2.285 – 3.400
Aspartic acid 4.18 – 4.99 c 3.808 – 5.122
Cystine 0.54 – 0.66 c 0.370 – 0.808

Glutamic acid 6.64 – 8.16 c 5.843 – 8.201
Glycine 1.60 – 1.87 c 1.458 – 1.997

Histidine 0.98 – 1.16 c 0.878 – 1.175

Isoleucine 1.65 – 1.95 c 1.539 – 2.077

Leucine 2.81 – 3.37 c 2.590 – 3.622
Lysine 2.47 – 2.84 c 2.285 – 2.839

Methionine 0.51 – 0.59 c 0.431 – 0.681
Phenylalanine 1.78 – 2.19 c 1.632 – 2.346

Proline 1.86 – 2.23 c 1.687 – 2.284

Serine 1.96 –2.28 c 1.106 – 2.484

Threonine 1.51 – 1.73 c 1.139 – 1.862
Tryptophan 0.56 – 0.63 c 0.356 – 0.502
Tyrosine 1.35 – 1.59 c 1.016 – 1.613

Valine 1.71 – 2.02 c 1.597 – 2.204
Isoflavones (ug/g DW)

Daidzein 219 – 1190 c 60.0 – 2453.5

Genistein 286 – 1380 c 144.3 – 2837.2
Glycitein 42.2 – 204a 15.3 – 310.4
Vitamin (mg/100g DW)

Vitamin E 1.09 – 2.84 g 0.19 – 6.17Monsanto Company 09-SY-200U Page 295 of 395 Table E-14. Literature and ILSI Database Values for Components in Conventional Soybean Seed and Forage (Continued)
Analytical Component (Units)1 Literature Range ILSI Range 2

Fatty Acid (% Total FA)

16:0 Palmitic 10.63 – 11.69 c 9.55 – 15.77

18:0 Stearic 3.85 – 4.55 c 2.70 – 5.88

18:1 Oleic 15.02 – 31.19 c 14.3 – 32.2

18:2 Linoleic 44.03 – 54.96 c 42.3 – 58.8

18:3 Linolenic 5.08 – 10.26 c 3.00 – 12.52

20:0 Arachidic 0.31 – 0.43 c 0.163 – 0.482

22:0 Behenic 0.46 – 0.59 c 0.277 – 0.595
Antinutrient (% DW, unless noted)
Phytic acid 1 – 2.74 i 0.634 – 1.960

Raffinose 0.4 – 1.8 h 0.212 – 0.661
Stachyose 3.08 – 4.13 f 1.21 – 3.50
Trypsin inhibitor (TIU/mg DW) 33.2 – 54.5 c 19.59 – 118.68
Lectin (H.U./mg FW) 0.8 – 2.4 c 0.105 – 9.038

FORAGE
Fiber (% DW)
Acid Detergent Fiber 32-38 i not available
Neutral Detergent Fiber 34-40 i not available

Crude Fiber not available 13.58-31.73
Proximate (% DW, unless noted)

Ash 8.8-10.5 i 6.718-10.782
Carbohydrates not available 59.8-74.7
Moisture (% FW) 74-79 i 73.5-81.6

Protein 11.2-17.3 i 14.38-24.71

Total Fat 3.1-5.1 i 1.302-5.132
1DW = dry weight; DM = dry matter; FW = fresh we ight; FA = fatty acid; TIU = trypsin inhibitor unit; H.U. = hemaglutinating unit.

2 ILSI-CCD, 2008. International Life Sciences In stitute Crop Composition Database. Version 3.0 http://www.cropcomposition.org. Search criteria soybean seed, all locations, all years, all proximates, amino acids, fatty acids, bio-actives, fi ber, dry weight other than moisture [Accessed June 3, 2008].

a Isoflavones Database (United States Department of Agriculture - Iowa State University, Version 1.3, 2002); b (Taylor et al., 1999); c (Padgette et al., 1996); d (Maestri et al., 1998); e (Hartwig et al., 1991); f (Grieshop et al., 2003); g (Guzman et al., 1986); h (Jacorzynski and Barylko-Pikielna, 1983); i (OECD, 2001).

Conversions: % dw x 10 4 = μ g/g dw; mg/g dw x 10 3 = mg/kg dw; mg/100g dw x 10 = mg/kg dw; g/100g dw x 10 = mg/g.Monsanto Company 09-SY-200U Page 296 of 395 E.6. Materials and Methods Used for the Compositional Analysis of MON 87769

Processed Fractions

E.6.1. Acid Detergent Fiber (ADF)

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. Lignocellulose fracti on was collected on the frit and determined gravimetrically. The limit of quantitation was 0.100%.

Reference USDA. 1970. Forage Fiber Analysis, Agriculture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

E.6.2. 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 amino acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids was accomplished through direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantitated using an automate d amino acid analyzer. The limit of quantitation for each amino acid was 0.100 mg/g.

Reference Standards

• Thermo Scientific/Pierce, K18, 2.5 μ mol/mL per constituen t (except cystine 1.25

μ mol/mL), Lot Number IJ115731

• Sigma, L-Tryptophan, 100%, Lot Number 076K0075

• Fluka, L-Cysteic Acid Monohydrate, > 99.9% (used as 100%), Lot Number 1157629

• Sigma, L-Methionine Sulfone, >99% (used as 100%), Lot Number 012H3349

Reference

AOAC. 2005. Method 982.30 in Official Met hods of Analysis. AOAC International, Gaithersburg, MD.

E.6.3. Ash

The sample was placed in an electric furnace at 550 ° C and ignited to drive off all volatile organic matter. The nonvolat ile matter remaining was quan titated gravimetrically and

calculated to determine percent ash. The limit of quantitation was 0.100%.

Reference

AOAC. 2005. Method 923.03 in Official Methods of Analysis, 18th ed. AOAC International, Gaithersburg, MD.Monsanto Company 09-SY-200U Page 297 of 395 E.6.4. Carbohydrates

The total carbohydrate level was calculated by difference using the fresh weight-derived data and the following equation:

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The limit of quantitation was 0.100%.

Reference USDA. 1973. Energy value of foods. Agri culture Handbook No. 74. United States Department of Agriculture, Washington, D.C.

E.6.5. 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 weighed. The limit of quantitation was 0.100%.

Reference

AOAC. 2005. Method 96.039 in Official Met hods of Analysis. AOAC International, Gaithersburg, MD.

E.6.6. Fatty Acid Profile

The lipid was extracted, saponified with 0.5N methanolic sodium hydroxide, and methylated with 14% BF 3 -methanol. The resulting methyl es ters of the fatty acids were extracted with heptane. An internal standard was added prior to th e lipid extraction. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation. The limit of quantitation was 0.005%-0.06% depending on the soybean fraction.

Reference Standards

• Nu Chek Prep GLC Reference Standa rd Hazelton No. 1, Lot Number JY19-R

• Nu Chek Prep GLC Reference Standard Hazelton No. 2, Lot Number M13-O

• Nu Chek Prep GLC Reference Standard Hazelton No. 3, Lot Number MA18-S

• Nu Chek Prep GLC Reference Standa rd Hazelton No. 4, Lot Number JY19-R

• Nu Chek Prep Methyl Gamma Linolenate, used as 100%, Lot Number U-63M-JY12-R

• Nu Chek Prep Methyl Tridecanoate, used as 100%, Lot Number N-13M-F5-S

• Nu Chek Prep Methyl Butyrate, used as 100%, Lot Number N-4M-J20-R

• Nu Chek Prep Methyl Hexanoate, us ed as 100%, Lot Number N-6M-A25-R

• Nu Chek Prep Methyl Erucate, us ed as 100%, Lot Number U-79M-AU3-Q

• Nu Chek Prep Methyl Lignocerate, us ed as 100%, Lot Number N-24M-F5-S

• Nu Chek Prep Methyl Docosapentaenoate , used as 100%, Lot Number U-101M-F18-S

• Nu Chek Prep Methyl Docosahexaenoate , used as 100%, Lot Number U-84M-AU28-RMonsanto Company 09-SY-200U Page 298 of 395 • Nu Chek Prep Methyl Eicosapentaenoate , used as 100%, Lot Number U-99M-JY17-R

• Cayman Chemicals Steariodonic Acid Met hyl Ester, used as 100%, Lot Number

182102-186015

• Nu Chek Prep Methyl Elaidate, us ed as 100%, Lot Number U-47M-JA18-R

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-F27-R

• Nu Chek Prep Methyl Palmitelaidate, used as as 100%, Lot Number U-41M-O26-R

• Monsanto Mono Trans SDA, 99%, Lot Number GLP-0707-18858-A

• Monsanto Mono Trans ALA, 96%, Lot Number GLP-0707-18857-A

• Larodan Methyl 6(z), 9(z)-Octadecadie noate, used as 100%, Lot Number LS-113

Reference

AOCS. Method Ce 1-63 in Official Methods and Recommended Practices of the AOCS, 5th ed. American Oil Chemists’ Society, Champaign, IL.

E.6.7. Isoflavones Analysis

The sample was extracted using a solution of hydrochloric acid and reagent alcohol heated on steam baths or hot plates. The ex tract 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 ultraviole t spectrophotometric quantitation and was compared against an exte rnal standard curve of known standards. The limit of quantitation for each component was 10.0 μ g/g.

Reference Standards Indofine, Daidzein, 99% , Lot Number 020508146 Indofine, Genistein, > 99% (used as 100% in calc ulations), Lot Number 0604043 Indofine, Glycitein, 99%, Lot Number 0704034

References

Seo, A. and C.V. Morr. 1984. Improved High-Performance Liquid Chromatographic Analysis of Phenolic Acids and Isof lavonoids from Soybean Protein Products

. Journal of Agricultural and Food Chemistry, 32(3):530-533.

Pettersson, H. and K-H Kiessling, K.-H. 1984. Liquid Chromatographic Determination of the Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official Analytical Chemists Journal, 67(3):503-506.

E.6.8. 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 dilution of the sample and lectin control was read by a spectrophotometer at 620 nm, using PBS to ze ro the instrument. One hemagglutinatingMonsanto Company 09-SY-200U Page 299 of 395 unit (H.U.) was defined as the level that caus ed 50% of the standard cell suspension to sediment in 2.5 hours. The limit of quantitation was 0.10 H.U./mg.

References Klurfeld, D.M. and D. Kritchevsky. 1987. Isol ation and Quantitation of Lectins from Vegetable Oils. Lipids 22:667-668.

Klurfeld, D. M., Pe rsonal communication.

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.

E.6.9. Moisture

The sample was dried in a vacuum oven at approximately 100 ° C to a constant weight. The moisture weight loss was determined a nd converted to percent moisture. The limit of quantitation was 0.100%.

Reference

AOAC. 2005. Methods 926.08 and 925.09. AOAC International, 18 th ed. AOAC Press, Gaithersburg, MD.

E.6.10. 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 grav imetrically. The limit of quantitation was

0.100%.

References AACC. 1998. Methods 32.20 in Approved Methods of the American Association of Cereal Chemists, 9th ed.

USDA. 1970. Forage Fiber Anlyses. Agri culture Handbook No. 379. United States Department of Agriculture, Washington, D.C.

E.6.11. Phytic Acid

The sample was extracted using 0.5M HCl with ultrasonication. Purification and

concentration was done on a silica based an ion exchange (SAX) column. Sample analysis was done on a polym er HPLC column PRP-1, 5 μ m (150 x 4.1mm) and a refractive index detector. The limit of quantitation was 0.100%.

Reference Standard

• Aldrich, Phytic Acid Dodecasodium Salt Hydrate, 97%, Lot Number 035K0590

• Sigma-Aldrich, Phytic Acid Dodecasodium Salt Hydrate from Rice, 95%, Lot Number

077K0693Monsanto Company 09-SY-200U Page 300 of 395 References Lehrfeld, J. 1989. High-performa nce liquid chromatography analysis of phytic acid on a ph-stable, macroporous polymer colum n. Cereal Chemistry 66(6):510-515.

Lehrfeld, Jacob, “HPLC Separation and Quantita tion of Phytic Acid and Some Inositol Phosphates in Foods: Problem and Solutions,” Journal of Agricultural Food Chemistry

, 42 :2726-2731, (1994).

E.6.12. Protein

Nitrogenous compounds in the sample were redu ced in the presence of boiling sulfuric acid and a mercury catalyst mixture to form am monia. The acid digest was made alkaline.

The ammonia was distilled and then titrated with a standard acid. The percent nitrogen was calculated and converted to protein usi ng the factor 6.25. Th e limit of quantitation

was 0.100%.

References

AOAC. 2005. Method 955.04 and 979.09 in Offici al Methods of Analysis of AOAC International, 18th ed. AOAC International, Gaithersburg, MD.

Bradstreet, R.B. 1965. The Kjel dahl Method for Organic Nitr ogen. Academic Press, New

York.

Kolthoff, I.M., and E.B. Sandell. 1948. Acidimetry and Alkalimitry in Quantitative Inorganic Analysis. The M acMillan Company, New York.

E.6.13. Phosphatides

The sample was extracted with a 98% CHCl 3 2% MeOH solvent. The extract is then analyzed on an HPLC system equipped with an evaporative light-scattering detector (ELSD). A calibration curve is used for quan tification. The Limits of Quantitation for these assays were as follows: L- alpha-Phosphatidic Acid 0.70%, L-alphaPhosphatidycholine 1.90%, L-alpha-P hosphatidylethanolamine 2.00%, and L-alpha-Phosphatidy linositol 1.20%.

Reference Standard

• (PA) – Avanti Polar Lipids, L-alpha-Phos phatidic Acid (sodium salt), 100%, Lot

Number SPA-19

• (PC) – Avanti Polar Lipids, L-alpha-Phospha tidylcholine, 100%, Lot Number PPC-116

• (PE) – Avanti Polar Lipids, L-alpha-Phos phatidylethanolamine, 100%, Lot Number

PPE-132b

• (PI) – Avanti Polar Lipids, L-alpha-Phospha tidylinositol, 100%, Lot Number PPI-149

Reference International Lecithin and Phospholipid Society, Analytical Method AM-101 HPLC, Determination of Lecithins, 1997.Monsanto Company 09-SY-200U Page 301 of 395 E.6.14. Raffinose and Stachyose

The sample was extracted with deionize d water and the extract treated with a hydroxylamine hydrochloride solution in pyridine, containing phenyl- β -D-glucoside as an internal standard. The resulting oxime s were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization de tector. The limit of quantitation was

0.0500%.

Reference Standards

• Sigma, Raffinose Pentahydrate, 99% / 84.0% after correction for degree of hydration,

Lot Number 035K1371

• Sigma, Stachyose, 98% / 96.4% after correct ion for degree of hydr ation, Lot Number

065K3775

References Mason, B. S., and H.T. Slover. 1971. A gas chromatographic method for the determination of sugars in foods. J. Agric. Food Chem. 19(3):551-554.

Brobst, K. M. 1972. Gas-liquid chromatography of trimethylsilyl derivatives in Methods in Carbohydrate Chemistry, Vol. 6. Academic Press, New York.

E.6.15. Trypsin Inhibitor

The sample was ground and defatted with petr oleum ether. A sample of matrix was extracted for 3 hours with 0.01N sodium hydroxide. Varying aliquots of the sample suspension were exposed to a known amoun t of trypsin and be nzoy1-DL-argininep-nitroanalide hydrochloride. The sample was allowed to react for 10 minutes at 37 °C. After 10 minutes, the reaction was halted by the addition of acetic ac id. The solution was centrifuged, then the absorbance was determined at 410 nm. Trypsin inhibitor activity as measured by trypsin inhibito r units (TIU) was determined by photometrically measuring the inhibition of trypsin’s reaction w ith benzoyl-DL-arginine-p-nitroanalide hydrochloride. The limit of quantitation was 1.00 TIU/mg.

Reference

AOCS. 1997. Method Ba12-75 in Official Me thods and Recommended Practices of the American Oil Chemists’ Society, 5th ed. Ch emists’ Society. AOCS Press, Champaign,

IL.

E.6.16. Vitamin E

The sample was saponified to break down any fat and release any vitamin E. The saponified mixture was extracted with ethyl et her and then quantitat ed directly by HPLC on a silica column. The limit of quantitation was 0.500 mg/100g.Monsanto Company 09-SY-200U Page 302 of 395 Reference Standard USP, Alpha Tocopherol, 100%, Lot Number M

References Cort, W.M., T.S. Vincente, E.H. Waysek, and B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high-performance liquid chromatographic Fluorescence. Journal of Agricultural Food Chemistry, 31:1330-1333.

Speek, A.J., J. Schijver, and W.H.P. Schreu rs. 1985. Vitamin E composition of some seed oils as determined by high-performance liquid chromatography with fluorometric quantitation. Journal of Food Science, 50(1):121-124.

McMurray, C.H., W.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction techniques on determination of α -tocopherol in animal feedstuffs. Journal of the

Association of Official Analytical Chemists

, 63(6):1258-1261.Monsanto Company 09-SY-200U Page 303 of 395Statistical Summary from the Combined-SiteAnalysis of MON 87769 Processed FractionsTable E-15. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Meal Fractionfrom Combined-Site for Amino Acids,Fiber, Proximates and AntinutrientsDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Amino Acid (% DW)Alanine 2.28 (0.025) 2.28 (0.025) 0.0053 (0.013)-0.029, 0.039 0.703 (2.23 - 2.39) 2.18 - 2.59a[2.24 - 2.32] [2.24 - 2.31] [2.05, 2.58]Arginine 4.34 (0.13) 4.17 (0.13) 0.16 (0.067) -0.68, 1.010.245 (3.80 - 4.32) 3.29 - 4.49a[4.18 - 4.51] [4.06 - 4.31] [3.36, 4.94]Aspartic Acid 6.10 (0.030) 6.00 (0.030) 0.10 (0.022) 0.044, 0.16 0.006(5.80 - 6.30) 5.18 - 6.83a[6.05 - 6.13] [5.94 - 6.04] [5.21, 6.97]Cystine 0.77 (0.0080) 0.77 (0.0080) 0.0036 (0.0084) -0.10, 0.11 0.740 (0.71 - 0.80) 0.6b- 0.92a[0.77 - 0.78] [0.75 - 0.79] [0.61, 0.93]Glutamic Acid 9.79 (0.031) 9.58 (0.031) 0.22 (0.043) 0.11, 0.320.002 (9.15 - 10.05) 8.05 - 11.21a[9.78 - 9.81] [9.50 - 9.68] [8.21, 11.33]Glycine 2.31 (0.023) 2.28 (0.023) 0.038 (0.015) -0.0017, 0.077 0.057 (2.23 - 2.36) 2.02 - 2.40a[2.28 - 2.34] [2.23 - 2.32] [2.06, 2.55]Histidine 1.42 (0.010) 1.40 (0.010) 0.021 (0.0059)0.0054, 0.036 0.017 (1.39 - 1.46) 1.32 - 1.63a[1.41 - 1.43] [1.38 - 1.42] [1.28, 1.57]Isoleucine 2.53 (0.050) 2.51 (0.050) 0.023 (0.046)-0.096, 0.14 0.642 (2.47 - 2.68) 2.11 - 2.74a[2.46 - 2.60] [2.38 - 2.59] [2.11, 2.98]Monsanto Company 09-SY-200U Page 304 of 395Table E-15. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Meal Fractionfrom Combined-Site for Amino Acids, Fiber,Proximates and Antinutrients (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Amino Acid (% DW)Leucine 4.13 (0.037) 4.08 (0.037) 0.047 (0.030)-0.030, 0.12 0.178 (4.04 - 4.29) 3.62 - 4.72a[4.07 - 4.19] [4.01 - 4.15] [3.64, 4.68]Lysine 3.34 (0.026) 3.31 (0.026) 0.027 (0.022) -0.030, 0.0840.284 (3.20 - 3.49) 2.97 - 3.69a[3.29 - 3.38] [3.26 - 3.36] [2.90, 3.82]Methionine 0.77 (0.0079) 0.76 (0.0079) 0.0038 (0.011) -0.044, 0.0520.767 (0.74 - 0.82) 0.5 - 0.9b[0.75 - 0.78] [0.75 - 0.78] [0.62, 0.92]Phenylalanine 2.78 (0.023) 2.74 (0.023) 0.042 (0.020) -0.0099, 0.094 0.091 (2.70 - 2.90) 2.39 - 3.19a[2.75 - 2.81] [2.69 - 2.78] [2.44, 3.15]Proline 2.66 (0.033) 2.62 (0.033) 0.034 (0.047)-0.17, 0.24 0.543 (2.50 - 2.75) 2.32 - 3.05a[2.64 - 2.68] [2.50 - 2.71] [2.15, 3.14]Serine 2.75 (0.034) 2.68 (0.034) 0.070 (0.048) -0.048, 0.19 0.197 (2.53 - 2.84) 1.97a- 3.3b[2.71 - 2.80] [2.58 - 2.77] [2.23, 3.27]Threonine 2.00 (0.016) 1.97 (0.016) 0.023 (0.022)-0.035, 0.080 0.361 (1.93 - 2.06) 0.80 - 2.24a[1.98 - 2.01] [1.91 - 2.01] [1.73, 2.28]Tryptophan 0.68 (0.020) 0.67 (0.020) 0.012 (0.0026) 0.0049, 0.019 0.006 (0.65 - 0.71) 0.60c- 2.08a[0.66 - 0.70] [0.65 - 0.69] [0.57, 0.77]Monsanto Company 09-SY-200U Page 305 of 395Table E-15. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Meal Fractionfrom Combined-Site for Amino Acids, Fiber,Proximates and Antinutrients (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Amino Acid (% DW)Tyrosine 1.81 (0.016) 1.78 (0.016) 0.027 (0.022) -0.027, 0.0810.265 (1.65 - 1.93) 1.68 - 2.17a[1.78 - 1.83] [1.73 - 1.82] [1.39, 2.25]Valine 2.67 (0.047) 2.64 (0.047) 0.031 (0.055)-0.11, 0.17 0.596 (2.61 - 2.79) 2.29 - 2.92a[2.59 - 2.74] [2.50 - 2.71] [2.37, 3.01]Fiber (% DW)Acid Detergent Fiber 5.18 (0.14)4.52 (0.14) 0.66 (0.19) 0.18, 1.13 0.014 (4.00 - 5.66) 5.2 - 6.7f[4.93 - 5.46] [4.22 - 4.96] [1.79, 7.28]Neutral Detergent Fiber 6.36 (0.13) 5.58 (0.13) 0.78 (0.19) -0.017,1.58 0.052 (4.41 - 6.10) 7.4 - 12.2f[6.19 - 6.56] [5.36 - 5.82] [2.46, 8.06]Monsanto Company 09-SY-200U Page 306 of 395Table E-15. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Meal Fractionfrom Combined-Site for Amino Acids, Fiber,Proximates and Antinutrients (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Proximate (% DW, unless noted)Ash 6.62 (0.27) 6.46 (0.27) 0.16 (0.14)-0.20, 0.52 0.299 (6.37 - 7.07) 5.2 - 9.1f[6.19 - 6.91] [5.96 - 6.83] [5.63, 7.91]Carbohydrates 38.00 (0.29) 40.05 (0.29) -2.06(0.39) -3.05, -1.06 0.003 (36.26 - 41.61) NA[37.49 - 38.38] [39.44 - 41.00] [28.99, 48.21]Moisture (% FW) 2.63 (0.56) 2.45 (0.56) 0.18 (0.64) -1.46, 1.81 0.794 (2.36 - 4.40) 5.58 - 11.7d[1.68 - 3.69] [1.76 - 4.12] [0, 7.38]Protein 54.22 (0.50) 52.64 (0.50) 1.57 (0.70) -7.28, 10.420.265 (50.03 - 55.23) 47.4 - 59.5a[53.28 - 55.50] [52.11 - 53.19] [44.73, 62.02]Total Fat 1.17 (0.39) 0.84(0.39) 0.32 (0.47) -5.70, 6.35 0.619 (0.82 - 2.38) 0.5e- 3.30f[0.50 - 2.00] [0.61 - 1.13] [0, 3.87]Antinutrient (% DW, unless noted)Phytic Acid 1.33 (0.051) 1.33 (0.051) 0.0034 (0.072) -0.17, 0.18 0.964 (1.22 - 1.51) 1.3 - 4.1d[1.19 - 1.52] [1.30 - 1.36] [0.87, 1.88]Trypsin Inhibitor (TIU/mg DW) 2.03 (1.23) 4.82 (1.23) -2.78 (1.75) -7.05, 1.49 0.162 (0.52 - 6.63) 3.8 - 17.9d[0.52 - 3.56] [2.31 - 9.10] [0, 16.57]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.3Literature/Historical range references:aKarr-Lilienthal et al., 2004,bGrieshop et al., 2003,cSoybean Meal Information Center, 2001.dPadgette et al., 1996.eOrthoefer, 1978,fOECD, 2001.Monsanto Company 09-SY-200U Page 307 of 395Table E-16. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean RBD Oil fromCombined-Site for Fatty Acids and Vitamin EDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Fatty Acid (% Total FA)14:0 Myristic 0.083 (0.0039) 0.082 (0.0039) 0.00064 (0.0016) -0.0036,0.0049 0.713 (0.067 - 0.084) NA[0.078 - 0.088] [0.078 - 0.089] [0.042, 0.11]16:0 Palmitic 12.10 (0.050) 11.48 (0.050) 0.61 (0.071) 0.44, 0.79 <0.001 (9.80 - 11.73) NA[11.98 - 12.23] [11.42 - 11.61] [7.68, 13.21]16:1 Palmitoleic 0.085 (0.0015) 0.091 (0.0015) -0.0065 (0.0021) -0.016,0.0027 0.093 (0.079 - 0.11) NA[0.083 - 0.087] [0.088 - 0.095] [0.044, 0.14]17:0 Heptadecanoic 0.10 (0.0094) 0.096 (0.0094) 0.0053(0.0031) -0.035, 0.045 0.337 (0.080 - 0.10) NA[0.090 - 0.11] [0.088 - 0.11] [0.057, 0.12]18:0 Stearic 4.18 (0.019) 4.08(0.019) 0.098 (0.027) 0.033, 0.16 0.010 (3.91 - 4.45) NA[4.13 - 4.20] [4.04 - 4.12] [3.00, 5.17]18:1 Oleic 16.02 (0.80) 19.25 (0.80) -3.23 (0.89) -14.51, 8.04 0.170 (19.57 - 23.22) NA[14.49 - 17.34] [19.02 - 19.74] [16.11, 26.19]18:2 6c,9c (Isolinoleic Acid) 0.091 (0.0055) 0.075 (0.0055) 0.015 (0.0078) -0.018, 0.049 0.184 (0.030 - 0.077) NA[0.089 - 0.094] [0.063 - 0.085] [0, 0.15]18:2 9c,12c Linoleic 25.66 (3.53)55.38 (3.53) -29.72 (4.54) -87.36,27.92 0.096 (54.06 - 56.98) NA[20.66 - 30.92] [54.82 - 55.87] [50.27, 60.17]Monsanto Company 09-SY-200U Page 308 of 395Table E-16. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean RBD Oil fromCombined-Site for Fatty Acids and Vitamin E (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Fatty Acid (% Total FA)18:3 9c,12c,15t (Trans-ALA) 0.51(0.020) 0.14 (0.020) 0.37 (0.020) 0.11, 0.63 0.035 (0.072 - 0.15) NA[0.47 - 0.54] [0.10 - 0.16] [0, 0.24]18:3 Linolenic 10.61 (0.55) 8.31 (0.55) 2.31 (0.47) -3.71, 8.32 0.128 (6.44 - 8.61) NA[10.34 - 10.95] [7.42 - 9.07] [3.48, 11.79]18:3 (Other 18:3 Trans) 0.064 (0.0091) 0.084 (0.0091) -0.019 (0.013) -0.075, 0.0360.271 (0.031 - 0.083) NA[0.031 - 0.078] [0.069 - 0.098] [0, 0.16]20:0 Arachidic 0.35 (0.0072) 0.31 (0.0072) 0.034 (0.0035) -0.011, 0.079 0.065 (0.30 - 0.35) NA[0.34 - 0.35] [0.30 - 0.33] [0.23, 0.42]20:1 Eicosenoic 0.18 (0.020) 0.17 (0.020) 0.014 (0.0016) -0.0061, 0.033 0.072 (0.15 - 0.22) NA[0.16 - 0.20] [0.14 - 0.19] [0.043, 0.31]22:0 Behenic 0.32 (0.019) 0.33(0.019) -0.0086 (0.022) -0.065,0.048 0.709 (0.32 - 0.40) NA[0.27 - 0.35] [0.29 - 0.36] [0.21, 0.51]24:0 Lignoceric 0.093 (0.015) 0.12 (0.015) -0.030 (0.0024) -0.036, -0.023 <0.001 (0.088 - 0.18) NA[0.076 - 0.11] [0.10 - 0.14] [0, 0.26]Monsanto Company 09-SY-200U Page 309 of 395Table E-16. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean RBD Oil fromCombined-Site for Fatty Acids and Vitamin E (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Vitamin (mg/100g FW)Vitamin E 8.61 (1.98) 7.14 (1.98) 1.48 (0.16) 1.06, 1.90<0.001 (3.56 - 15.35) 0.9 - 35.2a[6.56 - 10.90] [5.14 - 9.27] [0, 29.82]1FW = fresh weight; FA = fatty acid; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.3Literature/Historical range references:aCodex, 2005.Monsanto Company 09-SY-200U Page 310 of 395Table E-17. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Protein Isolatefrom Combined-Site for Amino Acids and MoistureDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Amino Acid (% DW)Alanine 3.70 (0.037) 3.83 (0.037) -0.13 (0.052) -0.36, 0.091 0.124 (3.56 - 3.82) -[3.63 - 3.81] [3.77 - 3.90] [3.33, 4.12]Arginine 7.78 (0.11) 7.85 (0.11) -0.072 (0.13) -0.41, 0.27 0.607 (7.54 - 8.08) 6.67a[7.61 - 8.09] [7.61 - 8.01] [6.76, 8.87]Aspartic Acid 10.98 (0.14) 11.07 (0.14) -0.087 (0.20) -0.59, 0.41 0.683 (10.72 - 11.48) -[10.70 - 11.34] [10.67 - 11.41] [9.79, 12.23]Cystine 1.14 (0.010) 1.12 (0.010) 0.022 (0.014) -0.013, 0.058 0.173 (1.09 - 1.18) 1.05a[1.12 - 1.16] [1.09 - 1.15] [0.98, 1.27]Glutamic Acid 18.80 (0.28) 18.83 (0.28) -0.024 (0.37) -0.99, 0.94 0.951 (18.16 - 19.56) -[18.42 - 19.65] [18.12 - 19.34] [16.29, 21.34]Glycine 3.93 (0.033) 4.00 (0.033) -0.069 (0.046) -0.18, 0.044 0.184 (3.71 - 4.01) -[3.88 - 3.97] [3.90 - 4.06] [3.45, 4.38]Histidine 2.40 (0.021) 2.45 (0.021) -0.048 (0.030) -0.12, 0.025 0.158 (2.33 - 2.50) 2.3a[2.37 - 2.44] [2.39 - 2.51] [2.15, 2.72]Isoleucine 4.43 (0.044) 4.57 (0.044) -0.13 (0.062) -0.40, 0.13 0.161 (4.39 - 4.68) 4.25a[4.33 - 4.50] [4.47 - 4.61] [3.98, 4.96]Monsanto Company 09-SY-200U Page 311 of 395Table E-17. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Protein IsolateFraction from Combined-Site for Amino Acids and Moisture (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Amino Acid (% DW)Leucine 7.28 (0.053) 7.47 (0.053) -0.19 (0.075) -0.37, -0.0062 0.044 (7.08 - 7.61) 6.78a[7.13 - 7.37] [7.38 - 7.59] [6.62, 8.11]Lysine 5.78 (0.040) 5.91 (0.040) -0.13 (0.056) -0.27, 0.0028 0.053 (5.68 - 5.95) 5.33a[5.70 - 5.86] [5.80 - 5.98] [5.41, 6.21]Methionine 1.21 (0.011) 1.22(0.011) -0.014 (0.015) -0.051,0.022 0.368 (1.13 - 1.25) 1.13a[1.20 - 1.23] [1.19 - 1.25] [1.02, 1.38]Phenylalanine 5.00 (0.046) 5.10 (0.046) -0.096(0.065) -0.25, 0.063 0.189 (4.88 - 5.24) 4.59a[4.88 - 5.10] [5.02 - 5.19] [4.54, 5.55]Proline 4.89 (0.13) 4.90 (0.13) -0.014 (0.18) -0.46, 0.43 0.939 (4.30 - 5.09) -[4.64 - 5.22] [4.57 - 5.08] [3.46, 6.04]Serine 4.96 (0.058) 5.01 (0.058) -0.048 (0.082) -0.40,0.30 0.614 (4.78 - 5.10) -[4.86 - 5.08] [4.82 - 5.11] [4.49, 5.54]Threonine 3.25 (0.052) 3.30 (0.052) -0.053 (0.074) -0.23, 0.13 0.497 (2.96 - 3.31) 3.14a[3.10 - 3.35] [3.21 - 3.39] [2.67, 3.78]Tryptophan 1.09 (0.011) 1.10 (0.011) -0.014 (0.0070) -0.032, 0.0043 0.107 (1.05 - 1.13) 1.12a[1.07 - 1.11] [1.09 - 1.12] [0.92, 1.25]Monsanto Company 09-SY-200U Page 312 of 395Table E-17. Summary of the Compositional Comparison ofMON 87769 vs. the Conventional Control Soybean Protein IsolateFraction from Combined-Site for Amino Acids and Moisture (Continued)Difference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Amino Acid (% DW)Tyrosine 3.43 (0.040) 3.52 (0.040) -0.087 (0.042) -0.19, 0.020 0.091 (3.33 - 3.60) -[3.33 - 3.52] [3.44 - 3.58] [3.03, 3.89]Valine 4.48 (0.061) 4.63 (0.061) -0.14 (0.086) -0.51, 0.23 0.236 (4.34 - 4.65) 4.1a[4.44 - 4.56] [4.51 - 4.74] [4.01, 5.03]ProximateMoisture (% FW) 3.22 (0.28) 2.73 (0.28) 0.50 (0.40) -0.47, 1.47 0.256 (1.97 - 3.28) 3.9 - 7.0c[2.56 - 3.64] [2.03 - 3.56] [0.47, 4.66]1DW = dry weight; FW = fresh weight; FA = fatty acid;S.E. = standard error; CI= Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.3Literature/Historical range references:aOECD, 2001,bMattil, 1974.Monsanto Company 09-SY-200U Page 313 of 395Table E-18. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soybean Lecithin Fractionfrom Combined-Site for PhosphatidesDifference (Test minus Control)Analytical Component (Units)1MON 87769Mean (S.E.)[Range]A3525Mean (S.E.)[Range]Mean (S.E.)95% CI(Lower, Upper)pValueCommercial(Range)[99% ToleranceInterval2]Literature/Historical Range3Phosphatide (%FW)L-alpha-Phosphatidic Acid 3.90 (0.43) 3.54 (0.43) 0.36(0.30) -0.40, 1.12 0.278(3.40 - 5.65) 0.2 - 14.0a[3.16 - 4.54] [2.74 - 4.12] [0.71, 8.07]L-alpha-Phosphatidylcholine 7.16 (1.16) 7.28 (1.16) -0.12 (1.02) -13.06, 12.83 0.926(5.66 - 8.54) 12.0 - 46.0a[6.37 - 8.16] [5.37 - 10.25] [2.16, 11.62]L-alpha-Phosphatidylethanolamine 5.66 (0.67) 5.48 (0.67) 0.18 (0.72) -9.01, 9.38 0.843(4.73 - 5.97) 8.0 - 34.0a[5.43 - 6.14] [4.40 - 7.42] [2.98, 7.63]L-alpha-Phosphatidylinositol 4.98 (0.37) 4.67 (0.37) 0.31(0.53) -1.96, 2.57 0.621(3.93 - 5.01) 1.7 - 21.0a[4.44 - 5.47] [4.00 - 5.89] [2.68, 6.61]1FW = fresh weight; FA = fatty acid; S.E. =standard error; CI = Confidence Interval.2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative limits were set to zero.3Literature/Historical range references:aSzuhaj, 2005.Monsanto Company 09-SY-200U Page 314 of 395 References Codex Standard for Named Vege table Oils, 2005. CODEX-Stan 210. http://www.fao.org/docrep/meeting/005/ X9919E/x9919e0e.htm .[Accessed May 11, 2006].

George, C., W.P. Ridley, J.C. Obert, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2004. Composition of grain and forage from corn rootworm-protected corn event MON 863 is equivalent to that of conventional corn ( Zea mays L .). Journal of Agricultural and Food Chemistry 52(13):4149-4158.

Grieshop, C.M., Kadzere, C.T., Clapper, G.M ., Flickinger, E.A., Bauer, L.L., Frazier, R.L., and Fahey, G.C., Jr., 2003. Chemical and nut ritional characteristic s of United States soybeans and soybean meals. J Agric Food Chem 51:7684-7691.

Guzman, G. J. and Murphy, P. A. 1986. Tocophe rols of soybean seeds and soybean curd (tofu). J. Agric. Food Chem. 34:791-795.

Hartwig, E.E. and T.C. Kilen. 1991. Yield and composition of soybean seed from parents with different protein, sim ilar yield. Crop Sci. 31:290-292.

Jacorzynski, B. and N. Barylko-Pikiel na. 1983. Quantitative and qualitative characteristics of sugars in the seeds of soybean cultivated in Poland. Acta Agrobotanica

36:41-48.

Karr-Lilienthal, L.K., Grieshop, C.M., Merchen, N.R., Mahan, D.C., and Fahey, G.C., Jr., 2004. Chemical composition and protein quality comparisons of soybeans and soybean meals from five leading soybean-produci ng countries. J Agri Food Chem 52:6193-6199.

Mattil,, K.F., 1974. Composition, Nutritional, and Functional Properties, and Quality Criteria of Soy Protein Concentrates and S oy Protein Isolates. J Am Oil Chemists’ Soc

51:81A-84A.

Maestri, D.M., D.O. Labuckas, J.M. Meriles, A.L. Lamarque, J.A. Zygadlo, and C.A. Guzman. 1998. Seed composition of soybean cultivars evaluated in different environmental regions. J. Sci. Food Agric. 77:494-498.

OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean:key food and feed nutrients and an ti-nutrients. Organizat ion for Economic Cooperation and Development, Evironmental Health and Safety Publications. Paris, France. ENV/JM/MONO (2001)15.

Orthoeffer, F.T., 1978. Processing and util ization. Page 239 in Soybean Physiology, Agronomy and Utilization. Academic Press, Inc.. New York, NY.

Padgette, S.R., Taylor, N.B., Nida, D.L., Ba iley, M.R., MacDonald, J., Holden, L.R., and Fuchs, R.L. 1996. The composition of glyphosate-to lerant soybean seeds is equivalent to that of conventional soybeans. J Nutr 126:702-716.Monsanto Company 09-SY-200U Page 315 of 395 Ridley, W.P., R.S. Sidhu, P.D. Pyla, M.A. Nemeth, M.L. Breeze, and J.D. Astwood. 2002. Comparison of the nutritiona l profile of glyphosate-tolera nt corn event NK603 with that of conventional corn ( Zea mays L.). Journal of Agricultural and Food Chemistry 50(25):7235-7243.

Soybean Meal Info Center. Copyright ©2001 by Iowa State Soybean Association. http://www.soymeal.org/worldlitarticles_ne w/baizejohn1999.html [accessed April, 19, 2006].

Szuhaj, B.F. 2005. Lecithins. Pages 361-441 in Bailey’s Industrial Oil and Fat Products. Sixth Edition. Shahidi, F. (ed). John Wiley & Sons, Inc, New York, NY

Taylor, N.B., R.L. Fuchs, J. MacDonal d, A.R. Shariff, and S.R. Padgette. 1999. Compositional analysis of glyphosate-tolerant so ybeans treated with glyphosate. J. Agric.

Food Chem. 47:4469-4473.Monsanto Company 09-SY-200U Page 316 of 395 Appendix F. Safety and Nutritional Impact Resulting from the Intended Fatty Acid Change in MON 87769

F.1. Safety and Nutritional Assessmen t of the Intended Changes in MON 87769

Beside the components typically present in soybean, MON 87769 is expected to contain SDA, GLA, and small amounts of trans-ALA and trans-SDA from the intended change. As expected, the intended change also decr eased the levels of LA in MON 87769. The safety and nutritional assessment of M ON 87769 will not be complete without a discussion of the safety and nutritional impact resulting from the intended changes. The following sections describe the safety and nu tritional impact of S DA, GLA, and the two

trans-isomers as well as the nutritional im pact from the reduced levels of LA in

MON 87769.

F.2. Safety and Nutritional Impa ct Assessment of SDA in MON 87769

SDA is an in vivo intermediate in the metabolism of ALA to long chain omega-3 fatty acids in mammals. There are many natural sour ces of SDA in the food supply. Fish oils contain levels of SDA ranging from 0.9 to 3.0 wt% (salmon, mackerel, cod, menhaden, herring, boal fish, and sardine). Many fish oils that contain SDA have previously received a Generally Recognized as Safe (G RAS) status (salmon, menhaden, sardine, and tuna oil). SDA is also present in certain edible algae species including Undaria pinnatifida and Ulva pertusa (16.3 to 26.3% of total fatt y acids) and the seed oil of several plant species, no tably black currant ( Ribes nigrum ) (2 to 4% of total fatty acids) and Echium ( Echium plantagineum ) (8 to 15% of total fatty acids). Echium oil, containing not less than 10% SDA, was recently authorized in the European Union as a novel food ingredient 

### ( EC, 2008 

### ) . Many dietary supplements made with fish oil, algae or plant species such as black currant are rich in SDA and are consumed as a source of omega-3 fatty acids. Thus, SDA has a hist ory of safe consumption in human foods across a range of sources.

The safety of SDA in MON 87769 is ba sed on: 1) its occurrence as an in vivo

intermediate in the metabolis m of ALA to long chain omega-3 fatty acids in mammals, 2) a long-standing history of safe consump tion of SDA from several marine and plant sources, 3) the GRAS status of four fish oils containing SDA, and 4) the positive confirmation from the European Food Safety Agency on the safety of Echium oil containing SDA. Furthermore, the safety of SDA was confirmed by several human, as well as animal studies, conducted with SDA a nd SDA soybean oil. These studies were conducted with SDA intake levels ranging from 0.8 to 62 mg/kg body weight/day for the human studies and up to 1.04 g/kg body weight/d ay for the rat studies with no adverse effects reported. Therefore, SDA is conc luded to be safe for human and animal consumption.

F.3. Safety and Nutritional Impa ct Assessment of GLA in MON 87769

GLA is an in vivo metabolite in the conversion of LA to arachidonic acid in mammals (Fan and Chapkin, 1998; Horrobin, 1992). It is essential that LA or GLA be present inMonsanto Company 09-SY-200U Page 317 of 395 the human diet 

### ( Fan and Chapkin, 1998; Horrobin, 1992 

### ) . GLA is present in oats, barley, and human breast milk (Horrobin, 1992). Sm all concentrations of GLA are found in meats, fish, and a variety of other foods (Horrobin, 1992). Human breast milk contains approximately 50 mg/l of GLA and breast feed ing infants appear to have the highest dietary intake among age groups (Kankaanpaa et al., 2001; Stoney et al., 2004; Thijs et al., 2000; Villamor et al., 2007). Breast fe d infants will consume 5-10 mg/kg/day of GLA, equivalent to an intake of 0.35-0.7 gr ams/day GLA intake by a 70 kg adult. GLA is also found in large concentrations in certa in plant oils (borage, echium, hemp, evening primrose, and black currant) (Horrobin, 1992) at levels which may exceed those found in

MON 87769.

The safety of GLA in MON 87769 is based on: 1) its occurrence as an intermediate in the

in vivo metabolism of LA to arachidonic acid in mammals, 2) a long history of consumption of foods that contain GLA, a nd 3) many human and animal studies using GLA-containing materials. A number of human clinical studies have examined chronic consumption of GLA with no safety issues reported. In published human studies, supplementation with GLA at doses of 1 to 5 g/day for periods of one to six months was well tolerated and without reports of serious ad verse effects. A number of animal studies have also demonstrated the safety of la rge doses of GLA consumption. There are no identified concerns at this time regarding excessive dietary consum ption of GLA (Health Canada, 2006; Horrobin, 1992). Thus, GLA is concluded to be safe for human and animal consumption.

F.4. Safety and Nutritional Impact of trans-SDA and trans-ALA in MON 87769

The primary source of trans-fatty acids (TFAs) in the human diet is the consumption of hydrogenated vegetable oils in liquid or soli d form resulting in food products that may

contain in excess of 30% TFA 

### ( Chardigny et al., 1996; Ledoux, 2007 

### ) . Naturally occurring trans-fats also arise as a result of bacterial reduction of unsaturated fatty acids in the gut of ruminant animals, with intake resulting from consump tion of meat and dairy products 

### ( Chardigny et al., 1996; Ledoux, 2007 

### ) . The TFA levels in non-hydrogenated vegetable oils are generally low by comparison, t ypically less than 1% of total fatty acids. The TFAs arise during oil refining, particul arly during heat-requiring processes such as

deodorization 

### ( Chardigny et al., 1996; Ledoux, 2007 

### )

. The total TFA levels in MON 87769 are approximately 0.6% of total fa tty acids. Total TFAs in MON 87769 are well within the range of the total TFA cont ent in samples of commercial soybean oil 

### ( Chardigny et al., 1996 

### ) . The contribution of the TF A from MON 87769 to the overall human dietary TFA intake will be minima l relative to commonly experienced human dietary TFA intakes. Thus, the trans-SDA and trans-ALA are concluded to not raise safety concerns.

F.5. Nutritional Impact from Reduced LA Levels in MON 87769

Due to the conversion of LA to ALA and GLA in MON 87769, the LA concentration is expected to be lower in MON 87769 (Figure VII-1). LA is c onsidered to be an essential fatty acid, and is necessary in the human diet to support health. However, the onlyMonsanto Company 09-SY-200U Page 318 of 395 recognized role of LA in mammalian species is as a precursor to GLA (Horrobin, 1992) and as a source of energy. In mammalian sp ecies, LA is first converted to GLA and subsequently GLA to arachidonic acid (Horro bin, 1992). Daily human LA intake greatly exceeds the amount of LA needed to support hea lth. Furthermore, LA can be replaced by GLA in the diet (Horrobin, 1992) and, due to the increased GLA concentration in MON 87769, it is anticipated that any decrease in LA intake will be compensated without any nutritional effect to humans or animals.

F.6. Safety and Nutrition Assessment of SDA Soybean Oil

The safety of SDA soybean oil is supporte d by the results of a published 90-day/onegeneration reproductive rat toxi city study in which a no obser vable adverse effect level (NOAEL) of 1 g SDA/kg body weight/day (4 g SDA soybean oil/ kg body weight/day)

was determined 

### ( Hammond et al., 2008 

### ) . Rats were fed diets supplemented with 1.5 or 4.0 g SDA soybean oil/kg body weight/day. Diet s containing control soybean oil derived from conventional soybean or menhaden oil (4.0 g/kg body weight/day) were provided to control groups. No statistically significan t dose-dependent test ar ticle-related adverse effects were reported in any of the parame ters evaluated, including clinical signs, behavior, mortality, body weight, organ weight s, macroscopic appearance of tissues, and histopathology. Statistically significant differences observed between the control and SDA-treated groups included: increased food consumption at Weeks 1 and 2 (low-dose SDA, females); increased basophils (high-dose SDA, females); decreased alanine transferase levels (low-dose SDA, females); decreased cholesterol levels (high-dose SDA, females); increased phosphorus levels (low -dose SDA, males); increased blood urea nitrogen levels (high-dose SDA, males); decreased triglycerides (high-dose SDA, males); and urine urobilinogen (low-dose SDA, males) . Given that these changes were slight, within historical limits of the testing labor atory, not dose-dependent , and/or also were observed in the menhaden oil-treated group ( i.e., considered typical responses for rats fed high doses of long-chain polyunsatur ated fatty acids), they were not cons idered to be of toxicological significance. Therefore, th e NOAEL was determined to be 4 g SDA soybean oil/kg body weight/day (providing 1,051 and 1,073 mg SDA/kg body weight/day in male and female rats, respectively), the highest dose test ed. Additional many published studies on SDA and GLA from othe r sources corroborate the safety of SDA soybean oil. The safety of SDA soybean oil is further supported by the regular dietary consumption of fats and oils containing ALA, LA, and palmitic acid, as well as by the permitted uses of LA and palmitic acid in food in the U.S.

Additionally, based on the known SDA:EPA conve rsion ratio of approximately 3:1 in humans (James et. al., 2003), the total intake of EPA and DHA (i.e ., resulting from the intake of SDA from the proposed food uses of SDA soyeban oil plus the baseline per capita daily intake) would not exceed the maximum FDA established intake level of 3 g/day per person combined EPA and DHA. 10,11 Therefore, SDA soybean oil is safe for human and animal consumption.

10 http://www.cfsan.fda. gov/~dms/ds-ltr11.htmlMonsanto Company 09-SY-200U Page 319 of 395 When viewed in its entirety, the scientific eviden ce presented above does not indicate any potential for adverse effects in humans fo llowing the consumption of SDA soybean oil under the conditions of intended use in foods. Following a critical evaluation of the scientific data generally availa ble in the public domain that pertain to the safety of SDA soybean oil, a panel of experts, qualified by scientific traini ng and experience to evaluate the safety of SDA soybean oil as a compone nt of food, unanimously concluded that the proposed uses of SDA soybean oil are safe and suitable and are GRAS, based on scientific procedures. Ther efore, Monsanto has concluded that SDA soybean oil is

GRAS under the intended conditions of use on the basis of scientific procedures.

F.7. Safety and Nutrition Assessment Conclusion

In conclusion, having demonstrated: (i) the compositional equi valence of MON 87769 seed (except for the intended fatty acid chan ge) and forage to seed and forage from conventional soybean already on the market, (i i) the safety of SDA soybean oil and the expected fatty acids resulting from the intended change, (iii) the history of safe use of the introduced proteins, and (iv) familiarity of the host organism from which the genes are derived, MON 87769 is as safe and nutritious as conventional soybean for food and feed

use with the added nutriti onal improvement of SDA, an omega-3 fatty acid. No additional information was considered to be necessary to support the safety and

nutritional assessment of MON 87769.

References Chardigny J.M., J.L. Sébédio, and O. Berde ux. 1996. Trans Polyunsaturated Fatty Acids: Occurrence and Nutritional Implicati ons. Ann. Appl. Lipid Res. 2:1-33.

EC. 2008. Commission of the European Commu nities. Commission Decision of 27 June 2008 authorising the placing on the market of re fined Echium oil as novel food ingredient under Regulation (EC) No 258/97 of the Eu ropean Parliament and of the Council (notified under document number C(2008) 3049) (Only the English te xt is authentic) (2008/558/EC). Off. J. Eur. Union. 51:17-19.

Fan, Y.Y. and R.S. Chapkin. 1998. Importance of dietary gamma-linolenic acid in human

health and nutrition. J. Nutr. 128:1411-1414.

Hammond, B.G., J.K. Lemen, G. Ahmed, K.D. Miller, J. Kirkpatrick, and T. Fleeman. 2008. Safety assessment of SDA soybean oil: Results of a 28-day gavage study and a 90- day/one generation reproduction feeding study in rats. Regul. Toxicol. Pharmacol.

52:311-323.

Health Canada. 2006. Gamma-Linolenic Ac id- Healthy Canadian Ingredient.

11 Substances Affirmed as Generally Recognized as Sa fe: Menhaden Oil. Federal Register. Vol 62, No 108. June 5, 1997, p 30751-30757.Monsanto Company 09-SY-200U Page 320 of 395 Horrobin, D.F. 1992. Gamma Linolenic Acid: An intermediate in essential fatty acid metabolism with potential as an ethical pharmaceutical and as a food. Rev. Contemp.

Pharmacother. 1:1-41.

James, M.J., V.M. Ursin, and L.G. Clela nd. 2003. Metabolism of st earidonic acid in human subjects: comparison with the metabo lism of other n-3 fatty acids. American

Journal of Clinical Nutrition 77:1140-1145.

Kankaanpaa, P., K. Nurmela, A. Erkkila, M. Kalliomaki, D. Holmberg-Marttila, S. Salminen, and E. Isolauri. 2001. Polyunsaturated fatty acids in maternal diet, breast milk, and serum lipid fatty acids of infants in relation to atopy. Allergy. 56:633-638.

Ledoux, M. J., P; Juaneda, and J.L. Sebe dio. 2007. Trans fatty acids: Definition and

occurence in foods. Eur. J. Lipid Sci. Technol. 109:891-900.

Stoney, R.M., R.K. Woods, C.S. Hosking, D.J. Hill, M.J. Abramson, and F.C. Thien. 2004. Maternal breast milk long-chain n-3 fatty acids are associated with increased risk of atopy in breastfed infants. Clin. Exp. Allergy 34:194-200.

Thijs, C., A. Houwelingen, I. Poorterman, A. Mordant, and P. Van den Brandt. 2000. Essential fatty acids in breast milk of at opic mothers: Comparison with non-atopic mothers, and effect of bora ge oil supplementation. Eur. J. Clin. Nutr. 54:234-238.

Villamor, E., I.N. Koulinska, J. Furtado, A. Baylin, S. Aboud, K. Manji, H. Campos, and W.W. Fawzi. 2007. Long-chain n- 6 polyunsaturated fatty acid s in breast milk decrease the risk of HIV transmission through br eastfeeding. Am. J. C lin. Nutr. 86:682-689.Monsanto Company 09-SY-200U Page 321 of 395 Appendix G. Materials, Methods, and Individual-Site Results of the Plant Phenotypic and Agronomic Assessmen t of MON 87769 from the 2006 and 2007

Field Trials

G.1. Materials

The materials for the 2006 and 2007 plant phenotypic assessments included MON 87769, the conventional soybean contro l (variety A3525), and 20 commercial reference soybean varieties. The list of the soybean materials planted in each site is presented in Tables G-1 and G-2. The presence or absence of MON 87769 in the te st and control seed was verified by event-specific polym erase chain reaction. The resu lts of these analyses were as expected.

G.2. Field Sites and Ex perimental Design

Phenotypic and agronomic data were coll ected from 21 field locations over two consecutive years: 17 locations in 2006 an d four locations in 2007. Table VIII-4 provides a list of the field site locations (i ncluding site codes). These locations provided a diverse range of environmental and agronomic conditions representa tive of major U.S. soybean-production regions. The researchers at each field site were familiar with the growth, production, and evaluation of the soybean characteristics.

In 2006, the field trial at each of the 17 site s was established in a randomized complete block design with thre e replications. At the IA2, IL2, IN1, IN2, KS, and MO1 sites, each plot consisted of four 20 ft long rows spaced approximately 30 inches apart. Phenotypic and agronomic data were collected from the second and third row of each plot. The plots were planted adjacent to each other and the entire plot area was surrounded by a four-row (approximately 10 ft) border of a commercial soybean va riety. At the IA1, IL1, MO2, and NE sites, each plot consisted of eight 30 ft long rows spaced approximately 30 inches apart. Phenotypic and agronomic data were collected from the second and third row of each plot. Each plot was surrounded by four rows (approximately 10 ft) of a commercial soybean variety. At the IA3, IA4, IL3, MI, NE 2, OH, and WI sites, each plot consisted of six 20 ft long rows spaced approximately 30 inches apart. Phenotypic and agronomic

data were collected from the fourth and fift h row of each plot. The plots within each replicate were separated by a pproximately two rows (approximately 5 ft) of a commercial soybean variety, and the entire plot area was surrounded by a four row (approximately 10 ft) border of a commercial soybean variety.

In 2007, the field trial at each of the four sites was establis hed in a randomized complete block design with three replications. At the IL, IN, MI, and WI sites, each plot consisted of eight 30 ft long rows spaced approxima tely 30 inches apart. Phenotypic and agronomic data were collected from the second and third row of each plot. Each plot was surrounded by at least four rows (approximately 10 ft) of a commercial soybean variety.Monsanto Company 09-SY-200U Page 322 of 395 G.3. Planting and Field Operations

Planting information, soil description, and croppi ng history of the study area at each site are listed in Table G-7. Ag ronomic practices used to prep are and maintain the trial at each site were characteristic of the respect ive region. All maintenance operations were performed uniformly over the entire study area.

G.4. Data Collection and Statistical Analysis

The following 14 phenotypic and agronomic character istics were evaluated at each site in 2006 and 2007: early stand count, seedling vi gor, plant growth st ages, days to 50% flowering, flower color, plant pubescence, plant height, lodging, pod shattering, final stand count, seed moisture, 100 seed weight, test weight, and yield. The description of the characteristics measured and the designa ted developmental stages where observations

occurred are listed in Table VIII-1 of Section VIII.A.

Analysis of the phenotypic and agronomic da ta from the 2006 and 2007 field trials were conducted separately. An analysis of vari ance was conducted on the data from each year according to a randomized complete block design using SAS (Release 9.1, 2002-2003 for the 2006 data; Release 9.2, 2002-2008 for the 2007 data). The level of statistical significance was set at the 5% probability level (p = 0.05). For each year, MON 87769 was compared to the control substance within e ach site (individual-site analysis) and in a combined-site analysis, in which the data we re pooled across all sites, for early stand count, seedling vigor, days to 50% flowering, plant height, lodging, shattering, final stand count, seed moisture, 100 seed weight, seed te st weight, and yield. Growth stage, flower color, and plant pubescence data were categori cal and were not statistically analyzed. For each assessed characteristic, the minimu m and maximum means were determined from among the reference soybean varieties (reference range) plante d at the sites to provide values that are repres entative of commercial soybean varieties. The following is a summary of the results from the 2006 a nd 2007 individual-site analyses of phenotypic and agronomic evaluations. Results from the 2006 and 2007 combined-site analyses are presented in Section VIII.D.2.1.

G.5. Individual Site Phenotyp ic and Agronomic Results

In the individual-site analysis of the da ta from the 2006 field trials, a total of 22 statistically significant differences were de tected out of 165 site × characteristic comparisons between MON 87769 and the c ontrol (Table G-3). These significant differences were distributed among eleven phenotypic characteristics. None of the significant differences detected in the individual-site analysis were detected in the combined-site analysis. This suggests these si gnificant differences we re not indicative of a consistent plant response associated with the trait and are unlikely to be biologically meaningful in terms of in creased plant pest or weed iness potential of MON 87769 compared to the control. All plants of MON 87769 and th e control had purple flowers and hairy pubescence at each site as expected (Table G-3). Furthermore, no differencesMonsanto Company 09-SY-200U Page 323 of 395 in plant growth stage were observed between MON 87769 and the control for any of the 126 observations conducted among the sites (Table G-4).

In the individual-site analysis of the data from the 2007 field tria ls, one statistically significant difference was detected out of 39 site × characteristic comparisons between MON 87769 and the control (Table G-5). Yield was lower for MON 87769 compared to the control at the MI site (43.0 vs. 52.5 bu/ac). Since the stat istical difference detected in the individual-site analysis was not detected in the combined-site analysis, this suggests

the difference was not indicative of a consistent response associated wi th the trait and is unlikely to be biologically m eaningful in terms of increa sed plant pest or weediness potential of MON 87769 compared to the control. All plants of MON 87769 and the control had purple flowers and hairy pubescence at each site as expected (Table G-5). Furthermore, no differences in plant grow th stage were observed between MON 87769 and the control for any of the 27 observations conducted among the sites (Table G-6).Monsanto Company 09-SY-200U Page 324 of 395 Table G-1. Test, Control, and Reference Mate rials Assessed in the 2006 Field Trials

Material Material
type 1 Sites 2,3

MON 87769 Test All

A3525 Control All

A3244 Reference IA1, IA3, IL2, KS, NE, NE2, OH, WI

ST3600 Reference IA1, IL2, IL3, KS

Stewart SB3454 Reference IA1, IL2, MO1

DKB34-51 Reference IA1, IN1, MO1

ST3608 Reference IA2, IN1, MO1

Pioneer 93M50 Reference IA2, IN1, MO1

Pioneer 93B82 Reference IA2, IL3, IN1, MO2

Lewis 372 Reference IA2, IA4, IN2, MI, MO2, NE2, OH

AG3505 Reference IL1, IN2, MO2 CST3461 (STS) Reference IL1, IN2, MO2

ST3300 Reference IA3, IA4, IL1, IN2, NE, OH

CST37002 Reference IL1, KS, NE

ST3870 Reference IL2, KS, NE

A2869 Reference IA3, NE2

ST2788 Reference IA4, WI

Lewis 392 Reference IL3

A2804 Reference MI

A2553 Reference MI, WI

1 The commercial reference materials were all c onventional soybean varieties with the exception

of DKB34-51, Pioneer 93M50, and AG3505, which were Roundup Ready soybean 40-3-2

varieties.

2 The test and control materials were planted at a ll field sites; the soybean reference varieties were

planted at specific sites.

3 IA1 = Jefferson County, IA; IA2 = Benton Co unty, IA; IA3 = Jefferson County, IA; IA4 =

Guthrie County, IA; IL1 = Stark County, IL; IL2 = Warren County, IL; IL3 = Clinton County, IL; IN1 = Parke County, IN; IN2 = Boone County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO1 = Macon County, MO; MO2 = Lincoln County, MO; NE = York County, NE; NE2 = York County, NE; OH = Fayette County, OH; WI = Walworth County, WI.Monsanto Company 09-SY-200U Page 325 of 395 Table G-2. Test, Control, and Reference Mate rials Assessed in the 2007 Field Trials

Materials Material type 1 Sites 2,3

MON 87769 Test All

A3525 Control All

DKB34-51 Reference IN, MI, Hoegemeyer 333 Reference IN, MI, CST3461 (STS) Reference IN, MI,

ST3600 Reference IN, MI,

AG3505 Reference IL

ST3300 Reference IL

Stewart SB3454 Reference IL

CST37002 Reference IL

Pioneer 93M50 Reference WI

Midland 363 Reference WI

A3244 Reference WI

ST3608 Reference WI

1 The commercial reference materials were all c onventional soybean varieties with the exception of DKB34-51, AG3505, and Pioneer 93M50, which were Roundup Ready soybean 40-3-2

varieties.

2 The test and control materials were planted at a ll field sites; the reference varieties were planted at specific sites.

3 Data were collected from the following field s ites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.Monsanto Company 09-SY-200U Page 326 of 395Table G-3. Phenotypic and AgronomicComparison of MON 87769 to the Conventional Control in the Individual-SiteAnalysis for the 2006 Field StudyPhenotypic Characteristic (units)Early stand count (#/plot)Seedling vigor(1-9 scale)Days to 50% flowering1Flower color2Plant pubescence2,3SiteMON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON87769 ControlMON87769 ControlIA1 241.3 (14.2) 263.8 (21.5)2.0 (0.0) 2.0 (0.0)201.7 (0.3) 202.0 (0.0)Purple Purple Hairy HairyIA2 274.0 (1.5) 279.3 (5.8)2.0 (0.0)†2.0 (0.0)192.0 (0.6) 192.0 (0.0)Purple Purple Hairy HairyIA3 225.7 (11.6)* 193.7 (9.7)2.3 (0.3) 2.3 (0.3)205.0 (0.6) 206.0 (0.0)PurplePurpleHairy HairyIA4 106.3 (8.4) 106.0 (14.7)4.0 (0.6) 4.0 (0.6)197.0 (0.0)†197.0 (0.0)PurplePurpleHairy HairyIL1 197.5 (1.2) 203.6 (5.8)1.0 (0.0)†1.0 (0.0)183.0 (0.0)†183.0 (0.0)Purple Purple Hairy HairyIL2 272.0 (12.4) 286.7 (24.3)1.0 (0.0) 1.0 (0.0)189.0 (0.0) 189.0 (0.0)Purple Purple Hairy HairyIL3 195.3 (5.7) 201.7 (12.7)3.0 (0.0) 3.3 (0.3)199.0 (0.0) 199.0 (0.0)Purple Purple Hairy HairyIN1 302.3 (1.2) 301.0 (6.1)2.0 (0.0)†2.0 (0.0)200.3 (0.3) 201.0 (0.6)Purple Purple Hairy HairyIN2 232.7 (5.5) 213.3 (45.7)5.0 (0.0) 5.7 (0.7)200.0 (0.0)†200.0 (0.0)Purple Purple Hairy HairyKS 212.7 (3.5) 202.7 (29.2)6.0 (0.6) 6.3 (0.3)188.7 (0.3) 188.3 (0.3)Purple Purple Hairy HairyMI 189.3 (4.4) 184.0 (4.2)4.7 (0.3) 4.3 (0.3)206.0 (0.0)†206.0 (0.0)Purple Purple Hairy HairyMO1 321.0 (3.1) 316.3 (2.4)4.7 (0.3) 4.7 (0.3)200.0 (0.0)* 202.3 (0.3)Purple Purple Hairy HairyMO2 134.5 (7.8) 128.0 (1.8)3.3 (0.3) 3.0 (0.0)193.0 (0.0)* 194.0 (0.0)Purple Purple — —NE 226.2 (5.0)* 216.0 (1.3)3.0 (0.0) 3.0 (0.0)179.3 (0.3) 179.3 (0.3)Purple Purple Hairy HairyNE2 132.7 (7.1) 139.7 (4.3)3.3 (0.3) 3.0 (0.0)189.0 (1.0) 187.0 (0.0)Purple Purple Hairy HairyOH 191.0 (6.9) 216.0 (2.6)2.7 (0.3)* 1.3 (0.3)191.7 (0.7) 192.3 (0.3)Purple Purple Hairy HairyWI 234.0 (16.0)* 282.7 (10.7)5.0 (0.6) 4.3 (0.9)207.0 (0.0)†207.0 (0.0)Purple Purple Hairy HairyNote: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Error.* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769)and conventional control (A3525).†No statistical comparisons were made due to lack of variability in the data.1Calendar day number when approximately 50% ofthe plants in eachplot were flowering.2Flower color and plant pubescence data were categorical and were not statistically analyzed.3Plant pubescence data from the MO2 sitewas eliminated from the study because the data were collected incorrectly.Monsanto Company 09-SY-200U Page 327 of 395Table G-3 (continued). Phenotypic and Agronomic Comparisonof MON 87769 to the Conventional Control in the IndividualSite Analysis for the 2006 Field StudyPlant Characteristic (units)Plant Height (in)Lodging(0-9 scale)Pod Shattering (0-9 scale)Final Stand Count (#/plot)SiteMON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)IA1 35.6 (0.6)* 37.3 (0.5) 0.3 (0.3) 1.3 (0.3) 0.0 (0.0)†0.0 (0.0) 188.7 (5.0)* 210.0 (4.7)IA2 46.5 (0.4) 44.7 (0.4) 3.0 (0.6) 3.7 (0.7) 0.0 (0.0)†0.0 (0.0) 245.3 (3.2) 253.7 (3.4)IA3 33.3 (0.2) 34.1 (0.4) 0.7 (0.3)* 1.7 (0.3) 0.0 (0.0)†0.0 (0.0) 202.7 (5.5) 204.0 (3.2)IA4 30.5 (0.4) 30.5 (0.5) 1.3 (0.3) 1.0 (0.0) 0.3 (0.3) 0.0 (0.0) 91.7 (6.5) 90.0 (11.7)IL1 45.2 (0.8) 43.7 (0.6) 1.7 (0.3) 1.7 (0.3) 0.0 (0.0) 0.0 (0.0) 287.8 (4.2) 298.7 (5.4)IL2 43.4 (0.9) 43.2 (1.6) 0.3 (0.3) 0.7 (0.3) 0.0 (0.0)†0.0 (0.0) 242.0 (13.0) 263.7 (23.2)IL3 27.2 (1.0) 29.5 (0.6) 0.0 (0.0)†0.0 (0.0) 0.0 (0.0)†0.0 (0.0) 190.3 (4.1) 196.3 (11.9)IN1 40.8 (0.3) 39.2 (0.7) 3.0 (0.8) 2.8 (0.7) 0.0 (0.0)†0.0 (0.0) 273.0 (3.0) 267.7 (7.2)IN2 37.6 (1.7) 36.5 (1.6) 0.7 (0.3) 1.0 (0.6) 0.0 (0.0)†0.0 (0.0) 176.0 (14.5) 136.0 (13.2)KS 30.9 (1.0) 31.8 (1.2) 2.3 (0.3) 2.7 (0.3) 0.0 (0.0)* 0.7 (0.3) 146.7 (9.8) 136.7 (1.2)MI 39.6 (2.0) 38.5 (2.4) 1.3 (0.9) 1.3 (0.7) 0.0 (0.0)†0.0 (0.0) 166.0 (4.4) 170.7 (5.2)MO1 26.1 (2.5) 26.8 (1.9) 1.3 (0.3) 1.3 (0.3) 0.3 (0.3) 0.0 (0.0) 298.7 (4.9) 284.3 (3.3)MO2 28.4 (0.2) 28.6 (0.7) 0.0 (0.0)†0.0 (0.0) 0.0 (0.0)†0.0 (0.0) 148.4 (9.8) 163.8 (8.2)NE 45.1 (2.0) 45.3 (0.8) 1.3 (0.3) 1.3 (0.3) 2.0 (0.6) 3.0 (0.0) 224.9 (5.8) 214.4 (6.0)NE2 39.7 (0.7) 39.6 (0.8) 1.3 (0.3) 1.3 (0.3) 1.0 (0.0) 1.0 (0.0) 128.3 (3.5) 143.3 (4.5)OH 28.1 (0.8) 26.1 (0.9) 0.0 (0.0)* 0.7 (0.3) 1.0 (0.0) 0.7 (0.3) 199.0 (11.0) 209.0 (5.8)WI 37.5 (0.4) 38.0 (0.6) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0)†0.0 (0.0) 183.3 (11.8)* 215.3 (5.8)Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Error.* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and conventional control (A3525).†No statistical comparisons were made due to lack of variability in the data.Monsanto Company 09-SY-200U Page 328 of 395Table G-3 (continued). Phenotypic and Agronomic Comparisonof MON 87769 to the Conventional Control in the IndividualSite Analysis for the 2006 Field StudyPlant Characteristic (unit)Seed Moisture (%)100 Seed Weight (g)Test Weight (lb/bu)1Yield (bu/ac)1SiteMON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)IA1 11.1 (0.2) 11.2 (0.1) 12.7 (0.5) 12.3 (0.4) 59.7 (0.3)* 57.3 (0.7) 37.3 (3.9) 44.0 (1.9)IA2 11.9 (0.0) 12.0 (0.0) 13.9 (0.7) 14.2 (0.1) 57.0 (0.0) 56.3 (0.3) 52.0 (4.0) 57.5 (1.4)IA3 11.2 (0.2) 11.1 (0.1) 14.2 (0.6) 14.3 (0.4) 57.7 (0.3) 56.3 (1.2) 50.9 (3.2) 52.6 (0.8)IA4 10.8 (0.4) 11.3 (0.4) 14.6 (0.3) 14.6 (0.1)— — — —IL1 13.2 (0.1) 12.9 (0.1) 15.4 (0.2) 15.8 (0.3) 53.7 (0.3) 54.0 (0.6) 58.2 (1.1) 59.2 (0.7)IL2 12.2 (0.5) 12.1 (0.2) 13.4 (0.3)* 14.4 (0.1) 57.0 (0.1) 56.8 (0.0) 65.6 (2.1) 68.8 (1.8)IL3 11.1 (0.1) 11.2 (0.2) 13.0 (0.5) 12.7 (0.7) 54.0 (0.6) 55.0 (0.6) 28.2 (1.9) 28.9 (3.0)IN1 11.8 (0.1)* 11.4 (0.1) 15.4 (0.1) 15.7 (0.5) 61.0 (0.1)* 60.1 (0.1) 59.9 (3.5) 65.4 (7.6)IN2 12.6 (0.3) 12.4 (0.1) 15.1 (0.1)* 16.4 (0.6) 60.0 (3.9) 61.1 (0.0) 61.3 (6.8) 65.0 (3.3)KS 8.7 (0.1) 9.0 (0.0) 15.2 (0.3) 14.5 (0.2) 57.9 (0.2) 57.7 (0.1) 60.0 (2.3)* 70.0 (6.9)MI 17.2 (0.2) 17.5 (0.3) 17.7 (0.2) 18.4 (0.2) 49.9 (0.2) 50.0 (0.3) 60.0 (1.9) 54.7 (1.5)MO1 15.3 (0.5) 15.1 (0.3) 15.3 (0.3) 15.7 (0.3) 52.0 (1.0) 51.7 (1.2) 47.2 (3.0) 47.2 (0.9)MO2 12.0 (0.1)* 11.3 (0.5) 18.7 (0.6) 16.7 (0.7) 59.8 (0.2) 60.1 (0.0) 35.8 (0.8) 39.7 (3.5)NE 11.4 (0.2) 10.8 (0.5) 15.0 (0.0) 15.3 (0.3) 56.3 (0.2) 55.7 (0.1) 52.4 (1.0) 51.2 (3.3)NE2 10.7 (0.0) 10.8 (0.0) 14.0 (0.0)* 15.0 (0.0) 57.3 (0.0) 57.2 (0.1) 64.8 (1.6) 67.7 (1.2)OH 14.2 (0.2) 14.0 (0.2) 18.2 (0.6) 18.8 (0.5) 54.0 (1.9) 52.3 (1.1) 47.7 (4.2) 48.6 (6.0)WI 12.3 (0.4)* 13.6 (0.1) 15.3 (0.7) 16.7 (0.7) 57.0 (0.6) 56.3 (0.3) 61.9 (1.1)* 70.1 (1.1)Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Error.* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and conventional control (A3525).1Test weight and yield data from the IA4 site were excluded from the data analysis because an inaccurate measuring device was used to collect the data.Monsanto Company 09-SY-200U Page 329 of 395Table G-4. Plant Growth Stages of MON 87769, the ConventionalControl, and Reference Varieties at 17 U.S. Sites in 2006Date and Range of Growth Stages Observed2Site Substance1Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5Obs. 6 Obs. 7 Obs.8 Obs. 9 Obs. 10IA1 06/28/2006 07/17/2006 07/31/2006 08/14/2006 09/05/2006 09/14/2006 09/29/2006 10/14/2006 — —MON 87769 V2 R1 R2 R3 R5 R6 R8 R8 — —Control V2 R1R2 R3 R5 R6 R8 R8 — —References V2 R1R2 R3 R5 R6 R7-R8 R8 — —IA2 06/03/2006 06/20/2006 07/11/2006 07/29/2006 08/18/2006 09/05/2006 09/20/2006 10/10/2006 — —MON 87769 VC V3 V6-R1 R3 R5 R6 R7 R8— —Control VC V3 V6 R3 R5 R6 R7 R8— —References VC V3 V6-R1 R3 R5 R6 R6-R7 R8— —IA3 07/03/2006 07/18/2006 07/27/2006 08/07/2006 08/23/2006 09/05/2006 09/19/2006 10/03/2006 — —MON 87769 V2 V6-V7 V10 V14 R4 R5 R6 R8 — —Control V2 V6-V7 V10 V14 R4 R5 R6 R8 — —References V2 V6-V7 V10 V14 R4 R5 R6 R8 — —IA4 06/23/2006 07/07/2006 07/25/2006 08/12/2006 08/28/2006 09/12/2006 09/27/2006 10/17/2006 — —MON 87769 V2 V5-V6 V14 R4 R5 R6 R6 R8 — —Control V2 V6 V14 R4 R5 R6 R6-R7 R8 — —References V2 V5-R1 V14 R3-R4 R5-R6 R6 R6-R7 R8 — —IL1 06/14/2006 06/28/2006 07/14/2006 08/3/2006 08/23/2006 09/07/2006 09/28/2006 10/19/2006 — —MON 87769 V2 V6 R1-R2 R2 R4 R6 R7 R8 — —Control V2 V6 R1-R2 R2 R4 R6 R7 R8 — —References V2 V6 R1-R2 R2 R4 R6 R7 R8 — —Obs. = Observation number.1Control = conventional variety A3525.2Month-day-year.— Information not available.Monsanto Company 09-SY-200U Page 330 of 395Table G-4 (continued). Plant Growth Stages of MON 87769, the Conventional Control, and Reference Varieties throughoutthe Growing Season at 17 Sites in 2006Date and Range of Growth Stages Observed2Site Substance1Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5Obs. 6 Obs. 7 Obs.8 Obs. 9 Obs. 10IL2 06/12/2006 06/30/2006 07/21/2006 08/03/2006 08/30/2006 09/15/2006 10/06/2006 — — —MON 87769 V2-V3 V5-V6 R2-R3 R5 R6 R7 R8 — — —Control V2-V3 V5-V6 R2-R3 R5 R6 R7 R8 — — —References V2-V3 V5-V6 R2-R3 R5 R6 R6-R7 R8 — — —IL3 06/29/2006 07/19/2006 08/09/2006 08/25/2006 09/15/2006 10/05/2006 — — — —MON 87769 V2 R1-R2 R3 R6 R7 R8 — — — —Control V2 R1-R2 R3 R6 R7 R8 — — — —References V2 R1-R2 R3 R6 R7 R8 — — — —IN1 06/27/2006 07/14/2006 08/03/2006 08/22/2006 09/06/2006 09/20/2006 09/26/2006 10/04/2006 10/18/2006 10/24/2006MON 87769 V3 V6 R2 R4-R5 R6 R6-R7 R7 R7-R8 R8 R8Control V3 V6 R2 R4-R5 R6 R6-R7 R7 R7-R8 R8 R8References V3 V5-V6 R2 R4-R5 R6 R6-R7 R6-R7 R7-R8 R8 R8IN2 06/26/2006 07/20/2006 08/18/2006 09/18/2006 ———— — —MON 87769 V2 R1-R2 R3 R7 ———— — —Control V2 R1-R2 R3 R7 ———— — —References V2 R1-R2 R3 R7 ———— — —KS 06/19/2006 07/12/2006 07/27/2006 08/11/2006 08/28/200609/11/200609/26/2006 — — —MON 87769 V2 R2 R3 R5 R6R7R8 — — —Control V2 R2 R3 R5 R6R7R8 — — —References V2 R2 R3 R5 R6R6-R7R8 — — —Obs. = Observation number.1Control = conventional variety A3525.2Month-day-year.— Information not available.Monsanto Company 09-SY-200U Page 331 of 395Table G-4 (continued). Plant Growth Stages of MON 87769, the Control, and Reference Varieties throughout the GrowingSeason at 17 Sites in 2006Date and Range of Growth Stages Observed2Site Substance1Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5Obs. 6 Obs. 7 Obs.8 Obs. 9 Obs. 10MI 06/23/2006 07/07/2006 07/21/2006 08/04/2006 08/18/2006 09/04/2006 09/17/2006 09/29/2006 10/14/2006 10/22/2006MON 87769 V2-V3 V5-V6 R1 R3 R5 R6 R6 R7 R8 R8Control V2-V3 V6 R1 R3 R5 R6 R6 R7 R8 R8References V2-V3 V6-V7 R1 R3R5 R6 R6 R6-R8 R7-R8 R8MO1 07/12/2006 08/02/2006 08/21/2006 09/15/2006 10/05/2006 —————MON 87769 V3 R2 R5 R6 R7 —————Control V3 R2 R5 R6 R7 —————References V3 R2 R5 R6 R7-R8 —————MO2 06/12/2006 06/27/2006 07/10/2006 07/26/2006 08/09/2006 08/24/2006 09/13/2006 09/30/2006 — —MON 87769 VC V3 R1 R3 R4 R5 R6 R8 — —Control VC V3 R1 R3 R4 R5 R6 R8 — —References VC V3 V6-R1R1-R3 R4 R5 R6 R8 — —NE 06/09/2006 06/23/2006 07/10/2006 08/03/2006 08/18/2006 09/08/2006 09/25/2006 — — —MON 87769 V2 V5-V6 R2 R5 R6 R7 R8 — — —Control V2 V5-V6 R2 R5 R6 R7 R8 — — —References V2 V5-V6 R2R5 R6 R6-R7 R8 — — —Obs. = Observation number.1Control = conventional variety A3525.2Month-day-year.— Information not available.Monsanto Company 09-SY-200U Page 332 of 395Table G-4 (continued). Plant Growth Stages of MON 87769, the Conventional Control, and Reference Varieties throughoutthe Growing Season at 17 Sites in 2006Date and Range of Growth Stages Observed2Site Substance1Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5Obs. 6 Obs. 7 Obs.8 Obs. 9 Obs. 10NE2 06/13/2006 06/28/2006 07/14/2006 08/04/2006 08/25/2006 09/08/2006 09/25/2006 — — —MON 87769 V2 V5-V6 R2 R4 R6 R6 R8 — — —Control V2 V5-V6 R2 R4 R6 R6 R8 — — —References V2 V5-V6 R2R3-R5 R6 R6-R7 R8 — — —OH 06/23/2006 07/12/2006 08/02/2006 08/29/2006 09/20/2006 10/24/2006 ————MON 87769 V2 V6-V7 R3 R6 R6-R7 R8 ————Control V2 V6-V7 R3 R6 R6-R7 R8 ————References V2 V6-V7 R3 R6 R6-R7 R8 ————WI 06/28/2006 07/12/2006 07/26/2006 08/07/2006 08/22/2006 09/07/2006 09/22/2006 10/18/2006 11/01/2006 —MON 87769 V2 R1 R2 R3 R5 R5 R6 R8 R8 —Control V2-V3 R1 R1-R2 R3 R5 R5-R6 R6 R8 R8 —References V2-V3 R1 R1-R2R3 R5 R5-R6 R6-R7 R8 R8 —Obs. = Observation number.1Control = conventional variety A3525.2Month-day-year.— Information not available.Monsanto Company 09-SY-200U Page 333 of 395Table G-5. Phenotypic and AgronomicComparison of MON 87769 to the Conventional Control in the Individual-SiteAnalysis for the 2007 Field StudyPhenotypic Characteristic (units)Early stand count (#/plot)Seedling vigor(1-9 scale)Days to 50% flowering1Flower color2Plant pubescence2SiteMON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON87769 ControlMON87769 ControlIL 458.0 (2.6) 463.3 (10.4)1.0 (0.0)†1.0 (0.0)204.7 (0.7) 204.7 (0.7)Purple Purple Hairy HairyIN 214.7 (7.1) 217.0 (18.8)4.0 (0.0)3.7 (0.3)207.0 (0.0)†207.0 (0.0)Purple Purple Hairy HairyMI 408.3 (18.6) 438.0 (27.6)3.7 (0.3) 3.3 (0.3)198.0 (0.0) 198.0 (0.0)Purple Purple Hairy HairyWI 350.3 (18.6) 298.3 (17.5)2.3 (0.3) 2.0 (0.0)212.0 (0.0)211.7 (0.3)Purple Purple Hairy HairyPlant Characteristic (units)Plant Height (in)Lodging(0-9 scale)Pod Shattering (0-9 scale)Final Stand Count (#/plot)SiteMON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)IL 46.4 (1.7) 45.6 (1.1)5.0 (0.0) 5.0 (0.0)0.0 (0.0)†0.0 (0.0)415.7 (2.3) 410.7 (12.9)IN 30.8 (1.9) 31.7 (1.2)0.3 (0.3) 0.3 (0.3)0.7 (0.3) 0.0 (0.0)261.7 (29.2) 320.3 (13.9)MI 32.0 (3.8) 38.1 (1.0)0.3 (0.3) 1.3 (0.3)0.0 (0.0)†0.0 (0.0)380.0 (7.0) 390.0 (15.3)WI 43.8 (2.5) 41.8 (1.2)1.0 (0.0) 0.7 (0.3)0.0 (0.0) 0.0 (0.0)269.0 (21.2) 237.3 (13.5)Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Error.†No statistical comparisons were madedue to lack of variability in the data.1Calendar day number when approximately 50% ofthe plants in each plot were flowering.2Flower color and plant pubescence data were categorical and were not statistically analyzed.Monsanto Company 09-SY-200U Page 334 of 395Table G-5 (continued). Phenotypic and Agronomic Comparisonof MON 87769 to the Conventional Control in the IndividualSite Analysis for the 2007 Field StudyPlant Characteristic (unit)Seed Moisture (%)100 Seed Weight (g)1Test Weight (lb/bu)Yield (bu/ac)SiteMON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)MON 87769Mean (S.E.)ControlMean (S.E.)IL 11.6 (0.1) 11.7 (0.2)15.3 (0.3) 15.3 (0.1) 53.7 (0.9) 53.7 (0.3)50.2 (1.0) 48.3 (1.7)IN 11.2 (0.2) 10.9 (0.4)16.0 (0.3) 15.6 (0.1) 49.9 (0.5) 51.4 (1.4)50.0 (4.1) 58.2 (1.6)MI 13.6 (0.1) 13.4 (0.2)20.6 (0.4) 20.2 (0.2) 56.7 (0.3) 56.0 (0.3)43.0 (2.9)* 52.5 (2.0)WI 10.7 (0.1) 10.7 (0.0)— — 56.7 (0.3) 57.0 (0.0)42.4 (0.9) 42.4 (3.0)Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Error.* Indicates a statistically significant difference (p≤0.05) between the test (MON 87769) and conventional control (A3525).1100 seed weight data from the WI site were excluded from the statistical analysis because the balance used to weigh the samplesdidnot have sufficient precision.Monsanto Company 09-SY-200U Page 335 of 395Table G-6. Plant Growth Stages of MON 87769, the Conventional Control, and Reference Varieties at Four U.S. Sites in 2007Date and Range of Growth Stages Observed2Site Substance1Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5Obs. 6 Obs. 7 Obs.8 Obs. 9 Obs. 10IL6/27/07 7/11/07 7/24/07 8/8/07 8/27/07 9/17/07 10/8/07⎯⎯⎯MON 87769V2 V3-V4 R1-R2 R3-R4 R5 R7 R7-R8⎯⎯⎯ControlV2V3-V4 R1-R2 R3-R4 R5 R7 R7-R8⎯⎯⎯ReferencesV2V3-V4 R1-R2 R3-R4 R5 R7 R7-R8⎯⎯⎯IN6/25/07 7/16/07 7/24/07 8/17/07 9/12/07 10/17/07⎯⎯⎯⎯MON 87769V3 R1 R1-R2 R3-R4 R5-R6 R8⎯⎯⎯⎯ControlV2-V3 R1 R1-R2 R3-R4 R6 R8⎯⎯⎯⎯ReferencesV2-V3 R1 R1-R2 R3-R4 R5-R6 R8⎯⎯⎯⎯MI6/20/07 7/5/07 7/18/07 8/1/07 8/14/07 8/29/07 9/12/07 9/26/07 10/9/07⎯MON 87769V2 V5-V6 R2 R3 R5 R6 R6 R7 R8⎯ControlV2V5-V6 R2 R3 R5 R6 R6 R7 R8⎯ReferencesV2V5-V6 R1-R2 R2-R3 R5 R6 R6 R7 R8⎯WI7/3/07 8/3/07 8/16/07 9/4/07 9/26/07⎯⎯⎯⎯⎯MON 87769V2 R1-R2 R23R4 R6⎯⎯⎯⎯⎯ControlV2R2 R2 R4 R6⎯⎯⎯⎯⎯ReferencesV2R1-R2 R2-R3 R4 R6⎯⎯⎯⎯⎯Obs. = Observation number.1Control = conventional variety A3525.2Month-day-year.3This value is representative of two of the three replicates asdata for one of the replicates was inadvertently not collectedor recorded.—Information not available.Monsanto Company 09-SY-200U Page 336 of 395Table G-7. Field Site Planting Information, Soil Description, and Cropping History2006 Field StudySitePlantingdate1Plantingrate(seeds/ft)Plantingdepth(in)Plotsize (ft)2Rows/plot Soil series, organic matter, pHCropping history2005 2004IA1 06/09/2006 9.0 1.5 20 × 30 8 Taintor loam, 3.7%, 6.7 Corn SoybeanIA2 05/23/2006 9.0 1.5 10 × 20 4 Tama-Muscatine silty clay loam, 3-4%, 6.3 Corn SoybeanIA3 06/10/2006 9.1 1.0 15 × 20 6 Taintor silty clay loam, 3.5%, 7.7 Soybean —IA4 05/29/2006 8.0 1.5 15 × 20 6 Carion loam, 4.0%, 6.5 Corn —IL1 05/24/2006 9.0 1.5 20 × 30 8 Plano silt loam, 3.3%, 6.7 Corn CornIL2 05/23/2006 9.0 2.0 10 × 20 4 Tama silt loam, 4.5%, 6.2 Weeds3Weeds3IL3 06/08/2006 8.0 1.0 15 × 20 6 Silt loam, 2.8%, 6.6 Corn —IN1 06/07/2006 9.0 1.0 10 × 20 4 Reesville silt loam, 2.0%, 7.4 Corn CornIN2 06/01/2006 9.0 1.5 10 × 20 4 Crosby silty clay loam, 2.9%, 6.4 Corn Weeds3KS 05/23/2006 9.0 1.5 10 × 20 4 Farnum loam, 1.2%, 7.9 Sorghum SoybeanMI 05/26/2006 6.0 1.5 15 × 20 6 Nester loam, 1.9%, 6.2 Soybean —MO1 06/15/2006 9.0 1.0 10 × 20 4 Gorin siltloam, 4.2%, 5.8 Fescue FescueMO2 05/29/2006 9.0 1.5 20 × 30 8 Hammond silt loam, 2.4%, 6.8 Corn SoybeanNE 05/19/2006 9.0 1.0 20 × 30 8 Hastings silt loam, 3.0%, 6.6 Soybean SoybeanNE2 05/23/2006 7.0 1.0 15 × 20 6 Silty clay loam, 2.7%, 6.5 Soybean —OH 05/29/2006 8.0 1.0 15 × 20 6 Crosbysilt loam, 2.0%, 6.6 Soybean —WI 05/29/2006 10.5 1.0 15 × 20 6 Radford silt loam, 2-4%, 5.6-7.8 Soybean —1Month/day/year.2Width × length.3This study area was previously used for weed research.— Information not available.Monsanto Company 09-SY-200U Page 337 of 395Table G-7 (continued). Field Site Planting Information, Soil Description, and Cropping History2007 Field StudySitePlantingdate1Plantingrate(seeds/ft)Plantingdepth(in)Plotsize(ft)2Rows/plot Soil series, organic matter, pHCropping history2006 2005IL 5/31/07 9.0 1.5 20 × 30 8 Plano silt loam, 3.8%, 7.1 Corn CornIN 5/25/07 9.0 1.75 20 × 30 8 Crosby silt loam, 2.9%, 6.4 Corn SoybeanMI 5/23/07 8.9 1.5 20 × 30 8 Nester loam, 2.1%, 6.5 Corn SoybeanWI 6/11/07 9.0 1.0 20 × 30 8 Radford silt loam, 1.9%, 6.7Winterwheat/turnipsSoybean1Month/day/year.2Width × length.Monsanto Company 09-SY-200U Page 338 of 395 Appendix H. Materials, Methods, and I ndividual-Site Results from the Seed Dormancy and Germination Assessment of MON 87769

H.1. Materials

Dormancy and germination characteristics were assessed on seed of MON 87769, the conventional soybean control, and the refe rence soybean varieties produced at the Jefferson County, IA; Stark County, IL; and B oone County, IN sites in the 2006 field trials (Appendix G). The field trial at each site was establis hed in a randomized complete block design with three replications. The seed of MON 87769, the co ntrol, and reference varieties were harvested from all three replicated plots at each of the three field sites and pooled to produce one seed lo t of MON 87769, the control, and each reference variety from each field site for dormancy and germination testing.

Material

Type 1 Seed Materials Produced at Each Site

IA IL IN

Test MON 87769 MON 87769 MON 87769

Control A3525 A3525 A3525

Reference A3244 AG3505 AG3505

Reference ST3600 CST3461 CST3461

Reference Stewart ST3300 ST3300

Reference DKB34-51 CST37002 Lewis 372

1 The test, control, and reference seed used to assess dormancy and germination characteristics were all produced from replicat ed field trials conducted in 2006 to assess phenotypic characteristics. None of the seed were obtained from commercial sources. The references were conventional soybean va rieties with the exception of DKB34-51and AG3505, which were Roundup Ready soybean 40-3-2 varieties.

H.2. Characterization of the Materials

Prior to pooling seed, the presence or abse nce of MON 87769 in the test, control, and

reference seed harvested from each of the th ree replicated plots among the three field sites was verified by event-specific polym erase chain reaction. The results of these analyses confirmed the presence of MON 87769 in the test seed and the absence of

MON 87769 in the control and reference seed with a few exceptions. Seed samples collected from two out of the nine control pl ots and from five out of a total of 36 reference plots across the three field tria ls contained between 0 and 1.84% MON 87769. In addition, seed samples collected from one of the reference pl ots at the IN site

contained between 0 and 5.65% MON 87769. Sin ce the seed of the conventional control and the reference varieties were all produced from the same replicated field trials as MON 87769, a low level of outcrossing can be ex pected between plots in close proximity.

It was determined that the levels of MON 87769 in the control and reference seed samples from the isolated plots were low and did not negatively affect the quality of the study or interpretati on of the results.Monsanto Company 09-SY-200U Page 339 of 395 H.3. Performing Facility and Experimental Methods

Dormancy and germination evaluations were conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigator wa s certified to conduc t seed dormancy and germination testing consistent with the st andards established by the Association of Official Seed Analysts (AOSA), an internat ionally recognized seed testing organization (AOSA, 2000; AOSA, 2006).

Seed materials of MON 87769, the control, and four referen ce varieties we re produced

from each of three sites and tested under six different temperature regimes. Thus, a total of 18 different seed lots (i.e., 6 soybean materials/production site × 3 production sites)

were tested. Each of six germination chambers used in the study were maintained dark under one of the following six temperature regimes: constant temperature of approximately 10, 20, or 30 o C or alternating temperatures of approximately 10/20, 10/30,

or 20/30 o C. The alternating temperature regimes were maintained at the lower temperature for 16 h and the higher temperat ure for 8 h. The temperature inside each germination chamber was monitored and reco rded throughout the duration of the study. For each seed lot, four repli cated germination towels (each containing a target of 100 seeds) were prepared per facility SOPs for each temperature regime. 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 assessed periodically during the study for normal germinated, a bnormal germinated, hard (viable and nonviable), dead, and firm-swollen (viable and nonviable) seed as defined by AOSA guidelines (AOSA, 2006). Each rolled germination towel in the additional temperature regimes (i.e., 10, 20, 30, 10/20, and 10/30 o C) was assessed period ically during the study for germinated, hard (viable and nonviable ), dead, and firm-swollen (viable and nonviable) seed.

H.4. Statistical Analysis

Statistical analyses were performed by th e Monsanto Statistics Technology Center. Analysis of variance was conducted accord ing to a split-plot design using SAS ® (SAS Release 9.1, 2002-2003). The whole- plot treatment was the seed production site arranged in a randomized complete block design. The su b-plot was the seed material arranged in a completely randomized design. The data we re pooled across seed production sites and MON 87769 was compared to the control for the following germination characteristics from each temperature regime: percent germinated (categorized as percent normal germinated and percent abnormal germin ated for the AOSA-recommended 20/30 °C temperature regime), percent viable hard, percent dead, a nd percent viable firm-swollen seed. The level of statistical significance was set at the 5% probabili ty level (p = 0.05). Seed from the three production sites were te sted within the same germination chamber for each temperature regime; thus, an analysis of the data pooled across sites was more

® SAS is a registered trademark of SAS Institute, Inc., Cary, NC.Monsanto Company 09-SY-200U Page 340 of 395 appropriate than an analysis within each site . However, if an in teraction between seed production site and seed material (i.e., MON 87769 and control material) had been detected, MON 87769 would have been compared to the control within sites. The means of MON 87769 and the control (across sites a nd within sites) and the results of the analysis of variance are reported. MON 87769 was not statistically compared to the

reference varieties. The minimum and ma ximum means were determined from among the nine reference varieties (reference rang e) to provide seed germination values representative of commercial soybean grown in the three locations.

H.5. Individual Site Seed Dorman cy and Germination Results

MON 87769, the control, and reference seed ma terials were produced at three sites to assess germination characterist ics of seed grown under variou s environmental conditions.

The individual site data in Tables H-1 a nd H-2 indicate that germ ination of MON 87769 and the control seed was greater than 99% across all production sites and temperature regimes. MON 87769 and control seed from th e IA and IL sites had relatively higher percent abnormal germination at the 20/30 o C temperature regime than MON 87769 and control seed from the IN site (Table H-1) . The values, however, are not uncommon for soybean. A total of two viable hard seeds were observed in this study (i.e., one in each of the 10 °C and 10/20 °C temperature regime s for MON 87769 produced from the IL site) (Table H-2). In soybean, it is not uncommon to observe low levels of hard seed (Mullin and Xu, 2001; Potts et al., 1978). Thus, th e observance of two hard seed was not unexpected. In the analysis of the data, no seed production s ite × seed material

interactions were detected fo r any characteristic in any temperature regime. Therefore, MON 87769 was comparable to the control ma terial across sites (Section VIII.D.1., Table VIII-3).

References

Association of Official Seed Analys ts. 2000. Tetrazolium Testing Handbook. AOSA, Lincoln, NE.

Association of Official Seed Analysts. 2006. Rules for testing seeds. AOSA, Lincoln, NE. 166 pp.

Mullin, J.W. and W. Xu. 2001. Study of s oybean seed coat components and their relationship with water abosorti on. J. Agric. Food Chem. 49:5331-5335.

Potts, H.C., J. Duangpatra, W.G. Hairston, and J.C. Delouche. 1978. Some influences of hardseededness on soybean seed quality. Crop Sci. 18:221-224Monsanto Company 09-SY-200U Page 341 of 395Table H-1. Germination Characteristicsof MON 87769, the Conventional Control,and the Reference Soybean Variety SeedProduced from each of Three Field Sites and Tested in the AOSA-Recommended Temperature RegimeMean % (S.E.)1IA IL INTemp.Regime2GerminationCategory3MON 87769 ControlRef.Range4MON 87769 ControlRef.Range4MON 87769 ControlRef.Range420/30 °C(AOSA)NormalGerminated81.5 (3.4) 82.3 (2.9) 93.8-96.5 88.8 (3.4) 94.5 (2.5) 92.8-98.3 97.0 (0.6) 99.5 (0.5) 98.3-99.5AbnormalGerminated18.0 (3.3) 17.5 (2.9) 3.5-6.0 11.3 (3.4) 5.0 (2.0) 1.8-7.3 2.8 (0.8) 0.5 (0.5) 0.5-1.5Viable 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 0.0 (0.0) 0.0 (0.0) 0.0-0.0Dead 0.5 (0.5) 0.3 (0.3) 0.0-0.5 0.0 (0.0) 0.3 (0.3) 0.0-0.8 0.3 (0.3) 0.0 (0.0) 0.0-0.3Viable Firmswollen0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0) 0.3 (0.3) 0.0-0.0 0.0 (0.0) 0.0 (0.0) 0.0-0.0Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed was arranged in germinationchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete blockand the sub-plot treatment wasseed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No production site ×seed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional control (A3525) across sites.1Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equaled 100.1% due tonumerical rounding of the means. S.E. = Standard Error.2Temperature Regime. In the alternating 20/30 °C temperature regime, the lower temperature was maintained for 16 hours and thehigher temperature for eighthours.3Germinated seed in the AOSA-recommended temperature regime were categorized as either normal germinatedor abnormal germinatedseed.4Minimum and maximum mean values from among four commercial reference soybean varieties produced from replicated field trials at each site.Monsanto Company 09-SY-200U Page 342 of 395Table H-2. Germination Characteristicsof MON 87769 and the Conventional ControlSeed Produced from each of ThreeField Sites and Tested in theAdditional Temperature RegimesTemperatureRegimeGermination CategoryMean % (S.E.)1IAILINMON 87769 Control MON 87769 Control MON 87769 Control10 °C Germinated100.0 (0.0) 100.0 (0.0)99.5 (0.3) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (0.0) 0.0 (0.0)0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead0.0 (0.0) 0.0 (0.0)0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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 °C Germinated100.0 (0.0) 100.0 (0.0)100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (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)0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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)30 °C Germinated99.8 (0.3) 99.3 (0.5)99.8 (0.3) 99.8 (0.3) 99.5 (0.3) 99.8 (0.3)Viable Hard0.0 (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) 0.8 (0.5)0.3 (0.3) 0.3 (0.3) 0.5 (0.3) 0.3 (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)Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed was arranged in germinationchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete blockand the sub-plot treatment wasseed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No production site ×seed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional control (A3525) across sites.1Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equaled 100.1% due tonumerical rounding of the means. S.E. = Standard Error.Monsanto Company 09-SY-200U Page 343 of 395Table H-2 (continued). Germination Characteristics of MON 87769 and the Conventional Control Seed Produced from eachof Three Field Sites and Tested inthe Additional Temperature RegimesTemperatureRegime1Germination CategoryMean % (S.E.)2IA IL INMON 87769 Control MON 87769 Control MON 87769 Control10/20 °C Germinated99.5 (0.5) 99.5 (0.3)99.8 (0.3) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (0.0) 0.0 (0.0)0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead0.5 (0.5) 0.5 (0.3)0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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)10/30 °C Germinated100.0 (0.0) 100.0 (0.0)100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard0.0 (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)0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)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)Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed was arranged in germinationchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete blockand the sub-plot treatment wasseed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized design. No production site ×seed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional control (A3525) across sites.1In the alternating temperature regimes,the lower temperature was maintained for 16hours and the higher temperature for eighthours.2Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equaled 100.1% due tonumerical rounding of the means. S.E. = Standard Error.Monsanto Company 09-SY-200U Page 344 of 395 Appendix I. Methods and Results from the Assessment of MON 87769 for Plant Interactions with Abiotic Stressors, Diseases, and Arthropods under Field

Conditions in 2006 and 2007

I.1. Abiotic stress response, disease damage, and arthropod damage

Plant response to abiotic stressors, disease damage, and arthropod damage were assessed

at each of the 17 field sites in 2006 and each of the four field sites in 2007. Three abiotic stressors, three diseases, and three arthropod pests were evaluated at approximately the V2 – V4, R1 – R2, R3 – R5, and R6 – R8 grow th stages. The observed abiotic stressors, diseases, and arthropod pests were “natural” (i.e., no artificial infestatio n or interference was used) and, therefore, often varied between observations at a site and between sites. During each observation, each plot was evaluated for the severity of injury caused by the abiotic stressor, disease, or arthropod pest . Plots were rated numerically using a continuous rating scale of increasing sy mptomology but the results are reported categorically (none, slight, moderate, or severe). Results from the 2006 and 2007

evaluations are summarized in Section VIII.D .2.2, while the data are presented in Tables I-1 through I-6.

The following 0 – 9 scale was used to rate pl ant response to abiotic stressors and disease damage in the 2006 and 2007 field trials and to rate arthropod damage in the 2006 field

trials.

Rating Extent 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)

In the 2007 field trials, ar thropod damage was assessed on the upper four nodes of ten representative plants per plot using one of the following 0 – 5 rating scales specific to the arthrodpod being evaluated:

Defoliating arthropods (e.g., corn earworm, bean leaf be etle, Japanese beetle, soybean looper) Rating Defoliation (%) Extent of plant damage 0 none none (no symptoms observed) 1 1 – 20 % slight (symptoms not damaging to plant development)

2 21 – 40% moderate (intermediate between slight and severe) 3 41 – 60%

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80%Monsanto Company 09-SY-200U Page 345 of 395 Pod feeding arthropods (e.g., corn earworm, bean leaf beetle, stink bugs, Lygus bugs on reproductive plant parts) Rating Damaged pods (%) Extent of plant damage 0 none none (no symptoms observed)

1 1 – 20 % slight (symptoms not damaging to plant development)

2 21 – 40% moderate (intermediate be tween slight and severe) 3 41 – 60%

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80%

Leafhoppers (e.g., potato leafhopper) Rating Foliar damage (%) Extent of plant damage 0 none none (no symptoms observed)

1 1 – 50% of foliage with leaf yellowing; no leaf puckering or leaf margin necrosis slight (symptoms not damaging to plant development)

2 1 – 50% of foliage with leaf yellowing, leaf puckering and/or leaf margin

necrosis moderate (intermediate between slight and severe)

3 > 50% of foliage with leaf yellowing; no leaf puckering or leaf margin necrosis

4 > 50% of foliage with leaf yellowing, leaf puckering, and/ or leaf margin necrosis severe (symptoms damaging to plant development) 5 > 50% of foliage with necrotic leaves (leaves dead due to leafhopper damage)

Aphids (e.g., soybean aphid) Rating Aphids present Extent of plant damage 0 none none (no symptoms observed)

1 1 – 100 aphids per plant; no leaf puckering slight (symptoms not damaging to plant development)

2 101 – 250 aphids per plant; no leaf puckering moderate (intermediate between slight and severe) 3 ≥ 250 aphids per plant with leaf puckering

4 ≥ 250 aphids per plant with leaf puckering and leaf yellowing and/or necrosis severe (symptoms damaging to plant development)

5 ≥ 250 aphids per plant with plant stuntingMonsanto Company 09-SY-200U Page 346 of 395 I.2. Arthropod abundance

The abundance of specific pest and beneficial arthropods were quantified at the IA1, IL1, MO2, and NE sites in the 2006 field trials an d the IL, IN, MI, and WI sites in the 2007 field trials. At these sites, ar thropods were collected from the plants in each replicate plot three times during the growing season at appr oximately the R1 – R2, R3 – R5, and R6 – R8 growth stages using a beat sheet samp ling method (Kogan and Pi tre, 1980). The beat sheet was a 36 × 42 inch vinyl sheet that was spread between the plants of two adjacent rows. Plants were shaken vigorously along the length of each side of the beat sheet to dislodge arthropods from the plants. A total of four sub-samples were collected in this way from each plot. Specifically, two sub-sa mples were collected from Rows # 5 and 6 of each plot (sub-samples 1 a nd 2) and two sub-samples were collected from Rows # 6 and 7 of each plot (sub-samples 3 and 4). The sub-samples collected from the same row were at least 3 ft from the edge of each pl ot. The four sub-samples were combined into

one container that was either filled with al cohol or was placed on dry ice to preserve the integrity of the collection. The samples were then shipped to the laboratory for arthropod

identification and enumeration.

A maximum of the six most abundant pest an d six most abundant be neficial arthropods

were determined for each colle ction from each individual site. These arthropods were then enumerated across all samples (i.e., one sample per plot) from a given collection at each individual site. The arthropods assessed of ten varied between coll ections from a site

and between sites due to differe nces in temporal activity a nd geographical distribution of

the taxa. Results from the 2006 and 2007 arthropod abundance evaluations are summarized in Section VIII.D.2.2, while the da ta are presented in Tables I-7 through

I-10.Monsanto Company 09-SY-200U Page 347 of 395 Table I-1. Abiotic Stressor Evaluations Us ing an Observational Severity Scale for

MON 87769 and the Conventional Control in the 2006 Field Trials

Abiotic stressor Number of observations across

the 17 sites 1 Number of observations

where no differences were

detected

Total 162 161

Cold stress 7 7
Compaction 7 7
Drought 40 40

Excess moisture 2 18 17*

Frost 3 3

Hail 16 16

Heat 28 28
Mineral toxicity 1 1
Nutrient deficiency 7 7

Wind 35 35

* Indicates an observed difference between MON 87769 and the conventional control (A3525) for symptom severity caused by excess moisture at the WI site (slight vs. none; Observation 1). Data were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2,

Observation 3 = R3-R5 and Observation 4 = R6-R8.

1 Data were collected from the following field sites: Jefferson County, IA; Benton County, IA; Jefferson County, IA; Guthrie County, IA; Stark County, IL; Warren County, IL; Clinton County, IL; Parke County, IN; Boone County, IN; Pawnee County, KS; Ottawa County, MI; Macon County, MO; Lincoln County, MO; York County, NE (2 sites); Fayette County, OH; Walworth County, WI.

2 Including wet soil and flooding.Monsanto Company 09-SY-200U Page 348 of 395 Table I-2. Abiotic Stressor Evaluations Us ing an Observational Severity Scale for

MON 87769 and the Conventional Control in the 2007 Field Trials

Abiotic stressor Number of observations across

the four sites 1 Number of observations

where no differences were

detected

Total 34 34

Cold stress 2 2
Compaction 2 2
Drought 8 8

Flood 3 3

Frost 4 4

Hail 7 7

Heat 4 4

Wind 4 4

Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2,

Observation 3 = R3-R5 and Observation 4 = R6-R8. No differences were observed between MON 87769 and the conventional control (A3525) for plant response to any of the assessed abiotic stressors. Data were not subjected to statistical analysis.

1 Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.Monsanto Company 09-SY-200U Page 349 of 395 Table I-3. Disease Damage Evaluations Us ing an Observational Severity Scale for

MON 87769 and the Conventional Control in the 2006 Field Trials

Disease Number of observations across

the 17 sites 1 Number of observations

where no differences

were detected

Total 208 207

Alternaria leaf spot 11 11

Anthracnose 11 11

Asian rust 4 4
Bacterial blight 13 13
Bacterial pustule 4 4
Brown spot 3 3

Brown stem rot 11 11
Cercospora leaf blight 2 4 4

Charcoal rot 3 3
Downy mildew 15 15
Frogeye leaf spot 14 13*

Fusarium 3 3
Phytophthora 3 17 17
Powdery mildew 6 6
Pythium 7 7

Rhizoctonia 5 5

Sclerotinia 4 4
Septoria 4 28 28
Soybean mosaic virus 10 10
Soybean rust 13 13

Stem canker 1 1

Sudden death 11 11

White mold 10 10

* Indicates an observed difference between MON 87769 and the conventional control (A3525) for disease susceptibility to frogeye leaf spot at the IA4 site (non e vs. slight; Observation 4). Data were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2, Observation 3 = R3-R5, and Observation 4 = R6-R8.

1 Data were collected from the following field sites: Jefferson County, IA; Benton County, IA; Jefferson County, IA; Guthrie County, IA; Stark County, IL; Warren County, IL; Clinton County, IL; Parke County, IN; Boone County, IN; Pawnee County, KS; Ottawa County, MI; Macon County, MO; Lincoln County, MO; York County, NE (2 sites); Fayette County, OH; Walworth County, WI.

2 Including Cercospora leaf disease.

3 Including Phytophthora root rot.

4 Including Septoria brown spot and Septoria leaf spot.Monsanto Company 09-SY-200U Page 350 of 395 Table I-4. Disease Damage Evaluations Us ing an Observational Severity Scale for

MON 87769 and the Conventional Control in the 2007 Field Trials

Disease Number of observations across

the four sites 1 Number of observations

where no differences

were detected

Total 48 48

Anthracnose 2 2

Asian rust 1 1
Bacterial blight 1 1

Brown stem rot 2 2
Cercospora leaf blight 2 1 1

Charcoal rot 1 1
Downy mildew 3 3
Frogeye leaf spot 2 2
Phytophthora 3 1 1
Powdery mildew 5 5
Pythium 2 2

Rhizoctonia 3 3

Sclerotinia 1 1
Septoria 4 4 4
Soybean cyst nematode 1 1
Soybean mosaic virus 1 1
Soybean rust 5 5

Sudden death 5 5

White mold 7 7

Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2,

Observation 3 = R3-R5 and Observation 4 = R6-R8. No differences were observed between MON 87769 and the conventional control (A3525) for plant damage caused by any of the assessed diseases. Data were not subjected to statistical analysis.

1 Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.

2 Including Cercospora leaf disease.

3 Including Phytophthora root rot.

4 Including Septoria brown spot and Septoria leaf spot.Monsanto Company 09-SY-200U Page 351 of 395 Table I-5. Arthropod Damage Evaluated Us ing an Observational Severity Scale for

MON 87769 and the Conventional Control in the 2006 Field Trials

Arthropod Number of observations across the 17

sites 1 Number of

observations

where no differences

were detected

Total 203 203
Aphid 2 35 35
Bean leaf beetle 53 53
Blister beetle 2 2

Corn rootworm 2 2

Flea beetle 1 1 Grasshopper 34 34
Green cloverworm 16 16 Japanese beetle 22 22 Leafhopper 3 15 15 Seed corn maggot 1 1 Soybean stem borer 3 3 Stink bug 4 10 10 Thistle caterpillar 2 2 Thrips 4 4 Velvetbean caterpillar 1 1

Wireworm 1 1 Woolybear caterpillar 1 1
Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2,

Observation 3 = R3-R5 and Observation 4 = R6-R8. No differences were observed between MON 87769 and the conventional control (A3525) for plant damage caused by any of the assessed arthropods. Data were not subjected to statistical analysis.

1 Data were collected from the following field sites: Jefferson County, IA; Benton County, IA; Jefferson County, IA; Guthrie County, IA; Stark County, IL; Warren County, IL; Clinton County, IL; Parke County, IN; Boone County, IN; Pawnee County, KS; Ottawa County, MI; Macon County, MO; Lincoln County, MO; York County, NE (2 sites); Fayette County, OH; Walworth County, WI.

2 Including soybean aphid.

3 Including potato leafhopper.

4 Including green and brown stink bugs.Monsanto Company 09-SY-200U Page 352 of 395 Table I-6. Arthropod Damage Evaluated Us ing an Observational Severity Scale for

MON 87769 and the Conventional Control in the 2007 Field Trials

Arthropod Number of observations across the

four sites 1 Number of

observations

where no differences

were detected

Total 48 48
Aphid 2 10 10
Bean leaf beetle 8 8

Corn rootworm 1 1 Grasshopper 7 7
Green cloverworm 3 3 Japanese beetle 7 7 Leafhopper 3 2 2
Leafroller 3 3
Mexican bean beetle 1 1 Spidermite 1 1 Stink bug 4 2 2 Thrips 1 1 White fly 1 1

Wireworm 1 1
Note: The experimental design was a randomized complete block with three replications. Observational data were collected at four crop development stages: Observation 1 = V2-V4, Observation 2 = R1-R2,

Observation 3 = R3-R5 and Observation 4 = R6-R8. No differences were observed between MON 87769 and the conventional control (A3525) for plant damage caused by any of the assessed arthropods. Data were not subjected to statistical analysis.

1 Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.

2 Including soybean aphid.

3 Including potato leafhopper.

4 Including green and brown stink bugs.Monsanto Company 09-SY-200U Page 353 of 395Table I-7. Abundance of Pest Arthropodsin Beat Sheet Samples Collected from MON 87769, the Conventional Control, and theReference Varieties in U.S. Field Trials in 2006Abundance of Pest Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeAphid IA1 2.0 (1.00) 0.7 (0.33) 0.0 – 1.7 — — — 116.7 (27.95) 96.7 (17.70) 47.3 – 103.0IL1 8.7 (1.20) 13.3 (12.33) 2.0 – 10.3 39.7 (16.95) 25.3 (10.97) 16.7 – 52.0 62.7 (8.69) 63.3 (21.87) 38.3 – 64.0MO2 1.3 (0.88) 15.3 (14.84) 0.0 – 1.7 0.0 (0.00) 1.7 (0.88) 0.0 – 2.7 45.3 (10.27) 65.0 (10.60) 55.7 – 82.7NE — — — 12.7 (10.68) 3.5 (0.50) 2.5 – 10.0 20.7 (6.17) 20.0 (7.00) 4.5 – 13.3Bean leafbeetleIA1 11.3 (2.19) 10.3 (2.33) 11.3 – 25.0 8.0 (2.08) 4.3 (1.45) 4.3 – 7.3 3.3 (0.88) 5.3 (1.20) 2.3 – 4.7IL1 13.7 (1.86) 12.3 (3.18) 10.0 – 16.3 27.3 (6.01) 17.7 (4.33) 26.0 – 31.3 22.3 (2.85) 14.0 (7.37) 16.3 – 22.0MO2 6.3 (1.33) 7.7 (2.33) 5.7 – 7.3 3.3 (0.88) 2.0 (0.58) 3.0 – 4.7 2.0 (1.00) 3.0 (0.00) 2.0 – 5.0NE 0.3 (0.33) 1.0 (0.00) 0.0 – 1.0 1.7 (0.67) 3.0 (0.00) 1.3 – 3.0 26.3 (7.62) 24.0 (2.00) 6.5 – 26.0GardenfleahopperIA1 — — — — — — 2.0 (0.58) 1.3 (0.88) 0.7 – 2.0IL1 — — — 0.3 (0.33) 0.0 (0.00) 0.3 – 2.0 0.0 (0.00)†0.0 (0.00) 0.0 – 0.0MO2 — — — 1.0 (0.58) 1.0 (0.58) 0.7 – 1.0 0.0 (0.00) 0.0 (0.00) 0.3 – 0.3NE — — — 0.0 (0.00) 0.0 (0.00) 0.0 – 0.5 2.3 (0.33) 1.5 (1.50) 0.5 – 0.7* Indicates a statisticallysignificant difference (p≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, and 3 occurred at theR1−R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number ofarthropods collected across three replications.S.E. = Standard Error.2IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.†No statistical comparisonswere made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 354 of 395Table I-7 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the ConventionalControl, and the Reference Varieties in U.S. Field Trials in 2006Abundance of Pest Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeGreencloverwormIA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3 13.3 (2.96) 11.0 (2.31) 7.3 – 14.0 1.7 (0.33) 1.7 (1.67) 0.0 – 2.3IL1 0.7 (0.33) 0.0 (0.00) 0.0 – 1.3 31.3 (2.96) 22.0 (3.00) 19.3 – 31.7 1.7 (0.33)* 0.3 (0.33) 1.3 – 2.0MO2 5.0 (2.65) 3.0 (1.15) 2.0 – 4.0 4.3 (1.45) 4.7 (0.33) 2.0 – 5.7 10.7 (4.41) 9.3 (3.93) 9.3 – 13.0NE 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3 0.3 (0.33) 0.0 (0.00) 0.0 – 0.5 1.3 (0.88) 0.0 (0.00) 0.0 – 0.5Leafhopper IA1 33.7 (5.46) 29.3 (8.69) 21.0 – 30.3 0.7 (0.67) 0.7 (0.33) 0.0 – 0.7 3.3 (0.88) 1.3 (0.67) 1.0 – 2.0IL1 55.0 ( 9.71) 94.7 (42.68) 58.3 – 86.3 0.3 (0.33) 0.7 (0.33) 1.0 – 2.7 0.3 (0.33) 0.0 (0.00) 0.0 – 0.7MO2 8.3 (3.76) 6.3 (2.03) 7.3 – 7.7 2.0 (1.00)* 0.3 (0.33) 0.3 – 2.0 0.0 (0.00) 0.3 (0.33) 0.0 – 0.7NE 12.3 (2.91) 7.0 (2.00) 4.0 – 8.0 8.7 (4.41) 1.0 (1.00) 2.3 – 7.7 0.7 (0.33) 1.0 (0.00) 0.0 – 1.0Looper IL1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.7 — — — — — —NE — — — 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)†0.0 (0.00) 0.0 – 0.0* Indicates a statisticallysignificant difference (p≤0.05) between MON 87769 and the conventional control (A3525).Collections 1, 2, and 3 occurred at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropodscollected across three replications. S.E. = Standard Error.2IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.†No statistical comparisons were made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 355 of 395Table I-7 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the ConventionalControl, and the Reference Varieties in U.S. Field Trials in 2006Abundance of Pest Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeStink bug IA1 0.0 (0.00) 0.3 (0.33) 0.0 – 0.7 1.0 (0.58) 1.3 (0.33) 0.3 – 1.7 2.0 (1.15) 5.3 (1.67) 0.3 – 3.7IL1 0.0 (0.00) 0.3 (0.33) 0.0 – 0.3 1.0 (0.58) 2.0 (1.15) 2.0 – 6.0 0.7 (0.33) 1.0 (0.58) 0.3 – 1.3MO2 0.7 (0.33) 0.0 (0.00) 0.0 – 2.3 1.3 (0.33) 1.0 (0.00) 0.0 – 2.3 3.7 (1.86) 6.3 (3.93) 1.7 – 6.3NE 0.7 (0.67) 0.5 (0.50) 0.3 – 0.5 3.7 (1.86) 0.5 (0.50) 0.3 – 1.3 4.3 (0.88) 2.0 (2.00) 2.3 – 5.0Tarnishedplant bugIA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.7 0.7 (0.67) 1.7 (1.20) 1.7 – 3.7 2.0 (1.53) 3.0 (1.53) 0.7 – 3.0IL1 1.0 (1.00) 0.0 (0.00) 0.0 – 0.7 6.3 (1.20)* 2.3 (1.45) 2.7 – 4.7 0.3 (0.33) 0.3 (0.33) 0.3 – 1.0MO2 0.0 (0.00) 1.3 (0.33) 0.3 – 4.0 0.0 (0.00) 0.0 (0.00) 0.0 – 1.0 0.0 (0.00)†0.0 (0.00) 0.0 – 0.0NE 0.0 (0.00)†0.0 (0.00) 0.0 – 0.0 0.7 (0.67) 1.5 (0.50) 0.0 – 1.3 0.0 (0.00) 0.0 (0.00) 0.0 – 1.0VelvetbeancaterpillarIA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3 — — — 1.3 (0.88) 0.3 (0.33) 0.0 – 0.7IL1 0.3 (0.33) 0.0 (0.00) 0.0 – 1.3 — — — — — —MO2 — — — — — — 0.0 (0.00)†0.0 (0.00) 0.0 – 0.0* Indicates a statisticallysignificant difference (p≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, and 3 occurred at theR1−R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number ofarthropods collected across three replications.S.E. = Standard Error.2IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.†No statistical comparisonswere made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 356 of 395Table I-8. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, the Conventional Control,and the Reference Varieties in U.S. Field Trials in 2006Abundance of Beneficial Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeBig-eyed Bug IA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.7 0.3 (0.33) 0.3 (0.33) 0.0 – 0.3 — — —MO2 8.0 (1.73) 7.0 (3.00) 3.0 – 10.3 24.3 (6.74) 12.7 (1.45) 13.3 – 18.7 5.7 (1.20) 6.0 (2.00) 7.0 – 12.7NE 1.3 (0.67) 0.0 (0.00) 0.0 – 0.5 — — — — — —LadybirdbeetleIA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.7 — — — 0.0 (0.00) 0.3 (0.33) 0.0 – 0.3IL1 0.3 (0.33) 1.3 (0.33) 0.3 – 1.0 0.0 (0.00) 0.3 (0.33) 0.0 – 0.3 4.3 (2.96) 7.7 (4.10) 1.3 – 7.7MO2 0.3 (0.33) 0.0 (0.00) 0.0 – 1.3 — — — 0.7 (0.33) 0.7 (0.33) 1.0 – 1.7NE — — — 0.7 (0.67) 0.5 (0.50) 0.0 – 2.0 0.3 (0.33) 0.5 (0.50) 0.0 – 0.5MicroparasitichymenopteraIA1 — — — 2.0 (1.15) 0.7 (0.67) 0.7 – 1.0 4.0 (0.58) 5.3 (2.33) 2.7 – 8.7IL1 1.3 (0.67) 0.7 (0.33) 0.0 – 0.7 3.7 (1.76) 3.0 (1.53) 1.0 – 2.7 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3MO2 — — — 0.0 (0.00) 1.0 (1.00) 0.0 – 1.7 4.3 (1.45) 3.7 (1.45) 4.7 – 14.3NE 0.0 (0.00) 0.5 (0.50) 0.0 – 0.7 1.7 (1.67) 0.0 (0.00) 0.7 – 3.3 0.7 (0.33) 0.0 (0.00) 0.7 – 3.0* Indicates a statisticallysignificant difference (p≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, and 3 occurred at theR−R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropods collected across three replications. S.E.= Standard Error.2IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.Dash (—) indicates arthropodwas not evaluated.Monsanto Company 09-SY-200U Page 357 of 395Table I-8 (Continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, theConventional Control, and the ReferenceVarieties in U.S. Field Trials in 2006Abundance of Beneficial Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeNabisIA1 0.7 (0.67) 1.3 (0.88) 1.3 – 2.3 2.3 (1.45) 3.3 (1.76) 2.7 – 5.0 8.7 (1.20) 12.0 (5.69) 12.3 – 19.7IL1 0.3 (0.33) 0.0 (0.00) 0.7 – 3.3 17.3 (1.76)* 6.3 (0.88) 11.3 – 21.7 1.3 (0.88)* 0.0 (0.00) 0.0 – 1.3MO2 2.7 (2.19) 3.0 (1.15) 0.7 – 4.3 2.0 (1.00) 4.0 (0.00) 2.0 – 7.7 1.7 (1.20) 0.3 (0.33) 1.0 – 5.0NE 2.0 (0.58) 1.0 (1.00) 0.7 – 3.0 8.7 (2.85) 6.5 (1.50) 6.7 – 9.5 1.7 (0.67) 1.0 (1.0) 2.0 – 5.0OriusIA1 2.7 (0.33) 3.0 (1.53) 1.3 – 3.7 7.3 (1.67) 5.3 (2.33) 4.3 – 7.7 16.0 (3.79) 16.3 (6.69) 16.7 – 29.0IL1 8.7 (1.76) 13.3 (2.33) 11.0 – 15.3 52.7 (3.48)* 33.0 (10.79) 42.0 – 62.3 8.7 (1.20) 4.0 (1.00) 3.7 – 6.0MO2 2.3 (1.45) 0.3 (0.33) 0.0 – 0.7 0.3 (0.33) 0.0 (0.00) 0.3 – 2.0 — — —NE 0.0 (0.00) 0.0 (0.00) 0.0 – 0.7 5.0 (2.52) 3.0 (0.00) 1.0 – 5.0 4.0 (1.73) 4.0 (1.00) 1.5 – 3.3Spider IA1 1.0 (1.00) 0.7 (0.67) 0.7 – 2.0 4.0 (1.00) 2.3 (1.33) 1.7 – 3.7 12.7 (3.18) 16.0 (4.93) 17.0 – 17.3IL1 0.7 (0.33) 1.3 (0.88) 1.0 – 2.0 8.7 (2.03) 6.3 (2.33) 7.7 – 9.7 4.0 (2.08) 1.3 (0.88) 1.3 – 4.0MO2 3.7 (0.88) 4.7 (1.86) 4.0 – 6.7 11.0 (2.52) 7.0 (1.00) 3.3 – 15.7 8.3 (2.96) 5.3 (1.45) 8.0 – 12.0NE 4.7 (1.45) 2.0 (2.00) 1.0 – 2.7 4.3 (2.85) 1.0 (1.00) 2.7 – 5.3 22.7 (2.67) 15.0 (3.00) 7.3 – 23.0* Indicates a statisticallysignificant difference (p≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, and 3 occurred at theR1−R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number ofarthropods collected across three replications.S.E. = Standard Error.2IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.Dash (—) indicatesarthropod was not evaluated.Monsanto Company 09-SY-200U Page 358 of 395Table I-9. Abundance of Pest Arthropodsin Beat Sheet Samples Collected fromMON 87769, the Conventional Control, andthe Reference Varieties in U.S. Field Trials in 2007Abundance of Pest Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeAphid IL 7.0 (2.6) 11.0 (5.9) 9.7 – 23.0 — — — 0.0 (0.0) 0.7 (0.7) 0.0 – 1.0MI 50.3 (26.3) 14.7 (11.3) 12.0 – 20.7 1540.0(761.7)3050.0(2241.8)650.0 – 1520.0 — — —WI 2950.0(907.4)1383.3(767.2)983.3 – 4283.3 — — — — — —Bean leafbeetleIL 11.0 (3.5) 12.0 (0.6) 6.3 – 16.0 6.3 (2.6) 4.7 (1.3) 2.3 – 8.0 24.7 (5.5) 25.3 (4.1) 2.0 – 20.0IN 2.0 (0.6) 2.0 (0.6) 0.7 – 2.0 0.3 (0.3) 0.3 (0.3) 0.7 – 1.3 23.7 (1.7) 23.0 (2.6) 20.3 – 40.7MI 0.3 (0.3) 0.7 (0.7) 0.0 – 0.7 0.0 (0.0)†0.0 (0.0) 0.0 – 0.0 1.3 (0.9) 0.3 (0.3) 0.0 – 2.7WI 2.3 (0.7) 3.0 (1.2) 2.0 – 2.7 0.0 (0.0) 0.7 (0.3) 0.0 – 0.7 6.0 (3.5) 3.3 (2.4) 5.0 – 8.0Corn fleabeetleIN— — — — — — 0.3 (0.3) 1.0 (1.0) 0.0 – 2.0GardenfleahopperWI— — — 10.3 (4.3) 3.7 (1.5) 7.0 – 12.3 1.3 (0.7) 2.7 (1.5) 0.3 – 3.7* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and the conventional control (A3525).Collections 1, 2, and 3 occurred at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropodscollected across three replications. S.E. = Standard Error.2Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.†No statistical comparisons were made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 359 of 395Table I-9 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the ConventionalControl, and the Reference Varieties in U.S. Field Trials in 2007Abundance of Pest Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeGreencloverwormIL 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 1.3 (0.7) 1.3 (0.9) 0.7 – 1.7 1.0 (1.0) 0.3 (0.3) 0.0 – 0.3IN 0.0 (0.0)†0.0 (0.0) 0.0 – 0.0 3.3 (0.9) 6.7 (4.8) 3.0 – 7.0 0.7 (0.7) 0.7 (0.7) 0.0 – 1.7MI 0.0 (0.0) 0.3 (0.3) 0.0 – 0.3 2.7 (0.3) 3.0 (0.6) 2.3 – 3.0 0.0 (0.0) 0.3 (0.3) 0.0 – 2.3WI 14.3 (1.2) 12.3 (1.8) 6.3 – 9.0 3.0 (1.5) 1.0 (0.6) 1.7 – 4.3 0.0 (0.0)* 1.0 (0.6) 0.0 – 0.3JapanesebeetleIN 0.7 (0.7) 0.7 (0.3) 0.0 – 1.3 2.7 (0.3) 2.7 (2.2) 0.3 – 5.3 — — —MI 1.7 (1.7) 2.0 (2.0) 3.0 – 4.3 0.3 (0.3) 0.3 (0.3) 0.3 – 3.0 — — —Leafhopper IL 57.0 (13.6) 70.0 (34.1) 39.0 – 119.0 — — — — — —IN 2.7 (1.8) 0.7 (0.7) 0.3 – 2.3 5.7 (1.5) 7.3 (3.4) 3.7 – 14.0 — — —MI 18.0 (6.1) 9.3 (0.3) 7.7 – 21.3 5.7 (2.4)* 0.3 (0.3) 2.3 – 10.0 — — —* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and the conventional control (A3525).Collections 1, 2, and 3 occurred at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropodscollected across three replications. S.E. = Standard Error.2Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.†No statistical comparisons were made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 360 of 395Table I-9 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the ConventionalControl, and the Reference Varieties in U.S. Field Trials in 2007Abundance of Pest Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeStink bug IL 0.0 (0.0) 0.3 (0.3) 0.0 – 0.7 2.3 (0.3) 2.0 (1.0) 1.3 – 3.7 2.3 (0.9) 2.7 (0.3) 1.0 – 2.0IN 0.0 (0.0)†0.0 (0.0) 0.0 – 0.0 0.0 (0.0) 1.3 (0.9) 0.0 – 2.0 5.0 (3.2) 2.0 (1.2) 2.0 – 4.0MI 3.0 (1.0)* 0.0 (0.0) 0.0 – 2.7 3.0 (0.6) 1.0 (1.0) 1.3 – 2.0 11.3 (3.3) 11.0 (3.8) 7.3 – 12.7WI 0.0 (0.0) 0.3 (0.3) 0.0 – 0.7 0.7 (0.7) 0.3 (0.3) 0.0 – 0.7 1.7 (0.9) 1.3 (0.3) 0.0 – 1.3Thrips IL 139.3 (61.9) 105.7 (61.8) 135.0 – 174.3 — — — — — —Tarnishedplant bugIN — — — — — — 0.0 (0.0) 0.7 (0.3) 0.0 – 0.7MI — — — — — — 11.0 (3.1)10.0 (5.1) 4.7 – 13.7WI 4.7 (0.3)4.0 (3.5) 1.7 – 4.7 3.7 (0.9) 3.3 (0.9) 2.0 – 3.7 0.7 (0.7) 0.7 (0.3) 0.0 – 1.0WesterncornrootwormIN 1.3 (0.9) 1.7 (1.2) 1.0 – 2.3 — — — — — —* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and the conventional control (A3525).Collections 1, 2, and 3 occurred at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropodscollected across three replications. S.E. = Standard Error.2Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.†No statistical comparisons were made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 361 of 395Table I-10. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, the ConventionalControl, and the Reference Varieties in U.S. Field Trials in 2007Abundance of Beneficial Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeCarabidbeetleIL — — — — — — 1.0 (0.0) 1.0 (0.6) 0.0 – 4.0Lacewing IL 0.3 (0.3) 0.3(0.3) 0.0 – 1.0 0.0(0.0) 0.0 (0.0) 0.3– 3.3 0.0 (0.0) 0.3 (0.3) 0.0 – 0.7IN — — — 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 — — —MI 0.3 (0.3) 1.0 (1.0) 0.0 – 1.0 3.0 (0.6) 3.0 (1.7) 2.0 – 7.7 2.3 (0.9) 4.7 (1.2) 1.0 – 5.7WI 5.3 (1.5) 3.0 (2.0) 2.0 – 4.7 0.7 (0.7) 1.3 (0.9) 0.3 – 2.0 — — —LadybirdbeetleIL — — — 0.0 (0.0) 0.3 (0.3) 0.0 – 3.7 2.0 (0.6) 0.7 (0.3) 0.3 – 1.0IN — — — 0.0 (0.0) 0.3 (0.3) 0.0 – 1.7 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7MI 0.0 (0.0) 0.0 (0.0) 0.0 – 0.7 7.3 (5.0) 3.7 (1.3) 2.3 – 5.0 32.7 (5.0)* 8.7 (4.2) 11.3 – 15.3WI 24.3 (5.2) 39.0 (7.8) 25.0 – 41.7 2.0 (1.0) 2.0 (0.6) 2.0 – 4.0 1.0 (0.6) 1.0 (0.6) 0.7 – 3.0* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and the conventional control (A3525).Collections 1, 2, and 3 occurred at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropodscollected across three replications. S.E. = Standard Error.2Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 362 of 395Table I-10 (Continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, theConventional Control, and the ReferenceVarieties in U.S. Field Trials in 2007Abundance of Beneficial Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMicroparasitichymenopteraIL 0.3 (0.3) 1.3 (1.3) 0.0 – 0.7 — — — — — —MI 0.7 (0.7) 1.3 (0.3) 0.3 – 1.3 2.0 (1.0) 1.3 (0.7) 0.3 – 2.0 1.0 (0.6) 1.0 (0.6) 0.3 – 3.0WI 5.3 (1.5) 7.0 (2.6) 2.3 – 4.3 2.0 (1.0) 3.0 (1.2) 0.7 – 2.3 3.7 (1.2) 1.7 (0.3) 2.7 – 8.3NabisIL 0.7 (0.7) 1.3 (0.3) 0.7 – 1.7 0.7 (0.3) 0.7 (0.7) 0.0 – 2.3 0.7 (0.3) 1.0 (0.6) 0.3 – 2.0IN 0.0 (0.0) 0.0 (0.0) 0.0 – 1.0 0.7 (0.7) 1.0 (0.0) 0.7 – 1.3 2.7 (0.9) 0.7 (0.3) 1.0 – 3.7MI 0.0 (0.0)* 1.7 (0.3) 0.0 – 1.0 3.7 (1.8) 2.0 (1.0) 1.7 – 5.7 9.3 (2.2) 9.0 (8.5) 3.0 – 14.0WI 3.3 (0.9) 4.7 (1.5) 2.0 – 5.7 0.7 (0.3) 3.3 (0.9) 2.3 – 7.3 6.7 (3.0) 4.0 (1.5) 5.7 – 9.7OriusIL 16.3 (7.8) 15.3 (12.9) 14.3 – 29.3 14.7 (7.2) 16.0 (2.5) 14.3 – 42.3 1.3 (0.3) 8.7 (2.0) 3.7 – 21.3IN 0.0 (0.0) 1.3 (0.7) 0.0 – 3.0 3.7 (1.5) 5.7 (3.0) 3.0 – 10.0 4.7 (1.7) 4.7 (1.5) 2.3 – 16.7MI 1.0 (0.6) 1.7 (0.9) 0.3 – 3.0 36.7 (16.5) 15.3 (6.1) 15.3 – 29.3 63.0 (20.1) 73.0 (16.8) 51.3 – 79.7WI 83.0 (11.7) 89.7 (8.0) 38.7 – 78.7 66.3 (2.7) 70.3(5.2) 50.7 – 92.7 62.0 (24.7) 29.7 (8.7) 39.7 – 109.7* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and the conventional control (A3525). Collections 1, 2, and 3 occurredat the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropods collected across threereplications. S.E. = Standard Error.2Data were collected from the followingfield sites: Stark County, IL; Boone County, IN; Ottawa County, MI; WalworthCounty, WI. Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 363 of 395Table I-10 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, theConventional Control, and the ReferenceVarieties in U.S. Field Trials in 2007Abundance of Beneficial Arthropod1Collection 1 Collection 2 Collection 3Arthropod Sites2MON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeMON 87769Mean (S.E.)ControlMean (S.E.)ReferenceRangeSpinedsoldier bugIL 0.0 (0.0) 0.3 (0.3) 0.0 – 0.7 — — — — — —IN — — — — — — 0.7 (0.7) 0.3 (0.3) 0.0 – 0.3Spider IL 1.7 (1.7) 2.7 (1.3) 1.0 – 2.7 1.3 (0.9) 1.0 (0.6) 0.0 – 1.0 0.7 (0.7) 0.7 (0.3) 0.0 – 1.3IN 0.7 (0.3) 0.3 (0.3) 0.3 – 1.3 0.7 (0.3) 2.3 (1.2) 0.3 – 1.0 0.0 (0.0) 0.0 (0.0) 0.7 – 1.7MI 0.0 (0.0)†0.0 (0.0) 0.0 – 0.0 0.3 (0.3) 1.3 (0.7) 0.3 – 1.3 0.7 (0.3) 0.7 (0.3) 0.0 – 1.7WI 9.0 (1.5) 8.3 (4.1) 3.3 – 5.3 9.0 (6.0) 6.7 (2.7) 1.7 – 8.0 4.3 (0.3) 6.3 (3.0) 4.7 – 11.3* Indicates a statisticallysignificant difference (p≤0.05) between the test (MON 87769) and the conventional control (A3525).Collections 1, 2, and 3 occurred at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively.1Test and control values represent mean number of arthropodscollected across three replications. S.E. = Standard Error.2Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa County, MI; Walworth County, WI.†No statistical comparisons were made due to lack of variability in the data.Dash (—) indicates arthropod was not evaluated.Monsanto Company 09-SY-200U Page 364 of 395 References

Kogan, M., and H.N. Pitre. 1980. General Sampling Methods for Above-Ground Populations of Soybean Arthropods. Pages 34-37 in Sampling Methods in Soybean Entomology. M. Kogan and D.C. Herzog (e ds.). Springer-Verlag, New York, United

States of America.Monsanto Company 09-SY-200U Page 365 of 395 Appendix J. Materials and Methods fr om the Pollen Viability and Morphology

Assessment of MON 87769

J.1. Materials and Methods

Plant Production Plants of MON 87769, the conventional soybean control, and four commercial reference soybean varieties were grown under simila r agronomic conditions in a randomized complete block design with three replications at a field site in Li ncoln County, Missouri. Each plot consisted of eight 30 ft long rows spaced approximately 30 inches apart.

Material

Type Material

Name 1

Test MON 87769

Control A3525

Reference Pioneer 93B82

Reference Lewis 372

Reference AG3505

Reference CST3461

1 The commercial references were all conventional soybean varieties with the exception of AG3505, which was a Roundup Ready soybean 40-3-2 variety.

Flower Collection Twenty flowers were collected from the four th row of each plot. One flower from the bottom of the plant, two flowers from the middle, and one flower from the top were collected from each of five representative plan ts per plot. Flowers were collected from all plots of the first replication on July 18, 2006 and from all plots of the second and third replication on July 19, 2006. Upon collection, a ll flowers from a plot were placed into a clean, uniquely labeled container. The containe rs were kept on wet ice for less than eight hours until the pollen was ex tracted and stained.

Pollen Sample Preparation Pollen samples were prepared in a laboratory. Clean microscope slides were labeled with the plot number. A circle of approximately 1 cm diameter was drawn in the center of the slide with a pap hydrophobic barrier pen. Tweezers were used to open each of the collected flowers from a plot and brush the polle n into the circle on the slide. The utensil was cleaned between extractions of the pol len from the flowers of another plot. Approximately 20 μ l of Alexander stain (Alexander, 1980) was added to the center of the circle containing the pollen. The pollen was st ained at ambient temperature for at least ten minutes prior to examination. All pollen gr ains from a replicate sample were stained and evaluated on the same day. Viability data were collected from a minimum of approximately 75-100 pollen grains per plot.Monsanto Company 09-SY-200U Page 366 of 395 J.2. Data Collection

All pollen samples were viewed under an Olympus Provis AX70 light/fluorescence microscope equipped with an Olympus DP70 di gital color camera. The microscope and camera were connected to a computer r unning Microsoft Windo ws 2000 Professional (© 1981-1999, Microsoft Corp.) and installe d with associated camera software [DP Controller v1.2.1.108 and DP Manager v1.2.1.107(© 2001-2003, Olympus Optical Co., Ltd.)] and imaging software [Imag e-Pro Plus v4.5.1.27 (© 1993-2002, Media Cybernetics, Inc.)].

Pollen Viability Pollen viability was evaluated for each samp le by counting the number of viable and nonviable pollen grains. When exposed to th e stain solution, viable pollen grains were stained red to purple due to the presence of living cytoplasm. Non-viable pollen grains were stained blue to green and may have appeared round to co llapsed in shape, depending on the degree of hydration. At leas t 75 individual pollen gr ains per plot were evaluated for viability from a random field of view under the microscope. Dense clusters of pollen or pollen grains adhe ring to flower parts were not counted because they may not have absorbed the stain solution uniformly.

Pollen Diameter Micrographs (400X magnification for re plication 1 and 500X magnification for replications 2 and 3) of ten representative pollen grains from each plot were taken and imported into the imaging software. The so ftware was used to measure pollen grain diameter along two perpendicular axes for ea ch selected pollen grain. Mean pollen diameter for each plot was calculated from the 20 total measurements (i.e., two axes per each of ten representative pollen grains from each plot).

General Pollen Morphology General pollen morphology was observed from micrographs of MON 87769, the control, and reference materials that were also used for pollen diameter measurements.

J.3. Statistical Analysis

An analysis of variance was conducted by the Monsanto Statistics Technology Center using SAS (SAS Release 9.1, 2002-2003) accord ing to a randomized complete block design with three replications. Statistical si gnificance was set at the 5% probability level (p = 0.05). MON 87769 was compared to the c ontrol for percent viable pollen and pollen diameter. No statistical comparisons we re made between MON 87769 and the reference varieties. A reference range for each measur ed characteristic was determined from the minimum and maximum mean values from among the four reference soybean varieties. General pollen morphology was qualitative; therefore, no statis tical analysis was

conducted on these observations . A summary of the results is provided in Section

VIII.D.3.Monsanto Company 09-SY-200U Page 367 of 395 Reference Alexander, M.P. 1980. A vers atile stain for pollen fungi, yeast and bacteria. Stain Technology. 55(1): 13-18.Monsanto Company 09-SY-200U Page 368 of 395 Appendix K. Materials and Methods fo r the Assessment of MON 87769 for Plant Interactions with Nitrogen-fixing Soil Symbionts

K.1. Initial Study

K.1.1. Materials

Seed of MON 87769, the conventional soybean control (variety A3525), and three commercial reference soybean varieties were used to assess the symbiotic association

between Bradyrhizobium japonicum and MON 87769 compared to that of conventional soybean.

Material Type Seed Materials Tested Phenotype

Test MON 87769 SDA trait

Control A3525 Conventional

Reference Stewart SB3454 Conventional

Reference ST3300-0 Conventional

Reference CST37002 Conventional

K.1.2. Characterization of Materials

The presence or absence of MON 87769 in the te st and control starting seed was verified by event-specific polymerase ch ain reaction. The results of these analyses were as expected. The identity of the reference subs tances was maintained via chain-of-custody documentation and seed bag labels.

K.1.3. Experimental Methods

Plants of each material were grown in a greenhouse with a 14-hour photoperiod, a target day-time temperature of 27 o C, and a target night- time temperature of 22 o C. At planting, eight replications (for each of two sampling periods) of 10-inch pots were filled with a nitrogen-deficient potting medium composed of primarily peat, vermicu lite, and perlite. Four seeds of MON 87769, the control, or reference variety were inoculated with approximately 1 x 10 8 cells of B. japonicum in phosphate-buffered saline and planted in each pot. An additional eight replications (for each of two samp ling periods) of pots were planted with uninoculated control material seed and in cluded in the st udy to validate the test system. Pots were arranged in eight re plicated blocks in the greenhouse for 4- and 6-week sampling periods using a random ized strip-plot design.

One week after emergence, all pots were thinned to one seed ling per pot and reinoculated with approximately 1 x 10 8 cells of B. japonicum

, with the exception of the uninoculated control pots. A solution of n itrogen-free nutrient solution (target of 250Monsanto Company 09-SY-200U Page 369 of 395 mL) was added weekly to each pot after plant emergence. The growth stage of each plant was evaluated and documented in the study notebook at least once per week.

Four and six weeks after thinning, plants were excised at the surface of the potting medium

and shoot and root materials were removed from the pots. The shoot material was cut into smaller pieces and placed in labeled bags. The plant roots with nodules were separated from the potting medium by washing with wa ter. Excess moisture was removed using absorbent paper towels and the roots plus nodules were placed in labeled bags. The nodules were later removed by hand from the roots of each plant and placed in weighing tins. Nodules from each plant were enumerat ed and the fresh weight determined. The

nodules were dried for a minimum of 48 hours at approximately 65 °C to determine dry weight. The fresh weight of the remaining root and shoot mass was determined for each plant. Root and shoot material from each plant was dried for a mi nimum of 48 hours at approximately 65 °C to determine dry weight . After drying, the shoot tissue was ground with a mortar and pestle prior to shippi ng to Agvise Laboratories (Northwood, ND) for total nitrogen analysis. Shoot total ni trogen was determined by combustion using a nitrogen analyzer.

K.1.4. Statistical Methods

An analysis of variance was conducted usi ng a randomized strip-plot design with eight replications for each test, control, and refere nce material at each sampling period. Data were analyzed using SAS (SAS Release 9.1, 2002-2003), and statisti cal significance was set at the 5% probability le vel (p = 0.05). MON 87769 was co mpared to the conventional control for five measurement endpoints from each of the two sampling periods (4- and 6- week): nodule number, nodule dry wt (g), shoot dry wt (g), root dry wt (g), and shoot total nitrogen (percent dry wei ght). The commercial refere nce varieties provided a range of values for each measured endpoint that are representative of commercial soybean varieties. A summary of the results is pr ovided in Section VIII.D.4. The nodule number and shoot total nitrogen from the inoculated and uninoculated control plants were also compared to each other and demonstrated that in the absence of the nitrogen-fixing symbiont, plant growth was limited in the nitr ogen-deficient test sy stem (Table K-1).Monsanto Company 09-SY-200U Page 370 of 395 Table K-1. Comparison of Uninoculated Conventional Control (A3525-UN) to the
Inoculated Conventional Control (A 3525) for Nodule Number and Shoot Total
Nitrogen

Measurement Endpoint Sampling

Period A3525 (Mean) A3525-

UN (Mean) Standard

Error p value Treatments

SSD 1

Nodule

Number (per plant) 4 week 68.29 3.25 0.46 <0.0001 S

6 week 140.50 2.38 0.70 <0.0001

Shoot Total

Nitrogen (% dry wt) 4 week 2.74 1.00 0.14 <0.0001 S

6 week 2.80 1.11 0.12 <0.0001

1 SSD, statistical significance of differences: S, significant at 5% level ( p < 0.05).

K.2. Subsequent Analysis Using Refined Methodology

K.2.1. Materials

Seed of MON 87769, the conventional soybean control (variety A3525), the negative isoline control, and six commercial reference soybean varieties were used to assess the symbiotic association between B. japonicum and MON 87769 compared to that of conventional soybean.

Material Type Seed Materials Tested Phenotype

Test MON 87769 SDA trait

Control MON 87769 negative isoline Negative isoline

Control A3525 Conventional

Reference Williams 82 Conventional

Reference A2553 Conventional

Reference 3585N Conventional

Reference CST 37002 Conventional

Reference A4324 Conventional

Reference 5989 ConventionalMonsanto Company 09-SY-200U Page 371 of 395 K.2.2. Characterization of Materials

The presence or absence of MON 87769 in the te st and control starting seed was verified by event-specific polymerase ch ain reaction. The results of these analyses were as expected. The identity of the reference subs tances was maintained via chain-of-custody documentation and seed bag labels.

K.2.3. Experimental Methods

Plants of each material were grown in a greenhouse with a 14-hour photoperiod, a target day-time temperature of 27 o C, and a target night- time temperature of 22 o C. At planting, eight replications of 6-inch pots were fill ed with a nitrogen-def icient potting medium composed of primarily peat, vermiculite, and perlite. Three s eeds of MON 87769, the conventional control, negative is oline, or reference soybean va riety were inoculated with approximately 1 x 10 7 cells of B. japonicum in phosphate-buffered saline and planted in each pot. Pots were arranged in the greenhouse in a randomized complete block design with eight replications for the 6-week samp ling period (plants were not sampled at four weeks).

One week after emergence, all pots were thinned to one seedling per pot. A solution of nitrogen-free nutrient solution (target of 250 mL) was added weekly to each pot after plant emergence. The growth stage of each plant was evaluated and documented in the study notebook at least once per week.

Six weeks after thinning, plants were excise d at the surface of the potting medium and shoot and root plus nodule material were remo ved from the pots. The shoot material was cut into smaller pieces and placed in labeled bags. The plant roots with nodules were separated from the potting medium by washing w ith water. Excess moisture was removed using absorbent paper towels and the roots plus nodules were placed in labeled bags. The nodules were later removed by hand from the roots of each plant and placed in weighing tins. Nodules from each plant were enumerat ed and the fresh weight determined. The

nodules were dried for a minimum of 48 hours at approximately 65 °C to determine dry weight. The fresh weight of the remaining root and shoot mass was determined for each plant. Root and shoot material from each plant was dried for a mi nimum of 48 hours at approximately 65 °C to determine dry weight . After drying, the shoot tissue was ground prior to shipping to A&L Great Lakes Labor atory (Fort Wayne, IN) for total nitrogen analysis. Shoot total nitrogen was determ ined by thermal conductance using a nitrogen analyzer.

K.2.4. Statistical Methods

An analysis of variance was conducted usi ng a randomized complete block design with eight replications. Data were analy zed using SAS (SAS Release 9.1, 2002-2003), and statistical significance was set at the 5% probability level (p = 0.05). MON 87769 was compared to the conventional control an d the negative isoline control for six measurement endpoints in separate pairwi se comparisons: nodule number, nodule dry wt (g), shoot dry wt (g), root dry wt (g), and shoot nitrogen (percent a nd total dry weight).Monsanto Company 09-SY-200U Page 372 of 395 The commercially-released reference soybean varieties provided a range of values for each measured endpoint that are representati ve of commercial soybean varieties. A summary of the results is pr ovided in Section VIII.D.4.Monsanto Company 09-SY-200U Page 373 of 395 Appendix L. Petitioner’s Environmental Assessment

L.A. Background

USDA-AHPIS has responsibility, under the Plant Protection Act (7 U.S.C. § 7701-7772), to prevent the introduction and dissemination of plant pests into the U.S. Additionally, APHIS must comply with the National Envi ronmental Policy Act (NEPA) when making the decision whether to gr ant nonregulated status to MON 87769. MON 87769 has been the subject of numerous field trials conducte d in the U.S. under APHIS notifications and permits since 2003. Information has been devel oped from these field trials, other tests, and the public literature to specifically asse ss whether the alteration in the soybean oil profile through the in troduction of two de saturase genes, Primula juliae Δ 6 desaturase ( Pj.D6D ) and Neurospora crassa Δ 15 desaturase ( Nc.Fad3 ) that encode for the Pj Δ 6D

and Nc Δ 15D proteins, the presence of steari donic acid (SDA) and/or the plant transformation process alte red MON 87769 in any way that would impart plant pest characteristics or cause significant environmen tal impacts, including cumulative impacts. The purpose of this section is to provide relevant, trait-specific information regarding the potential for reasonably foreseeable, significant environmental impacts.

An analysis of the potential impact of deregulation of MON 87769 on current soybean agronomic production systems, and related acti vities such as soybean processing, food and feed uses as well as marketing of soybean and soybean products is presented in this section. Factors evaluated as part of th e assessment include potential impacts to:

• land use patterns, non-agricultural la nds, farming practices, commodity and specialty soybean production,

• marketability of soybean seed for planti ng and seed for specialty and commodity markets, and

• public health, impacts to non-target organi sms, threatened or endangered species and biodiversity.

The analysis conducted considers current conditions, the potential to impact these conditions, and potential cumulative impacts. In most cases, there are no impacts to current conditions (e.g., no differences betw een deregulation versus continuing to regulate). Where differences were noted, these differences are described and their significance evaluated.

L.B. Purpose and Need

Monsanto Company (Monsanto) is submitting to APHIS this petition for the determination of nonregulated status for MON 87769 plants genetically modified to produce stearidonic acid (SDA), which is not found in conven tional soybean but which is found in other plants, fish and marine micro and macroalgae. MON 87769 is a sustainable alternate source of an omega-3 fatty acid to help meet th e needed intake of long chain omega-3 fatty acids. The Am erican Heart Association (AHA) has recommended a dietary intake of 1 gram/day of omega-3 fatty acid for heart patients andMonsanto Company 09-SY-200U Page 374 of 395 the incorporation of two servings of fatty fi sh per week for health y individuals (or 500 mg/day of omega-3 fatty acid in the diet). Due in large part to food preferences, the current average intake of omega-3 fatty acids in the diet is well below these recommendations (Kris-Etherton et al., 2002, 2003). MON 87769 is intended to be a value-added specialty soybean crop commerci alized as an altern ate source of omega-3 fatty acid, the oil of which will replace non-hydrogenated soybean oil and/or be added to select food products such as margarine, mayonnaise, shortenings, salad dressings and ready-to-eat foods.

Common sources of omega-3s include AL A found in canola, soybean and other vegetables oils, flax and walnuts, fatty fish and algae. Studies have shown that the

conversion of ALA to the more desirable long chain PUFAs, EPA and DHA, found in fatty fish and algae, is inefficient in humans. As a result, studies have shown that it can take 14-20 grams of ALA to convert to 1 gr am EPA (James et al., 2003). SDA is an eighteen carbon fatty acid with four double bonds ( 18:4n-3) that is naturally found in fish and fish/algal oil products and some plants. SDA is the intermediate in the biochemical pathway between ALA and EPA and does not requi re the action of delta-6 desaturase for

conversion to EPA. As a resu lt, conversion of SDA to EPA is much more efficient (see Appendix F).

The development of MON 87769 involved the introduction of two de saturase genes, Primula juliae Δ 6 desaturase ( Pj.D6D ) and Neurospora crassa Δ 15 desaturase ( Nc.Fad3 ) that encode for the Pj Δ 6D and Nc Δ 15D proteins. The genes and subsequent enzymes responsible for production of SDA in M ON 87769 are derived from plant and algal

sources and are not from fish.

Soybean plants lack the Δ 6 desaturase gene, which is a minimal requirement for the production of SDA. However, Δ 6 desaturase also converts LA to GLA. The addition of

a Δ 15 desaturase with temporal expression similar to the Δ 6 desaturase increases the flux

of ALA to SDA and lowers the substrate pool for GLA production. To produce SDA in soybean, the conventional soyb ean variety A3525 was transfor med with a vector that

contained the Nc.Fad3 and the Pj.D6D genes driven by promoters that are known to be spatially and temporally active only in th e developing soybean seed. The oil from MON 87769 contains 20% to 30% SDA (wei ght % of total fatty acids).

Oil derived from MON 87769 (referred to as SDA soybean oil) is expected to help food manufacturers develop foods th at can assist consumers in meeting the American Heart Association’s (AHA) goals for dietary intake of omega-3 lo ng chain fatty acids and to improve heart health. SDA ha s been utilized in several human clinical studies. For example, James et al. (2003) found that by in corporating SDA in the diet at 1500 mg/day,

EPA levels in red blood cells increased si gnificantly and conversion was several-fold

more efficient for SDA than for ALA. Harri s et al (2007) found that SDA enriched the red blood cells and cardiac lipi ds with EPA in dogs. Likewi se, Harris et al. (2008) found in a human clinical study using SDA soybean oil over a 16 week peri od, that there was a significant increase in red blood cell EPA levels whereas none was observed for participants using only conventiona l soybean oil containing ALA.Monsanto Company 09-SY-200U Page 375 of 395 Fish oil and plant oils rich in omega-3 fatty acids are also currently used in certain feed applications especially in aquaculture and poultry feeds. SDA soybean oil may eventually be used in aquacultu re and feed applications, as an alternative to fish oil and other omega-3 rich feed components.

L.C. Soybean Production

The following section describes the setting for the proposed deregulation and provides the context for evaluating the intensity of the impact due to APHIS granting deregulated status to MON 87769. The propos ed deregulation would be relevant to the production of an intensively cultivated row crop – soybean. Soybean is grown as a commercial crop in over 35 countries. In the United States, it is generally grown on greater than 70 million

acres in at least 27 states w ith over a million acres grown in each of the following states: IA, IL, MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, NC, KY, and TN (USDANASS, 2006a-b). On the basis of predicte d demand for food and feed applications with omega-3 fatty acids, MON 87769 is expected to remain a specialty soybean product that is projected to be grown on less than 5% of the approximately 70 million acres of soybean grown annually in the U.S.

Soybean is the dominant oilseed in the U.S. and in global markets. Commodity soybean is grown on the majority of U.S. soybean acrea ge for its meal which is largely used for animal feed and for oil that is widely used in commercial processe d foods (Sonka et al., 2004). Approximately 12% of soybean gr own is specialty soybean produced for a specific market or use 12 . Examples of specialty soybean products include nonbiotechnology-derived, certified seed, organic food-grade, low saturated fat, clear hilum, tofu, natto, high sucrose, high oleic, low li nolenic and high protein (ASA, 2009). These soybean varieties are specified by buyers a nd end-users of soybean for production, and premiums are paid for delivering a product that meets purity and quality standards of that soybean variety. The specialty, value-adde d, product may be the whole soybean or, in the case of MON 87769, a processe d fraction such as the oil. Product differentiation and market segmentation in the specialty soybean industry includes mechanisms to keep track of the soybean (traceability), methods for id entity preservation (IDP), including closedloop systems, and quality assurance proce sses (e.g., ISO9001-2000 cer tification), as well as contracts between growers a nd buyers that specify delivery agreements within product specifications.

This deregulation is being sought in an environment that has rapidly adopted biotechnology-derived soybean varietie s (James, 2008). Thirteen different biotechnology-derived soybean crop products have been deregulated by USDA since 1994. Biotechnology-derived herbicide-tole rant soybean varieties were grown on approximately 69 million of the 75 million acres of soybean grown in the U.S. in 2008 (USDA-ERS, 2008). Thus, soybean breeder s, seed manufacturers, and soybean

12 http://usb.adayana.com:8080/usb/jsp/login.jsp . Percent U.S. Soybean acreage estimate based on U.S. Domestic Consumption by Segment – 2008/09. D. Ludwig, personal communication, 2009.Monsanto Company 09-SY-200U Page 376 of 395 producers have developed practices and system s that allow for the concurrent breeding

and manufacture of biotechnol ogy-derived and specialty soybean and are capable of breeding, manufacturing seed, and producing harves ted seed to meet th e needs of various markets. MON 87769 is the first SDA-producing soybean crop product for which deregulated status is being requested. However, APHIS is currently reviewing a high oleic acid soybean product (E vent DP-305423-1) and has gran ted nonregulated status to Laurate canola (Events pCGN 3828-212/86 18 & 23) with modifi ed oil characteristics, and there are other modified oil soybean products produced by c onventional breeding on

the market.

In recent years, there has been an increased demand by c onsumers and food processers for soybean and other oilseed crops that have specific physical or chemical characteristics to meet specific food or feed needs. Traditi onal plant breeding has be en used to alter the fatty acid profiles of oil pr oducing plants such as soybean. For example, soybean varieties with lower levels (1-3%) of the pol yunsaturated linolenic fatty acid have been commercially introduced (Fehr, 2007) and programs to develop high oleic soybean through conventional breeding have resulted in soybean varieties with an oleic content of >70% (Alt et al., 2005). MON 87769 has been developed to meet a demand for a landbased, sustainable source of omega-3 fatty acid for food and possibl y feed use. As a replacement for omega-3 rich oils or omega- 3 fatty acid containing products, use of SDA soybean oil would be limited to incorporatio n into food applications where it is: 1) functionally suitable (discussed below); 2) provides acceptable sensory properties (reduced off-flavors relativ e to longer chain omega-3 fatty ac ids) to foods and feeds; and 3) gives an appropriate stabil ity profile and shelf life during food and feed production and storage. As expected, the SDA soybean o il from MON 87769 contains high levels of SDA (approximately 20-30 weight % of total fatty acids), a polyunsaturated fatty acid. Vegetable oils containing hi gh levels of polyunsaturated fa tty acids, such as oil from MON 87769, are known to undergo rapid oxidati on during frying applications and will impart undesirable taste and odor to fried foods . Therefore, SDA soybean oil will not be suitable for all food applications and may not be suitable as a substitute for saturated fats in food recipes. However, SDA soybean oil can partially replace unhydrogenated soybean oil or other oils in many food cat egories. The curren t anticipated food applications for SDA soybean oil are in bake d goods and baking mixes, breakfast cereals and grains, cheeses, dairy pr oduct analogs, fats and oils, fish products, frozen dairy desserts and mixes, grain products and pastas , gravies and sauces, meat products, milk products, nuts and nut products, poultry pro ducts, processed fruit juices, processed vegetable products, puddings and fillings, sn ack foods, soft candy, and soups and soup mixes (GRAS Notice No. GRN 000283).

Hydrogenation of vegetable oils makes the oil more stable relative to unhydrogenated oil and thus prolongs the shelf life of products c ontaining these oils. Through the process of hydrogenation, the chemical structure of the fa tty acids are changed. In the case of SDA soybean oil, hydrogenation woul d convert SDA (18:4) to ot her fatty acids present in soybean oil (e.g. 18:3, 18:2, and 18:1) and ther eby eliminate the bene fits of the long chain PUFA produced by MON 87769. Thus, as is the case with fish oils, and for the same reasons, MON 87769 oil will not be hydrogenated.Monsanto Company 09-SY-200U Page 377 of 395 Because oil produced from MON 87769 is not suita ble for all applications that currently use soybean oil, and because MON 87769 oil will be more valuable than commodity soybean oil, it is Monsanto’s intent that MON 87769 will be marketed and produced in a manner similar to other value-added, specialty soybean products (e .g., low linolenic, high protein, and high oleic). Thes e value-added products are t ypically produced using IDP systems that include contracts between all ha ndlers of soybean (seed suppliers, growers, handlers, and processors), tr aceability, product tracking, proce ss verification and separate channeling to minimize commingling. Monsanto has introduced the value-added low

linolenic Vistive ® soybean, developed via conventiona l breeding, to the marketplace and will utilize similar management practices in order to identity preserve the SDA soybean oil produced from MON 87769.

L.D. Potential Environmental Impacts

The soybean production environment and MON 87769 are described in detail throughout this petition. The discussion of impacts below is focused on specific areas of the production environment and related activi ties including land use patterns, farming practices, commercial activitie s such as seed production and marketing of soybean and impacts to the quality of agricultural products.

L.D.1. Impacts on Land Use

Soybean is grown as a commercial crop on over 75 million acres in at least 27 states in the U.S. (USDA-NASS, 2009). Soybean acreag e in the past five years has been relatively stable varying from 64.7 to 75.7 m illion acres with a 10-year average of 73.3 million acres. Fluctuations in soybean acreag e are due to environmental, agronomic and

economic factors 13 , as well as government programs su ch as the crop reserve program (CRP) or ethanol mandates imposed by the U.S. government. Soybean fields are typically highly managed agricultural areas that can be expected to be dedicated to crop production for many years and cultivation of MON 87769 is not expected to differ from typical soybean cultivation practices. The modified oil trait in MON 87769 provides growers with an option to produce a valu e added soybean and potentially greater profitability in their farming operation. MON 87769 will likely be used in common rotations on land previously used for agricultural purposes.

Deregulation of MON 87769 is not expected to cause any significant impact to land devoted to agricultural uses. The cumulative land area in the U.S. planted to principal crops, which include corn, sorghum, oats, barley , winter wheat, rye, durum, spring wheat, rice, soybean, peanuts, sunflower, cotton, dry ed ible beans, potatoes, canola, proso millet, and sugar beets, has remained relatively cons tant over the past 25 y ears. From 1983 to 1995, the average yearly acreage of principal crops was 328 million. This average is statistically unchanged at 326 million acres since the introduction of biotechnology-

® Vistive is a registered trademark of Monsanto Technology LLC.

13 2008 NASS crop acreage report [http://www.nass.usda.gov/Newsroom/2008/06_30_2008.asp ] accessed

6/2009Monsanto Company 09-SY-200U Page 378 of 395 derived crops in 1996 14 . In fact, the total U.S. cropl and acres have been relatively constant since the 1940s ( Vesterby and Krupa, 1997) . Therefore, there is no indication that the introduction and widespread adoption of biotechnology-derived cr ops, or specialty soybean varieties, has resulted in a significant change to the total U.S. cropland acres. As with these other products, introduction of MON 87769 is not expected to have a significant impact on total U.S. cropland acres.

MON 87769 will likely be introduced into the Midwest/Great Plains region. This region is responsible for production of over 75% of the soybean grown in the U.S. On the basis of projections for use in food and possibl y feed, it is expected that MON 87769 will remain a low acre “specialty” soybean produced on less than 5% of the U.S. soybean acreage. Granting nonregulat ed status to MON 87769 is not expected to have a significant impact to land use for the follo wing reasons. MON 87769 will not alter the range of soybean cultivation as the new trait (SDA production) does not change the growth habits compared to conventiona l soybean. MON 87769 will be grown on land already devoted to soybean production replac ing existing commodity or other specialty soybean production. As discussed above, soybean acreage fluctuates annually based upon the dynamics of the marketplace and other environmental and agronomic factors. Thus, assuming that demand for commodity and other specialty soybean remains the same, if MON 87769 achieves maximum adoption (3.5 million acres), soybean acres could slightly increase. However, the increase in acreage is well within the range of normal acreage fluctuations observed for s oybean. Based on these reasons, Monsanto does not consider that deregulation of M ON 87769 will result in significant impacts to

land use.

A decision to deregulate MON 87769 similarl y is not expected to result in any cumulative impacts to land use. It is fore seeable that MON 87769 could and likely will

be stacked with MON 89788 which is tolerant to glyphosate. This herbicide tolerance trait is not expected to change the demand for SDA oil and is not considered a factor impacting the acreage devoted to MON 87769 production. Numerous specialty soybean

varieties have been introduced over the past 10 years, incl uding non-biotechnologyderived, certified seed, organi c food-grade, low saturated fa t, clear hilum, tofu, natto, high sucrose, high oleic, low linolenic and high protein (ASA, 2009). Adoption and continued use of these specialty soybean varie ties has not correlated with an increase in the total acreage for soybean.

L.D.2. Potential for Non-crop and Non-agricultural Impacts

Soybean does not grow in the w ild in the U.S. (Hymowitz and Singh, 1987; CFIA, 1996; OECD, 2000). Soybean does not grow and pers ist in unmanaged habitats and would not be expected to invade and/or persist in the natural environment, including streams, lakes, oceans or other aquatic environments. With the exception of production of the Pj Δ 6D

and Nc Δ 15D proteins and the presence of SDA and GLA as components of the fatty

14 Calculated from datasets at http://usda.mannlib.cornell.edu/usda/current/htrcp/ by comparing the total

acres from 1983 to 1995 and from 1996 to 2007.Monsanto Company 09-SY-200U Page 379 of 395 acids in the seed, MON 87769 is similar to ot her commercial soybean varieties currently grown in the U.S. including both conventiona l and biotechnology-derived, and would be expected to similarly have no significant impact to non-agricultural lands or aquatic systems. MON 87769 could be produced on la nd where soybean may be grown. There are no changes in agricultural inputs needed to produce varieties containing MON 87769 relative to commodity or specialty soybean and specifically no altered pest or disease susceptibility. Hence, pesticide applica tions on MON 87769 that may result in drift impacting non-crop or nonagricultural lands would be comparable to those used on conventional or currently availa ble soybean varieties.

L.D.3. Potential Impacts to Agricultural Practices

MON 87769 has been shown to be no differe nt from conventional soybean in its agronomic and ecological characteristics, and ha s the same levels of resistance to insects and diseases as current commercial soybea n. A summary of agronomic practices for soybean production is presented in Section IX of this petition. Agricultural practices for MON 87769 are expected to be similar to those used for production of commodity or specialty soybean. Specifi cally, MON 87769 requires no change s to current cultivation practices, including seedi ng, tillage, fertilizer and pesticid e applications and crop rotation practices. Growers will utiliz e best practices associated with production and identity preservation (IDP) of harvested soybean si milar to those currently used for other specialty soybean varieties.

Potential Impact to Soybean Commodity and Specialty Soybean Production

Deregulation of MON 87769 would allow fo r unconfined commercial planting of varieties containing MON 87769 a nd would provide an altern ative commercial use of soybean oil. It would also provide grower s with a higher value cr op resulting in greater profit potential for U.S. soyb ean producers. Soybean gr owers and processors are accustomed to accommodating a diversity of sp ecialty soybean varieties in their normal handling of commodity soybean. Currently sp ecialty soybean varieties are produced on approximately 12% of the U.S. soybean acreag e. Soybean growers ha ve demonstrated an ability to provide customers with their soybean product of choice. MON 87769 will be produced using IDP practices consistent with those used for other specialty soybean varieties. IDP practices will mitigate potential significant impacts to commodity or other specialty soybean. MON 87769 will be se gregated from commodity soybean by producers as part of their contract and to maintain the value of the crop. Because soybean is primarily a self-pollinated crop w ith minimal gene movement and due to the identity preservation production system, no si gnificant impact to commodity or other specialty soybean production is anticipated, should APHIS gr ant nonregulated status to

MON 87769.

No cumulative impacts to commodity or othe r specialty soybean pr oduction are expected from deregulation. Soybean producers have demonstrated thei r ongoing ability to produce multiple specialty soybean varie ties in a diverse marketplace.Monsanto Company 09-SY-200U Page 380 of 395 Potential Impact to Certified Seed Production

Certified seed production is a carefully mana ged process. MON 87769 is not expected to impact certified seed production practices or production of other cer tified conventional, specialty or organic soybean seed for the reas ons described in this section. As discussed elsewhere, MON 87769 is expected to be a small acre specialty pr oduct grown on less than five percent of total soybean acres. The amount of acreage n eeded to supply seed for five percent of the total soybean acr eage is approximately 100,000 acres. Thus, MON 87769 would occupy a small percentage of the roughly 1.9 to 2.3 million annual soybean seed producing acres.

If MON 87769 is deregulated, seed productio n could occur within production systems already developed by seed producers for certif ied seed varieties. MON 87769 has been thoroughly characterized and (with the exception of the modified fatty acid profile) is not phenotypically different. The implementation of management practi ces to avoid pollen from a biotechnology-derived crop in organic, specialty or conventional soybean seed or commodity seed production operations is dire ctly impacted by the nature of soybean pollination. Soybean is a highly self-pollinate d species that exhibits very low levels of outcrossing. When soybean plants were grow n in very close proximity to each other (15 cm), average cross-pollination rates were 1.8% (Ray et al., 2003). At greater distances, cross-pollinations rates we re 0.41 and 0.03% at 0.9 and 5.4 m, respectively. Hence, certified soybean seed producers can and have effectively implement ed practices (e.g., isolation distances during the growing s eason, equipment cleaning during harvest, and post-harvest separation of harvested seed) th at allow them to maintain commercially acceptable levels of varietal purity.

No cumulative effects are anticipated to cer tified seed production from deregulation of MON 87769. The use of management practi ces that prevent trait movement and comingling of soybean varieties has resulted in production of soyb ean varieties with improved genetics over time. As a result of the implementation of these management practices, growers today can choose from nume rous varieties of soybean including those used to produce commodity and sp ecialty soybean varieties.

Potential Impacts to Organic Soybean Production

Organic soybean markets typically enjoy a market premium offsetting the additional production and record-keeping co sts associated with this production method. Organic farming operations as described by the Nationa l Organic Program, which is administered by USDA’s Agricultural Marketing Service, requires organic production operations to have distinct, defined boundaries and buffer zones to prevent unin tended contact with prohibited substances or products of exclude d methods from adjoining land that is not under an organic production management plan. Organic production operations must also develop and maintain an organic production system plan approved by an accredited certifying agent. This plan enables the production operati on to achieve and document compliance with the National Organic Standards, including the prohibition of the use of

excluded methods. Excluded methods include a variety of methods used to genetically engineer organisms or influence their grow th and development by means that are notMonsanto Company 09-SY-200U Page 381 of 395 possible under natural conditions or processes. The use of biotechnology such as that used to produce MON 87769 is an exclude d method under the National Organic Program [7 C.F.R. § 205.2].

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

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. The unintentiona l presence of the pr oducts of excluded

methods will not affect the status of an or ganic product or operation when the operation

has not used excluded methods and has taken reasonable steps to a void contact with the products of excluded methods as detailed in an approved organic system plan. Organic

certification certifies that or ganic production and handling pr ocesses have been followed, not that the product itself is “free” from any particular substance.

Production systems designed prior to the intr oduction of MON 87769 or even prior to the introduction of biotechnology-derived soybean have allowed for production of soybean to

meet varied customer demands. In addition to the market segments that produce organic or conventional soybean, distinct identity-preserv ed specialty soybean with such traits as clear hilum or high protein have also been grown and successfully marketed for specific food uses in domestic and export markets fo r many years (Zhanglin et al., 2004). The choice to grow biotechnology-derived, orga nic or conventional soybean depends on many factors and the dynamics of the market place. The dynamics of the marketplace, choice between various varieties of soybea n, and the existing production practices will not be impacted by the introduction of MON 87769.

Organic soybean producers utilize production prac tices designed to sp ecifically avoid the presence of both soybean products using co nventional herbicide or other pesticide treatments, as well as biotechnology-derived crops. These well esta blished practices to

avoid “excluded methods” will continue with the introduction of MON 87769 varieties. They include isolation zones, use of buffe r rows surrounding the organic crop, adjusted planting dates and varietal selection (www.attra.ncat.org). The implementation of management practices to avoid pollen from a biotechnology-derived crop in organic or conventional soybean production operations is facilitated by the nature of soybean pollination. As noted previously in this petition, soybean is a highly self-pollinated species and exhibits very low level of out crossing. Hence, organic or conventional soybean producers can and have effectively implemented practices (e.g., isolation during the growing season, equipment cleaning during harvest, and post-harvest separation of harvested seed) that allow them to avoid the presence of biotechnology-derived soybean and maintain organic or c onventional production status.

Currently, biotechnology-derived herbicide tolerant soybean is planted on 92% of the soybean acreage (USDA-ERS, 2008) and th e Roundup Ready soybean system – that is, planting Roundup Ready soybean a nd applying glyphosate in crop – has become the mostMonsanto Company 09-SY-200U Page 382 of 395 widely used weed control program in U.S. soybean production. Despite the high adoption rates of Roundup Ready soybean by gr owers, organic and conventional soybean production remains an option for farmers who choose to produce using these production practices or varieties of soybean. The d ecision to grow organi c, conventional, or biotechnology-derived soybean varieties is typically an economic one based on market dynamics. Organic soybean producers and those growing convent ional soybean for sensitive non-biotechnology markets typically enjoy a market premium offsetting the additional production and record-keeping cost s. While the widespread adoption of Roundup Ready soybean has reduced the number of conventional soybean varieties that are available, conventional and organically produced soybean seed is currently available from numerous seed suppliers (Table L-1). Additional information on organic seed sources is provided though the U.S. Agri cultural Marketing Service (AMS) at: www.ams.usda.gov. Thus, growers ha ve a choice in the soybean variety they plant, and this is not expected to change with the introduction of MON 87769.

Buyers recognize that where there are biot echnology-derived crop varieties on the market, as with soybean, a guarantee that a commod ity crop is 100% “free” of biotechnologyderived material is not feasible based on the limitations of testing and sampling methodology (Born, 2005). While the presence of a biotechnology-derived product like MON 87769 is unlikely in instances wher e producers utilize production practices designed to avoid biotech presence, in some instances buyer allowa nces between 0.1 to 5% biotechnology-derived commodity soybean in organic soybean are specified. This

also is consistent with the USDA Nationa l Organic Program allowing for detectable residues of excluded methods (including biotech nology-derived crop prod ucts) as long as the producer has taken steps to avoid thos e methods (www.ams.usda.gov/nop/Q&A.html). Similarly, international regul atory organizations have r ecognized that testing and sampling methodologies limit the ab ility to confirm that comm odity or specialty soybean is 100% free of biotechnology-de rived material. Thus, they ha ve set allowable tolerances

for this material in conven tional products to su pport food labeling and traceability laws. These tolerances allow from 0.9% (European Un ion) up to 5% (Japan ) of the food to be biotechnology-derived in products consider ed “conventional.” Levels above the threshold may trigger sp ecial labeling. Thus, de minimis levels of approved biotechnology-derived soybean are allowabl e in certified organic or conventional soybean.Monsanto Company 09-SY-200U Page 383 of 395 Table L-1. Organic and Conventional Soybean Seed Sources

Organic Soybean Seed Sources* : Conventional Soybean Seed Sources

Albert Lea Seed House AgVenture Seeds (modified oil)

Blue River Hybrids Campbell Seed (modified oil)

Golden Grains Becks Hybrids (food grade)

Great Harvest Organics Monsanto (Asgrow)

Greis Seed Farm Schillinger Seed

Lancaster Ag Products Pioneer

Lawler Farm Center Soy Genetics

Prairie Gold Seeds Stewart Seed (modified oil)

Superior Organic Grains, Ltd Stine Seed

Walter Seed and Honey Co Syngenta - multiple brands

Terral Seed
Various State Crop Improvement

Or g anizations

* From: www.organicgrains.ncsu.edu

L.D.4. Potential Impacts to Raw or Processed Agricultural Commodities

With this petition, extensive data have been presented relating to plant growth parameters, disease susceptibility, insect susceptibili ty, and forage and seed composition of MON 87769 compared to conventional soybean va rieties. These data indicate that there are no biologically relevant differences between MON 87769 and conventional soybean, except for the deliberate change in the fatty acid composition of the oil derived from

MON 87769. Monsanto has initiated a volun tary consultation with the FDA, and information has been provided to the agen cy supporting the food and feed safety of soybean, forage and SDA soybean oil derived from MON 87769 (BNF 00117, GRAS Notice No. GRN 000283).

From a grain quality standpoint (e.g., fore ign material, damaged grain, etc; see www.gipsa.usda.gov for soybean grain sta ndards), the soybean produced from MON 87769 varieties is expected to be of co mparable quality to soybean commercially produced in the U.S. for commodity markets based on the data presented herein. The intended use of MON 87769 will be for proc essing for the production of SDA soybean oil within an IDP system. MON 87769 will be grown under a contract with growers that will direct harvested soybean to its intended purpose. If for any reason a load of MON 87769 soybean is not suitable to be processed within the IDP system for the production of SDA soybean oil, the soybean will be directed to appropriate uses.

Soybean is typically processed into two ma jor fractions, the oil and meal, and several minor fractions including lecithin and protei n isolate. With the exception of the oilMonsanto Company 09-SY-200U Page 384 of 395 fraction, the other processed fractions produced by MON 87769 are compositionally equivalent to, and will be put to the same us es as commodity soybean processed fractions. The meal, and other non-oil proc essed fractions are intended to be distributed into the commodity stream and will be marketed and so ld in the commodity market. Because of their substantial equivalence, no significan t impact will occur to these commodity soybean processed fractions when APHIS grants deregulated status to MON 87769.

As described elsewhere in this petition, MON 87769 and SDA soybean oil produced from MON 87769 will be produced and processed u tilizing an identity preservation (IDP) system for use in specific food applications. If for any reason, the oil from MON 87769 is not used for the intended food applications, it would be directed to an appropriate use.

The fatty acid profile of SDA soybean oil produced from MON 87769 would not be suitable for all of the food applications th at currently use commodity soybean oil. Vegetable oils containing high levels of polyunsaturated fatty acids, such as SDA soybean oil, are known to undergo rapid oxidati on during frying applications that impart

undesirable taste and odor to the fried foods. For this re ason, undiluted SDA soybean oil

would not be suitable for us e in frying applications.

It is not expected that the introduction of MON 87769 will cause a significant impact to processed soybean oils (e.g., commodity or ot her specialty) due to the IDP system under which it will be commercialized, handled and used, and because of the systems employed by the soybean processing and us e chain to specify the charac teristics of oils, and to identify, segregate and test thos e oils for different uses.

Before implementing an IDP system, Monsanto will identify relevant stakeholders in the value chain and engage them in dialogue to describe the intended us es and benefits of SDA soybean oil, to discuss and understand th e potential impacts on commodity oils and processed soybean products from the ID P production, handling and use of MON 87769 soybean and SDA soybean oil. Based on the dialogue and the resulting assessment, Monsanto will take the following steps, where applicable and appropriate:

1. refine plans for production, handling and processing of MON 87769 soybean and SDA soybean oil to address valid concerns raised by the stakeholders,

2. assess potential impacts on processed soybean products due to the presence of SDA in commodity oil, and collaborate with stakeholders to support research required for the evaluation of those impacts and the le vels of MON 87769 soybean or of the SDA soybean oil at which they occur,

3. develop management, mitigation and response plans to address potential

instances where the trait is present at levels, if a ny, that are not compatible with commodity soybean and processing streams, and

4. engage in industry outreach and education regarding the MON 87769 soybean/SDA soybean oil IDP system, including product handling and use, and the implementation of any necessary risk management, mitigation and response plans.Monsanto Company 09-SY-200U Page 385 of 395 Based on Monsanto’s assessment, it is unlikely that there will be significant impacts on raw or processed soybean commodities due to APHIS granting nonre gulated status to

MON 87769.

Based on this assessment, Monsanto does not anticipate any significant cumulative impacts on either raw or processed soybean commodities resulting from the deregulation of MON 87769. Soybean does not survive outs ide of cultivation and does not readily survive as a volunteer in agricultural fields. Hence, the only source of SDA soybean oil

will be from MON 87769 branded varieties produced under contract. The use of well established breeding and seed pr oduction methods assures the pur ity of soybean varieties. These same methods will be used to produce soybean varieties that contain MON 87769. Similarly, these methods will be used to minimize the presence of MON 87769 in other soybean varieties. Food ingredient and f ood manufacturers routinely manage and handle a wide variety of specialty oils derived from oilseed crops (includi ng other modified oils produced by other deregulated events) with no adverse or cumulative effects on their manufacturing processes or on the quality of their products. Similarly, food manufactures will integrate SDA soybean oil into their existing handling and manufacturing process, and are expected to have the equivalent experience with SDA soybean oil, resulting in no adverse or cumulative effects due to APHIS granting deregulated status to MON 87769.

L.D.5. Potential Impacts on Commercial Use of Soybean

The decision to deregulate MON 87769 would allow for breeding of this product with conventional and biotechnology-derived soybe an varieties and would make MON 87769 available to breeders, certified seed producer s, and growers. Like other biotechnologyderived traits, it is expected that breed ers and certified seed producers would use MON 87769 to supply seed for planned commer cial markets in the U.S. Monsanto anticipates that commercial use of M ON 87769 may include export of soybean and soybean products, and has described import approval submission plans elsewhere in the petition.

L.D.6. Potential Impacts to Human Health and Safety

Most human interactions with soybean occu r through agricultural production, industrial operations, or through consumption. Therefore, this health and safety discussion will focus on food and feed safety as well as safety of workers exposed to MON 87769. SDA soybean oil derived from MON 87769 is intend ed to be commercialized as an omega-3 oil that will replace non-hydrogenated soybean oi l and/or is added to select food products such as margarine, mayonnaise, shortenings, salad dressings and ready-to-eat foods. SDA soybean oil may also be used as a food/feed ingredient as a replacement for fish oil, flax seed oil, vegetable oils or animal fats.

Under the Federal Food, Drug, and Cosmetic Ac t (FFDCA), it is the responsibility of

food and feed manufacturers to en sure that the products they ma rket are safe and properly

labeled. Food and feed derived from M ON 87769 must be in compliance with all applicable legal and regulato ry requirements. Biotechnolo gy-derived crops for food andMonsanto Company 09-SY-200U Page 386 of 395 feed use undergo a voluntary consultation proc ess with the FDA prio r to release onto the market. Although a voluntary process, Monsanto completes a consultation with the FDA prior to placing a new biot echnology derived crop product on the market. A list of completed consultations on biotechnology-deri ved crop products is available at the FDA website. As part of the consultation process for MON 87769, Monsanto provided product characterization and safety data to the FDA on March 23, 2009 (BNF 00117).

Monsanto has also completed an assessmen t of the safety of the oil produced by MON 87769 and a panel of independent expert s has concluded that SDA soybean oil is generally recognized as safe (GRAS) for its intended use in foods. Following a critical evaluation of the scientific data generally av ailable in the public domain that pertain to the safety of SDA soybean oil, a panel of experts, qualified by scie ntific training and experience to evaluate the safety of SDA soybean oi l as a component of food, unanimously concluded that the proposed uses of SDA soybean oil ar e safe and suitable and are GRAS, based on scientific procedures . Therefore, MON 87769 is as safe and

nutritious as conventional s oybean for food and feed use with the added nutritional improvement of the SDA omega-3 fatty acid. The GRAS assessment has been submitted to the FDA for review (GRAS Notice No. GRN 000283).

Soybean produced by MON 87769 contain approxi mately 20% to 30% stearidonic acid (SDA) and 7% gamma linolenic acid (G LA) (see Appendix F). Oil produced by MON 87769 represents a sustaina ble alternate source of an om ega-3 fatty ac id to help meet the needed dietary intake of long chain omega-3 fatty acids. The health benefits of omega-3 fatty acid consumption are widely recognized (James et al., 2003). SDA is naturally present in some plants, fish and ma rine algae and has a l ong standing history of safe consumption in humans (see Table X-1). The safe ty of SDA soybean oil is supported by the results of a 90-day/one ge neration reproductive toxicity rat study (Hammond et al., 2008). Furthermore, no advers e effects attributable to SDA soybean oil were reported in a published human study (H arris et al., 2008). Additional published

studies on SDA and GLA from other sources co rroborate the safety of SDA soybean oil. The safety of SDA soybean oil is further s upported by the regular dietary consumption of fats and oils containing the ot her primary fatty acids present in SDA soybean oil, namely ALA, LA, and palmitic acid.

As part of the consultation with the F DA regarding MON 87769, Monsanto has provided the agency with information on the identity, function, and characterization of the genes, as well the expression levels of the gene pr oducts. Monsanto also provided information on the potential allergenicity and toxicity of the expressed Pj Δ 6D and Nc Δ 15D proteins. The development of MON 87769 involves the introduction of two proteins (Pj Δ 6D and

Nc Δ 15D) produced by genes from plant and fungal sources (see Section VI for details). These two proteins are not cu rrently present in soybean and the safety of each was assessed for both food and feed applications . A summary of the safety assessment conducted on these two proteins is provided in Section VI of this petition. These

assessments lead to the conclusion that there is no meaningful risk to animal or human

health from dietar y exposure to the Pj Δ 6D and Nc Δ 15D proteins produced in

MON 87769.Monsanto Company 09-SY-200U Page 387 of 395 Extensive compositional analyses of forage and seed were conducted on samples from replicated, multi-site field trials conducted in the U.S. to compare the composition of MON 87769 to a conventional soybean control and to commercially available soybean varieties. The compositional analyses of MON 87769 demonstrated it to be compositionally equivalent to conventional soybean, except for the modified fatty acid profile. Composition data support the conclusion that there are no biologically relevant changes in MON 87769 due to the genetic mo dification, outside of the modified fatty acid profile.

On the basis of the assessment of laboratory data and scientific literature, it is reasonable

to conclude that MON 87769 is as safe and nutritious as conventional soybean for food

and feed use with the added nutritional improvement of the SDA omega-3 fatty acid. Therefore, a decision to grant nonregul ated status to M ON 87769 would have no significant impact on human or animal health. Possible beneficial effects could occur to

human heart health if MON 87769 is deregulated. The ava ilability of products that contain SDA from SDA soybean oil may incr ease the intake of omega-3 fatty acids thereby providing a health benefit. These e ffects are not considered significant because

there are other sources of SDA and other hear t healthy omega-3 fatty acids available in existing foods such as from fish, other plants and marine micro and macroalgae. MON 87769 provides an additional sustainable source of omega-3 fatty acid from which food processors and manufacturers can choose.

Worker Safety

During agricultural production of soybean, grow ers may be exposed to pesticides during application to the crop. Downstream proce ssing and manufacturi ng workers deal with hazardous equipment and chemicals during the processing of soybean into food and feed

and also for industrial uses. As discussed in Section IX, MON 87769 does not change the agronomic practices, or use of chemicals such as pesticides, associated with soybean production. Thus, deregulation of MON 877 69 will have no significant impact to agricultural workers. Similarly, there are no changes to industrial processing of MON 87769 relative to the processi ng of commercial soybean.

SDA soybean oil derived from MON 87769 is expected to substitute for other oils currently used in foods and possibly in f eeds. The composition of SDA soybean oil contains fatty acids that are already used in the food and fe ed industries. Since workers are already accustomed to handling various oi ls for formulation into foods, there are no significant impacts identified to processors or food formulators either.

L.D.7. Potential Impacts to Plant, Anim al and Microbial Co mmunities Including Threatened or Endangered Species and Biodiversity

In assessing the potential impact to plant a nd animal communities, th e potential for gene movement and introgression from MON 8776 9 was evaluated because movement and establishment of the gene and trait to related species could have indi rect impacts to plant and animal communities that extend beyond the original recipient organism. MonsantoMonsanto Company 09-SY-200U Page 388 of 395 considered two primary issues : 1) the potential for gene flow and introgression, and 2) the potential impact of introgression. The genus Glycine has approximately nine species,

with G. max being placed in the subgenus Soja along with one other species, G. soja

. G.

max is sexually compatible with only G. soja and no other Glycine species. G. max is the only Glycine species located in the United States. Therefore, the probability of gene flow and introgression of MON 87769 into other species in the U.S. is essentially zero (Stewart et al., 2003); thus, the potential impact of introgression of MON 87769 to sexually compatible relatives on plant and an imal communities is nonexistent if APHIS were to grant the petition fo r nonregulated status.

Animals

Soybean production systems in agriculture ar e host to many animal species. Mammals and birds may seasonally use grain, and inve rtebrates can feed on the plant during the entire growing season. Agronomic pr actices used to produce MON87769 will be substantially the same as those used to pr oduce many types of specialty or conventionally grown soybean, thus this discussion fo r MON 87769 on animals will focus on the potential effects of the tw o introduced proteins (Pj Δ 6D and Nc Δ 15D) and the modified fatty acids in MON 87769. Animals that feed primarily on soybean 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 harvest.

Information included in this petition on the characterization of MON 87769 and safety of

MON 87769 for food and feed uses is relevant to an assessment of potential impacts to animals that may visit soybean fields cont aining MON 87769. As discussed previously, there is no meaningful risk to animal or human health from dietary exposure to Pj Δ 6D

and Nc Δ 15D from MON 87769. There are no toxic pr operties associated with either the Pj Δ 6D or Nc Δ 15D protein or with the fatty acids in the seed produced by MON 87769. The composition of the seed produced by MON 87769 is (with the exception of the intended change in fatty acid profile) comparab le to that of conventional soybean seed. Furthermore, forage produced by MON 87769 is compositionally equi valent to forage produced by conventional soybean. This inform ation indicates that there would be no negative effects to animals that forage on MON 87769 or consume seed. Additionally, animals have been previously exposed to S DA in the environment, as SDA is found in some plants, fish, and marine algae (James et al., 2003). During field trials, no changes in insect feeding damage were observed, furthe r supporting the lack of potential impact to animals. On the basis of this analysis , deregulation of MON 87769 will not result in significant impacts on animals, including insects that live n ear or in soybean fields containing MON 87769.

Plants

Soybean production systems in agriculture are host to many plant sp ecies as well. The landscape surrounding a soybean field varies de pending on the region. In certain areas, soybean fields may be bordered by other s oybean, corn (or any other crop); fields mayMonsanto Company 09-SY-200U Page 389 of 395 also be surrounded by wooded and/or pasture/gr assland areas. Therefore, the types of vegetation around a soybean field depend on th e area where the soybean is planted.

If MON 87769 is granted nonregulated status, ag ricultural practices that are used for the production of commodity soybean would be used for plant management during the

cultivation of MON 87769. MON 87769 does not ex hibit characteristic s associated with weedy growth and will not compete with plan ts found outside of agricultural production.

Weeds within fields of MON 87769 will be managed using mechan ical, cultural, and chemical control measures, similar to curre nt management practi ces for commodity and many types of specialty soybean. As MON 87769 has no toxic effects on animals (see discussion above), pollinators of other plants in or aro und fields cultivated with MON 87769 will not be impacted and, likewise, no indirect e ffects are expected to other plants that rely on pollinators. Thus, granting nonregulated st atus to MON 87769 will have no significant impact to plant communities.

Threatened and Endangered Species

No significant impact to threatened and e ndangered species is expected due to APHIS granting nonregulated status to MON 87769. As previously noted, consumption of the Pj Δ 6D and Nc Δ 15D proteins has shown no toxicity in laboratory testing with mice and it

has been demonstrated that there has been previous environmental exposure to SDA with no documented adverse effects. MON 87769 does not express any additional proteins, natural toxicants, etc. that ar e known to directly or indirectly affect a listed threatened or endangered species (TES) or species proposed for listing by the U.S. Fish and Wildlife

Service. MON 87769 is not se xually compatible with any federally listed TES or a species proposed for listing. The only TES anim al listed that occupies habitat that is likely to include soybean fiel ds and that might feed on soybean is the federally Endangered Delmarva Peninsula Fox Squirrel ( Sciurus niger cinereus ), found in areas of

the mid-Atlantic Eastern seaboard. It is known to utilize certain agri cultural lands readily, but its diet includes acorns, nuts/seeds of hickory, beech, wa lnut, and loblolly pine; buds and flowers of trees, fungi, insects, fruit, and an occasional bird egg. Given all these

factors and the lack of noted adverse effect s of the introduced proteins or SDA soybean oil on mammalian species (mice, rats) and the lack of environmental interactions of MON 87769 with other non-target organism s in the field, it is concluded that

MON 87769 will not have an effect on th e Delmarva Peninsula Fox Squirrel.

No significant impact to any threatened or e ndangered plant species is expected from the

cultivation of MON 87769. G. max has no sexually-compatible relatives in the U.S. and does not survive outside of cultivation. Thus, there is no opportunity for MON 87769 to interbreed with any plant species or displace natural vegetation in the U.S.

Soil Microorganisms

On the basis of the biochemical and physiochemical characteristics of the Pj Δ 6D and

Nc Δ 15D proteins, no accumulation of these pr oteins is expected in soils where MON 87769 will be grown. Information ha s also been presen ted in the petition demonstrating the established presence of S DA in the environment. No adverse effectsMonsanto Company 09-SY-200U Page 390 of 395 on soil microorganisms are associated with SDA nor do the characteristics of the modified fatty acid profile merit concern to soil microorganisms because SDA will be degraded in the soil like other PUFAs. M onsanto presented data in this petition demonstrating the lack of impact to symbiotic microbes associated with soybean plants.

The B. japonicum -soybean symbiosis of MON 87769 was not altered as a result of the introduction of the Pj Δ 6D and Nc Δ 15D proteins compared to a conventional soybean

control. On the basis of these observati ons and in conjunction with related phenotypic measurements for MON 87769, no impact on soil microorganisms and other soil arthropods is expected due to deregulation of MON 87769.

Biodiversity

Analysis of available information indicates that MON 87769 exhibits no traits that would cause increased weediness, th at its unconfined cultivation should not lead to increased weediness of other sexually compatible relatives (of which there are none in the United States), and it is likely to have no effect on non-target or ganisms common to agricultural ecosystems or threatened or endangered specie s. Based on this analysis, it is concluded that deregulation of soybean varieties c ontaining MON 87769 would have no significant impact on biodiversity.

No impacts to plant and animal communities or threatened and endangered species have

been identified for MON 87769. No cu mulative impacts to plant and animal communities or threatened and endangered species have been identified.

L.E. Other Cumulative Effects

Conventional Breeding with Other Biotechnology-derived or Conventional Soybean

Varieties

As previously mentioned, several biotechnolo gy-derived soybean products have been deregulated or are under consideration for dere gulation. A list of the events deregulated or are under consideration for deregulation by USDA-APHIS is presented in Table L-2. MON 87769 may be bred with these deregu lated biotechnology-derived soybean crop products as well as with conve ntional soybean, creating new improved varieties. APHIS

has determined that none of the biotechnol ogy-derived individual s oybean products it has deregulated display increased plant pest characteristics and that any progeny derived from crosses of these soybean varieties with other conventional or biotechnology-derived soybean are unlikely to exhibit new plant pest properties.

An assessment of the stabil ity of the genetic insert in MON 87769 was conducted, and data have been presented in this petition demonstrating that MON 87769 is stable in progeny. Having established that the genetic material is stable and that MON 87769 is inherited in a Mendelian fashion, and based on experience with MON 87769 in Monsanto’s plant breeding program, it can be concluded that the m odified oil phenotype present in MON 87769 is likewise stable. Conventional breeding has an established history of safe use, and use of MON 87769 in breeding programs is expected to behave

similar to other conventional traits and biotech nology-derived traits. For example, in anMonsanto Company 09-SY-200U Page 391 of 395 assessment by McCann et al. ( 2005), it was shown that duri ng three years of breeding into multiple varieties, the composition of glyphosate-tolerant soybean remained equivalent to that of conventional soybean. Given that there have been no plant pest characteristics associated with MON 87769, or with any of the previously deregulated events listed below, no significant impact s are expected to other soybean varieties through the use of MON 87769 in breeding program s and in combination with any of the previously deregulated soybean crop products.

All biotechnology-derived soybean products on the market today have satisfactorily completed the FDA consultation process expre ssly established to review the safety of whole foods derived from biotechnology- derived crops for human and animal consumption (see Table L-2). No impacts to public health (e.g., food or feed safety) are expected due to combination of these even ts through conventional breeding because the deregulated events have a history of safe us e and on the basis of knowledge of the type of modifications made to each of the deregulat ed events, the biochemical pathways are not likely to unexpectedly interact or result in the production of novel constituents. As expected, breeding with other modified o il soybean products on the list below would predictably alter the fatty acid composition of MON 87769 (i.e ., the combined trait will have an altered fatty acid profile that is a combination of the individual trait characteristics).

Breeders use standard testing and assessment procedures to further examine and confirm the equivalence of combined trait products to th e single event produ cts in terms of phenotype, agronomic characteristics, and the effi cacy of the traits. Th is testing is a part of the process to develop any new soybean va riety. The use of conventional breeding to produce combined trait or combined event products would identify off-types and nonperforming germplasm during development of new varieties and they would be removed from further development.Monsanto Company 09-SY-200U Page 392 of 395 Table L-2. Deregulated and Submitted Biotechnology-derived Soybean Products 1

Phenotype ID Code(s) Institution Date Deregulated

Herbicide Tolerant NA 2 DOW Ag Submitted

Herbicide Tolerant FG72 Bayer CropScience Submitted

Modified Oil MON 87705 Monsanto Submitted

Insect Resistant MON 87701 Monsanto Submitted

Herbicide Tolerant BPS-CV127-9 BASF Submitted

High Oleic Acid DP-3Ø5423-1 Pioneer Submitted

Glyphosate/ALS

Tolerant DP-356043-5 Pioneer October, 2007

Glyphosate Tolerant MON 89788 Monsanto February, 2007

Phosphinothricin

Tolerant GU262 AgrEvo October, 1998

Phosphinothricin

Tolerant A5547-127 AgrEvo May, 1998

Altered Oil Profile G94-1, G94-19, G-168 DuPont May, 1997

Phosphinothricin

Tolerant W62, W98, A2704-12, A2704-21, A5547-35 AgrEvo August, 1996

Glyphosate tolerant 40-3-2 Monsanto May, 1994

1 Source: http://www.aphis.usda.gov/brs

2 Event ID code not available - not listed on USDA website

L.F. Highly Uncertain, Unique or Unknown Risks

MON 87769 has been thoroughly characterize d and data submitted in the petition demonstrate that it does not pose an increased plant pest risk compared to conventional soybean. APHIS has previously deregulated 14 biotechnology-derived soybean products. MON 87769 represents an additional soybean product with a modified fatty acid profile in the soybean oil. Introduction of previ ous biotechnology-derived cr ops have resulted in no unexpected effects on the quality of the human environment as defined under NEPA and have provided benefits to growers, consumer s and the environment. In this respect, a decision to deregulate a ne w biotechnology-derived soybean product is not precedent setting nor are the effects to the quality of the human environment highly uncertain or unpredictable.

L.G. Summary

The extensive body of information presented in Sections I through X of this petition demonstrates that MON 87769 does not present a plant pest risk, a nd has no significant impact on threatened or endangered specie s or biodiversity. S oybean produced from

MON 87769 has been demonstrated to be safe and wholesome for food and feed purposes. MON 87769 will not significantly impact the comm ercial interests of soybean producers or those involved in the marketing and sa le of soybean and soybean products. TheMonsanto Company 09-SY-200U Page 393 of 395 amount of land devoted to soybean productio n or crop production in general has not changed with the introduction of biotechnol ogy-derived soybean products. Similarly, no significant change in the use of agricultural land or amount of land devoted to farming would be expected to occur with th e commercial introduction of MON 87769.

MON 87769 will utilize common cultivation practices typically employed for production of commodity soybean and many types of sp ecialty soybean, and management practices typically used for specialty soybean (i.e., ID P production practices). Hence, agricultural practices for MON 87769 are not likely to change.

The introduction and adoption of specialty soyb ean varieties in the past have provided differentiated high value soybean and soybean products that benefited U.S. growers and the end users of these products. The opportu nity for growers to produce a land-based omega-3 fatty acid is a benefit from an ec onomic and human health perspective. For these reasons, the proposed action to grant nonregulated status to MON 87769 does not represent a significant impact to the environment.

References Alt, L.J., W.R. Fehr, G.A. Welke, and J. G. Shannon. 2005. Transgressive segregation for oleate content in three soybean populations. Crop Science 45:2005-2007.

ASA. 2009. Grower Opportunities for Ident ity Preserved Value-Added Soybeans. American Soybean Associat ion, St. Louis, Missouri.

Born, H. 2005. Marketing organic grains. ATTRA publication. Av ailable on-line at: http://www.attra.ncat.org/attra- pub/PDF/marketingorganicgrains.pdf [Accessed February 8, 2009].

CFIA. 1996. The biology of Glycine max (L.) merr. (soybean). Biology Document BIO1996-10. Canadian Food Inspection Agency, Ontario, Canada.

Fehr, W.R. 2007. Breeding for modified fatty acid composition in soybean. Crop Science 47(S3):S73-S87.

Hammond, B.G., J.K. Lemen, G. Ahmed, K.D. Miller, J. Kirkpatrick, and T. Fleeman. 2008. Safety assessment of SDA soybean oil: Results of a 28-day gavage study and a 90- day/one generation reproduction feeding st udy in rats. Regulatory Toxicology and Pharmacology 52:311-323.

Harris, W.S., M.A. DiRienzo, S.A. Sands, C. George, P.G. Jones, and A.K. Eapen. 2007.

Stearidonic acid increases the red blood cell and heart eicosa pentaenoic acid content in dogs. Lipids 42(4):325-33.

Harris, W.S., S.L. Lemke, S.N. Hansen, D.A.a Goldstein, M.A. DiRienzo, H. Su, M.A.Nemeth, M.L. Taylor, G. Ahmed, and C. George. 2008. Stearidonic acid-enriched soybean oil increased the omega-3 index, an em erging cardiovascular risk marker. Lipids

43:805-811.Monsanto Company 09-SY-200U Page 394 of 395 Hymowitz, T., and R.J. Singh. 1987. Ta xonomy and speciation. Pages 23-48 in Soybeans: Improvement, Production, and Uses. Boerma. H.R., and J.E. Sprecht (eds). American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, Wisconsin.

James, M.J., V.M. Ursin, and L.G. Clela nd. 2003. Metabolism of st earidonic acid in human subjects: comparison with the metabo lism of other n-3 fatty acids. American

Journal of Clinical Nutrition 77:1140-1145.

James, C. 2008. Global Status of Co mmercialized Biotech/GM Crops: 2008. ISAAA Brief No. 39. ISAAA: Ithaca, NY.

Kris-Etherton, P.M., W.S. Harris, and L.J. Appel. 2002. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106(21):2747-2757. Kris-Etherton, P.M., W.S. Harris, and L.J. Appel. 2003. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular di sease: New recommendations from the

American Heart Association." Arterioscl erosis, Thrombosis and Vascular Biology 23(2):151-152.

McCann, M.C., K. Liu, W.A. Trujillo and R.C. Dobert. 2005. Glyphosate-tolerant
soybeans remain compositionally equivalent to conventional soybeans (Glycine max. L.)
during three years of field te sting. Journal of Agricult ure and Food Chemistry 53:5331-

5335.

OECD. 2000. Consensus document on the biology of glycine max (L.) merr. (soybean)..

OECD ENV/JM/MONO(2000)9. Organization for Economic Co-operation and Development, Paris.

Ray, J.D., T.C. Kilen, A.C. Abel, and R.L. Paris. 2003. Soybean natural cross-pollination

rates under field conditions. E nvironmental Biosafety Research 2:133-138.

Sonka, S.T., K.L. Bender, and D.K. Fisher. 2004. Economics and Marketing. Pages 919- 947 in Soybeans: Improvement, Production, and Uses American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison

.

Stewart, C.N., M.D. Halfhill, and S.I. Warwick. 2003. Transgence introgression from genetically modified crops to their wild relatives. Nature 4:806-817.

USDA-ERS. 2008. Agricultural Biotechnology: Adoption of Biotechnology and its Production Impacts. http://www.ers.usda.gov/ briefing/biotechnology/chapter1.htm [Accessed Feb. 10, 2009]

USDA-NASS. 2006a. Crop production 2005 summ ary, United States Department of Agriculture, Washington, D.C.Monsanto Company 09-SY-200U Page 395 of 395 USDA-NASS. 2006b. Quick Stats (Soybean): 1996-2005 Reports. United States Department of Agriculture National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2008. Crop Values: 2007 Summ ary. United States Department of Agriculture - National Agricultural St atistics Service, Washington, D.C.

USDA-NASS. 2009. U.S. & All States Data - Soybeans. in Quick Stats.

Vesterby, M. and K.S. Krupa. 1997. Major Uses of Land in the United State. USDAEconomic Research Service. . Statistical Bulletin No. 973. http://www.ers.usda.gov/publications/sb973/sb973.pdf

Zhanglin, C., A.T. James, S. Miyazaki, R.F. Wilson, and T.E. Carter Jr. 2004. The breeding of specialty soyb eans for traditional and new soyfoods. Pp 264-322 in Proceedings of the American Oil Chemists' Society.Page 1 of 25



## Petition for the Determination of Nonregulated Status for

MON 87769

ADDENDUM

Potential Market Impact of MON 87769

Submitted Ju

ly 1 st 

## , 2011

OECD Unique Identifier: MON

- 87769

- 7

Mon santo Petition Number: 09

- 183

- 01pPage 2 of 25



### 1. Introduction

Monsanto has developed biotech nology

- derived soybean MON 87769 (SDA soybean) that

contains stearidonic acid (SDA)

, a sustainable alternate source of an omega

- 3 fatty acid to help
meet the need for increased dietary intake of l ong chain omega

- 3 fatty acids. In mammals, SDA

is a metabolic intermediate in the production of eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) from alpha linolenic acid (ALA), a common dietary constituent.
Refined oil produced from MON 87769 contains ap proximately 20 to 30% SDA (w t% of total

f atty ac ids) and can be included in a range of food products for health benefits

. The oil from
MON 87769 ( SDA soybean oil ) is Generally Recognized As S afe (GRAS) in the United States

and can be used for the production of products such a s margarine , shortenings, salad dressings,
ready

- to

- eat foods , and other food categories specified in the GRAS notification 1

.
MON 87769 is a specialty trait soybean, not developed for the commodity soybean market, and
it is anticipated that MON 87769 wil l be a low acreage product planned initially for production

in North America. In bringing this product to market, Monsanto will adhere to the
Biotechnology Industry Organization‟s (BIO‟s) Product Launch Stewardship policy 2 , including
its Annex 2: “Specialty Use Traits in Commodity Crops.”



### Market and Trade Assessment :

Monsanto has evaluated the potential impacts from the introduction of MON 87769 into the
marketplace. This assessment which is hereafter referred to as „ma rket and trade assessment‟ has
taken into consideration the potential impacts which could be anticipated within the value chain
due to the introduction of SDA soybean

. The defatted soybean meal from MON 87769 is
compositionally similar to other commodity defatted soybean meal and will be used in a manner similar to commodity soybean meal. The oil derived from MON 87769 , however, has a unique
fatty acid profile and will be identity preserved to maintain its value and assure its use in
appropriate food appl

ications

. Compared to commodity soybean oil, SDA soybean oil contains
two additional fatty acids, SDA and GLA. SDA soybean oil is intended for use only in certain
markets. The presence of this oil in markets where it is not intended could resu l t in pote ntial
market and trade impacts.

At the time SDA soybean oil is first introduced to the market, w e do not expect that MON 87769 will have approvals in all key soybean export markets with functioning regulatory systems.

Until s uch approvals are received, MO N 87769 will be grown and handled in a closed loop
stewardship s ystem (CLSS)

.

Monsanto will uti

lize and implement the CLSS throughout the initial product introduction phase

, at least until approvals are received from key soybean export countries with funct ioning
regulato ry systems. After such approvals are received, Monsanto will, b ased on the experiences

1 http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000283.pdf

2 http://www.bio.org/letters/Product_Launch_Stewarship_12_10_09.pdfPage 3 of 25

and learning gained during the initial product introduction phase under the CLSS, refine and
update the stewardship system for long

- term implement ation. This long

- term system will

include measures to preserve the identity of this high value product and mitigat ion measures to

minimize any impact resulting from the inadvertent comingling of SDA soybean s or oil with
commodity or other specialty soybean produ cts

.

This document describes the intended uses for SDA soybean oil, current processes for handling
vegetable oils

, and potential impacts to commodity soybean oil as well as other vegetable oils

from the commercial introduction of SDA soybean oil

. T his d ocument also describes the CLSS

which Monsanto will implement during the initial phase of the product introduction

.



### 2. Intended U ses SDA Soybean

MON 87769 is a sustainable source of omega

- 3 fatty acid for food use. SDA has fewer double

bonds than other omega

- 3 fatty acids such as EPA (20:5) and DHA (22:6) . Therefore, SDA
soybean oil is more stable to oxidation (i.e., less prone to fishy or rancid odors and taste)
compared to other oils containing EPA or DHA

, thereby expanding the potential formula tion
options for food companies and consumers. SDA soybean oil is generally recognized as safe in

the United States 3

.
SDA is a metabolic precursor to the long chain omega

- 3 fatty acids

, EPA and DHA , in humans
and animals and is found in pro ducts such as fish and fish/ algal oils. Although the benefits of

omega

- 3 fatty acid consumption are widely recognized, typical Western diets contain very little
fatty fish, while at the same time current foods and supplements provide limited alternatives to
satisfy co nsumer demand for long chain omega

- 3 fatty acids. An alternative approach to
increase omega

- 3 fatty acid intake is to provide a wider range of foods that are enriched in

omega

- 3 fatty acids so that people can choose foods that suit their usual dietary hab its. Human
and animal studies have shown that 1 g dietary SDA is approximately equivalent to 200

- 300 mg dietary EPA in terms of increasing tissue concentrations of EPA. Thus, MON 87769 can serve
as an alternate source of an omega

- 3 fatty acid to help meet the need for increased dietary intake
of long chain omega

- 3 fatty acids in food

. SDA soybean oil can be used for the production of
margarine , shortenings, salad dress ings, ready

- to

- eat foods , and other food categories described

in the GRAS notification 4

. The use of SD A soybean oil in selected food categories could
provide a wide range of dietary alternatives for increasing omega

- 3 fatty acid intake.

Given t he target ed commercial application of SDA soybean as an alternate source of omega

- 3
fatty acids in food it is anticipated that MON 87769 will be a low acreage product planned
initially for production in North America. The oil derived from MON 87769, however, has a
unique fatty acid profile and will be identity preserved to maintain its value and assure its use in
appropriate food applications. The co

- product

, soybean meal

, derived from MON 87769 has

3 http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm185688.htm

4 http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000283.pdfPage 4 of 25

been shown to be compositionally comparable to other commodity soybean meal and will be

used in a manner similar to commodity soybean meal.

As a food ingredient

, SDA soybean oil use s would be limited to those applications that

demonstrate (1) functional suitability for incorporation into food products, (2) acceptable sensory properties (no off

- flavors or bad taste) to the food, and (3) appropriate stability profile and shelf

life of food products containing SDA soybean oil during food production and storage. As
expected, SDA soybean oil from MON 87769 contains high l evels of SDA (approximately 20

- 30

wt % of total fatty acid s) . Vegetable oils containing high levels of polyunsaturated fatty acids

(PUFU s) like SDA are known to undergo rapid oxidation during frying applications and will
impart undesirable taste and od o r t o the fried foods. Therefore

, SDA soybean oil will not be

suitable for all food applications and may not be suitable as a substitute for saturated fats in food
recipes. However, SDA soybean oil can partially replace commodity soybean oil or other oils in
many food categories. As is the case with fish oils, containing omega

- 3 fatty acids,
hydrogenation of SDA

- containing oil would be without purpose, as hydrogenation would
eliminate the benefits of long chain poly unsaturated fatty acids (LC PUFA s )

. Thus,
hydrogenation is not applicable to SDA soybean oil from MON 87769. The current anticipated
food applications for SDA soybean oil are in baked goods and baking mixes, breakfast cereals
and grains, cheeses, dairy product analogs, fats and oils, fish produc ts, frozen dairy desserts and
mixes, grain products and pastas, gr avies and sauces, milk products, nuts and nut products,
poultry products, processed fruit juices, processed vegetable products, puddings and fillings,
snack foods, soft candy, and soups and soup mixes 5

.



### 3. Market Potential of SDA Soybean

Consumer demand for long chain omega

- 3 fatty acids through food and supplements is expected
to continue to grow

. As of 2008, the United States ome ga

- 3 ingredient market for marine oils

was estimated at 26,447 metric tons (MT) per year, with 13.8% of that volume being consumed

in functional foods. Furthermore, the projected average annual growth rate for this i ndustry

between 2008 and 2013 i s 13.4 % (Strategic Analysis of the North American Marine and Alg ae Oil Omega

- 3 Ingredients Market, Frost & Sullivan, March 2010 6 ). The value added product

derived from MON 87769 is SDA soybean oil. SDA s oybean oil provides increased alternatives
for food companies to formulate omega

- 3 fatty acids into a wider range o f foods. Because SD A
soybean oil can be used in wider food categories, Monsanto estimates , for the purposes of this
analysis that the industry growth of om ega

- 3 oils in functional foods c ould increase by 26.6%.

For the purposes of this analysis, Monsanto assumed that SDA soybean oil could capture 100%
of that incremental growth, which, given market dynamics, may over estimate the demand for

this oil. SDA soybean oil will typically contain 20% SDA, the expected rela tive conversion of

SDA to EPA i

n humans is estimated at about 5 :1 and marine oils in average may contai n about
30% long chain omega

- 3 fatty acids ( James et al., 2003 and Lemke et al., 2010 ) . As a result

, it is

5 FDA Food classifications 21 CFR §170.3(n).

6 http://www.frost.com/prod/servlet/report

- brochure.pag?id=N73E

- 01

- 00

- 00

- 00Page 5 of 25

expected that about 7.5 times the volume of S DA soybean oil might be needed to satisfy the EPA
equivalent amount currently provided by other marine oils. Under such a scenario, and assuming
this growth is sustained at that constant level through 2016 the incremental amount of SDA
soybean oil needed to satisfy that 26.6 % in dustry growth would be approximately 20 ,000 MT.
Furthermore, assuming an average oil yield of 0.2 MT/acre for soybean, this would equate to

around 1 00,000 acres per year of SDA soybean needed to be grown to satisfy the U.S. demand

for SDA soybean oil derived from MON 87769. It is foreseeable that global market demands for

omega

- 3 containing foods may augment the U.S. soybean acres grown with MON 87769 for the

purpose s of SDA soybean and/or SDA soybe an oil exports. The United States is t he largest
producer of soybeans and regularly exports soybean and soy bean products to global markets. In

20 10 approximately 75 million acres of soybean crop was grown in the U.S. producing
approximately 87 million MT of soybeans.



### 4. S tewardship of SDA S oybean

Monsanto is committed to product stewardship 7 and adhere to the B IO Product Launch
Stewardship Policy 8

. In compliance with B IO 's Product Launch Stewardship Policy

, Monsanto
considered Annex 2 “Special Use T raits in Commodity Crops” to deve lop launch plans for SDA
soybean including: (1) identifying relevant stakeholders for the trait and crop and engaging them

in dial ogue and outreach regarding use of SDA soybean , (2) conducting a market and trade

as sessment to evaluate the potential impact from the introduction of MON 87769 to the market
place

, and (3) i

mplementing a closed loop stewardship system to direct MON 87769 and derived
products to their proper channels and developing a risk mitigation plan

.



### 4.1. Stakeholder Dialogue and Outreach

Monsanto is committed to dialogue with key industry stakeholder groups and has held several
meetings with the National Oil Processors Association (NOPA) as well as other key industry

associations such as the North America n Export Grain Association (NAEGA), National Grain
and Feed Association (NGFA), National Agri

- Marketing Association (NAMA), American
Bakers Association (ABA), and Grocery Manufacturers Association (GMA). Soybean grower
organizations consulted include : Ame rican Soybean Association (ASA), United States Soybean

Board (USB), and many state soybean associations. Additionally, Monsanto has kept

QUALISOY , a collaborative program sponsored by USB that serves as an independent third
party resource for information on trait

- enhanced soybean oils, informed of the plans for this
product, along with dietary and nutrition thought leaders. Monsanto continues to have

conversations with , and considers the input of several soybean and food industry key
stakeholders regardin g the oil composition, stewardship plan and perfor mance of MON 87769

oil

.

7 http://www.monsanto.com/ourcommitments/pages/product

- stewardship.aspx

8 http://www.bio.org/letters/Product_Launch_Stewarship_12_10_09.pdf ;Page 6 of 25

Monsanto has been marketing specialty Vistive low linolenic soybeans to growers since 2005.
As grower and market demand for this specialty soybean product has grown, we have continu ed
to work with processors to assure adequate testing methods and quality processes are applied to
assure that the commodity soybean supply stream is not impacted

. A t a minimum, the same
outreach, education, and quality principles will be applied as we fu rther develop SDA soybean
stewardship and commercialization plan

. These quality systems are described in the following

section s

.



### 4.2 Market and Trade Assessment

Approximately 85 million MT of soybeans are produced in the U.S. annually and the U.S. is a
leading exporter of soybean , soybean meal and soybean oil to global markets

. A pproximately
12% of soybean grown is specialty soybean produced for a specific market or use 9

. These
soybean varieties are specified by buyers and end

- users of s oybean for production, and premiums
are paid for delivering a product that meets purity and quality standards of that soybean variety.
The specialty, value

- added, product may be the whole soybean or, in the case of MON 87769, a
processed fraction such as the oil. Product differentiation and market segmentation in the
specialty soybean industry includes mechanisms to keep track of the soybean (traceability),
methods for identity preservation (I D P), including closed

- loop stewardship systems, and quality

ass urance processes (e.g., ISO9001

- 2000 certification), as well as contracts between growers and
buyers that specify delivery agreements within product specifications

.

Monsanto has conducted a market and trade assessment to anticipate and consider the po t e n t i al
impacts within the value chain from the introduction of SDA soybean

, and to commodity
soybean oil as well as other vegetable oil in the market place

. Soybean is a globally traded
commodity with the U.S. being the top global p roducer 10 . Biotechnology

- de rived crops are
subject to regulation in many countries. In order to support free trade in soybean, Monsanto is
pursuing r egulatory approval for MON 87769 and any stacked products intended for

commercialization in all key soybean ex port countries with a f unctioning regulatory system.
International regulatory authorities are evaluating the food and feed safety of MON 87769 as well as the processed fractions derived from MON 87769. It is expected that uses of SDA
soybean oil would be similar on a global ba sis. Defatted soybean meal from MON 87769 is
similar to commodity soybean meal and therefore it can be comingled with commodity soybean
meal upon gaining regulatory approva l from all key soybean export countries that have
functioning regulatory systems

.
The stewardship system will be designed to prevent the comingling of SDA soybean or oil with
commodity products. The market and trade assessment supports the conclusion that i n the
unlikely event that comingling of SDA soybean or oil in the commodity stre am did occur, the
impact would be minimal. Sections 4.2.1, 4.2.2 and 4.2.3 below discuss in detail the potential

9 http://usb.adayana.com:8080/usb/jsp/login.js p . Percent U.S. Soybean acreage estimate based on U.S. Domestic Consumption by Segment

– 2008/09. D. Ludwig, personal communication, 2009.

10 http://www.soyatech.com/oilseed_statistics.htmPage 7 of 25

impacts of inadvertent comingling of SDA soybean

, oil

, and meal with commodity soybean

, oil

, and meal in the market place and show that any such comingling would have only minimal
impact to current soybean and vegetable oil markets. As dis cussed earlier, the SDA soybean and
oil are for a specialty market and are not meant to be part of the commodity chain. The
comingling scen arios and impact s are prese nted as worst case situations

. Oilseed processors and
users of vegetable oils are accustomed to the presence of numerous vegetable oils that are
currently available for various food applications. The comingling levels where an impact would

occ ur could only happe n if there were a major deviation in the system which is highly unlikely as
routine analytical methods are in place. Additionally, there is economic motivation, legal
contracts, standard operating procedures (seed quality to end user), and demonstrated
competency handling other vegetable oils of similar fatty acid profiles.



### 4.2.1 Potential Impact to Commodity Soybean in the Market Place

The impact from comingling was assessed based on the following

, scenario : An inadvertent
unloading of a tr uck containing 100% SDA soybean into the commodity stream at an elevator or
processor wher e the event goes unnoticed. This scenario was chosen because it represents a

“worst case” situation. This is unlikely to occur because a farmer producing SDA soybe ans is
financially motivated to identity preserve the SDA soybean from commodity soybean during
harvest to realize the premium paid upon delivery. Further

, comingling can be identified via
routine fatty acid analysis or QC checks before the oil is shipped from the processing facility.
All soybean oil is analyzed by gas chromatography (GC) to determine the fatty acid profile prior
to shipping. This information is placed on the “Certificate of Analysis” (CofA) accompanying
the shipment. Any commingling of commodity oil with SDA soybean oil should be identified
prior to loading for export shipment, which minimizes commingled oil arriving at an exporting

country.

For the purposes of this analysis, the following assumptions were made: t he load limit on roads
in most soybean producing states is 80,000 lbs gross weight. The empty weight of most semi
trucks is at or below 35,000 lbs, leaving a load capacity of 45,000 lbs (the difference). At 60 lbs
per bushel, a standard semi truck will haul less than 800 bushel s of soybeans. Thus, t his
commingling scenario assumes 800 bushels of SDA soybean commingled with commodity
soybean.

Upon processing of the comingled soybeans

, 800 bushels of SDA soybeans would yield
approximately 8000 lbs of refined SDA soybean oil. The capacity of the smal lest soybean oil
holding tank at U.S. processing plants is approximately 200,000 lbs. Thus the a ccidental
comingling of the truck load of SDA soybean will result in a 25X dilution of SDA soybean oil in
a 200,000 lb tank of commodity s oybean oil. In this situation, 8000 lbs of 20% SDA soybean oil

will be diluted to 200,000 lb resulting in a final SDA concentration of 0.8% SDA in commodity
soybean oil

. The presence of 0.8% SDA in commodity soybean oil will have no impact toPage 8 of 25

human health because the oil has been through the food safety assessment 11 . Impacts to food
labeling and oil performance are discussed in Section 4.2 of this document.

Another scenario would involve delivery of 800 bushels of SDA soybean to a grain eleva tor. In

this case

, the SDA soybean would be diluted with commodity soybean in a grain st orage bin that
typically h olds 100,00 0 to 500,000 bushels of soybean

, resulting in 125X to 625X dilution of
SDA soybean in commodity soybean

. The commodity soybean oi l produced from this
comingling scenario will only have SDA ranging from 0.03% to 0.16% of total fatty acids.

Soybeans are exported as whole beans or as processed fractions. S oybean oil is commonly
exported via cargo ships in containers with a capacity of approximately 40,000 lbs. These oil
filled containers are transferred from the cargo ships to trucks at the importing ports. If a
container of SDA soybean oil unintentionally ends up in a port where it is not suppose to be, it

can be identified and diver ted back to the country of origin or to another importing country. Due
to the CofA and the high value of the product, the chance of SDA in soybean oil entering into the
export market uni

ntentionally is very small.

Com m odity soybean shipped for export are typically loaded into unit trains (360,000 bu shels ) or
barges (50,000 bu shels /barge) that eventually are loaded into 2 million bushel ocean going

vessels. Before a shi

p loaded with commodity soybean reaches a destination country, the
inadvertent delivery of a truck contain in g 800 bushels would be mixed and diluted within 2

million bushels. The amount of SDA soybean in the vessel would be less than 0.04% of the tot al

amount, or 2500X dilution. The soybean oil produced from this commingled soybean will have
SDA at level below 0.008% of total fatty acids, which is the more than six time below the
CODEX limit of quantitation for fatty acids in vegetable oil (0.05%).



### 4.2.2 Potential Impact to Commodity So ybean Meal in the Market Place

Monsanto provided information to USDA in the petition demonstrating that the composition of
the meal from MON 87769 is equivalent to meal derived from conventional soybean and safe
and wholesome for food or feed applications as commodity soybean meal

. Defatted SDA
soybean meal contains approximately 1% residual oil. According to the National Oil Processors
Association defatted soybean meal should contain a minimum of 0.5% oil to meet quality
standards and guidelines for soyb ean meal from domestic and intern ational shipping (NOPA,

2006). With the exception of the presence of minor amounts of the Pj 6D and Nc 15D proteins

and de minimi s amounts of residual oil, the soybean meal and other non

- oil processed fractions

used for an imal feed and human food applications are unchanged from commodity processed
soybean fractions. Upon receiving regulatory approval for MON 8776 9 in all major soybean

export countries, SDA soybean meal can be comingled with commodity soybean meal in the

11 http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm185688.htmPage 9 of 25

ma rket place. Therefore no trade impact is anti cipated from the commingling of SDA soybean

mea l with commodity soybean meal.



### 4.2.3 Potential Impact to Commodity Soybean Oil in the Market Place

Monsanto has assessed the impac t due to the presence of SDA soybean o il on commodity and
other vegetable oils

. Areas of potential impact identified include the following : (1) food i ngredient labeling, (2)
nutritional facts panel labeling (3 ) functionality of the oil, and (4 ) sensory evaluation

. T hi s

assessment has been shared with key stakehol ders such as NOPA

, NA E GA

, ASA

, QUALISOY

, USB and GMA

.

Food Ingredient Labeling:

T he impact due to the presence of SDA soybean oil in commodity soybean oil from the
perspective of soybean oil ingredient labeling was considered. SDA soybean oil contain s two

additional fatty acids (SDA and GLA) that are not present in commodity soybean oil. Therefore

, we evaluated the potential imp act to food ingredient labeling if SDA soybean oil was
inadvertently present in the soybean oil that is added to food.

The U.S. Food and Drug Administration require s that all ingredients p resent in a food to be

declared with a few exceptions. Although the regulations do not explicitly define “ingredient”
they do discuss a class of “incidental additives” that are not considered ingredients and yet can be present in a food. An “incidental additive” is exempt if, according to the regulations, it is
present at “insignificant levels” and has no “technical or functional effect” in a food 12

. Residual
product from a previous soybean oil processing run may occasionally be mixed into a different
product in a new run. Such re sidual product would fit the concept of “i ncidental additive” 13 and

need not be included on the label. Thu s
, the unintended presence of insignificant amounts of
SDA soybean oil in soybean oil is tolerated under the food label laws and should not impact the
ingredient labeling of soybean oil or foods containing such oil.

Nutrition Facts Panel Labeling :

The FDA requires that information on fats to be declared on the nutrit i on facts panel of foods

. They are (1) t otal fat, (2) t rans fat, and (3) saturated fat. I t is voluntary for a food company to list
the amounts of monounsaturated fat and polyunsaturated fa t on the nutrition facts panel

. The
nutrition facts panel of SDA soybean oil will be similar to commodity soybean oil because the

total fat, saturated fat and trans fa t co ntent of SDA soybean oil is similar to commodity soybean

oil. M any vegetable oil pr oviders voluntarily list also monounsaturated and polyunsaturated
fatty acids present in the oil . Since i n SDA soybean oil one polyunsaturated fatty acid (LA) is
replaced with two other (SDA and GLA) polyunsaturated fatty acids ; the impact of comingling

o n nutrition fact s panel labeling from listing mono and polyunsaturated fatty acid is negligible.

12 21 CFR § 101.100

13 21 CFR § 101.100 (3)iPage 10 of 25

Even though bottled SDA soybean oil is not an intended use at this time

, a worst case assessment using 100% commo dity soybean oil was consider ed to assess the potential impact of SDA
soybean oil comingling with commodity soybean oil on nutritional facts panel labeling of bottled
soybean oil . Table 1 shows the amount of SDA soybean oil that would be needed to be
comingled with commodity bottled soybea n oil in order to significantly impact the n utrition facts

p anel for major fatty acids on bottled soybean oil

. Based on this analysis even 2 5% of SDA
soybean oil co mingled with commodity soybean oil will not significantly impact the nutritional

fact s pane l label for soybean oil. This example illustrates that any incidental presence of SDA
soybean oil in commodity soybean oil will not have any impact on the nutritional labeling of
soybean oil or SDA soybean oil containing food products

. Figure 1 shows the typical nutrition

fact s panel for commodity soybean oil (100 % soybean oil) and a theoretical nutrition facts panel

for SDA soybean oil.

Table 1

. Impact of Comingling SDA S oy bean Oil with Commodity Soybean O il on Nutritional Facts Panel Labeling for Bottled Soybean Oil

Major Fatty Acids SBO 1 SDA

SBO 1% SDA

SBO in

SBO 5% SDA

SBO in

SBO 10% SDA

SBO in

SBO 25% SDA

SBO in

SBO

Saturated Fat (g) 2.11 2.27 2.11 2.12 2.13 2.15

Polyunsaturated Fat (g) 8.35 9.05 8.36 8.38 8.42 8.52

Monounsaturated Fat (g) 3.19 2.15 3.18 3.14 2.98 2.93

1 SBO=Soybean Oil

Note: The impact of mixing SDA soybean oil (SDA SBO) on nutritional facts labeling for
bottled soybean oil (SBO) is depicted in the table above. The impact to nutritional facts
panel labeling was assessed at various percentages of SDA soybean oil mixed with
commodity soybean oil.

14 g serving size and FCC rounding rules applied. GLA and SDA included in
polyunsaturated fatty acid calculation. Mono and polyunsaturated fatty acid labelin g is
voluntary. No change in trans fat.Page 11 of 25

Fig ure 1. Nutrition Facts Panel for Commodity Soybean O il and Theoretical Nutrition

Facts Panel for SDA Soybean Oil

Note: Nutrition facts panel generated using Genesis SQL R&D software (© 2011 ESHA
Research), the same as used typically by food companies. Commodity soybean oi l exists

in the database. SDA soybea n o il‟s typical fatty acid composition was utilized to develop

a theoretical nutrition facts panel.

Oil Functionality :

Oxidative Stability Monsanto conducted a n assessment of the im pact of the oxidative stability of
commodity soybean oil comingl ed with SDA soybean oil

. Figure 2 demonstrates that
comingling of SDA soybean o il with commodity soybean oil de creases the Oxidative Stability

I ndex (OSI) of commodity soybean oil

. The OSI stability index is an indicator of soybean oil
stability. All oils and fats have a resistance to oxidation, which depends on the degree of
saturation, antioxidant and pro

- oxidant concentration, and prior abuse. Oxidation is slow until
this resistance is overcome, at which point oxidation accelerates and becomes more rapid. ThePage 12 of 25

length of time prior to the acceleration of oxidation is referred to as the „induction period

, ‟ and
the point of maximum rate change is referred to as the OSI , and is reported in hours. The

o xidative stability of soybean oil is significant ly influenced by the proportion of

monounsaturates to polyunsaturates.

To assess the impact of comingling on the OSI, the OSI of 21 commercial ly available soybean

oil as well as blends of commodity s oybean oil with SDA soybean oil were evaluated

. Commod ity/SDA soybean o il blends were prep ar ed in duplicate (wt. /wt.) using SDA soybean oil

in retail salad oil (soybean oil) obtained from a local St. Louis market. Neither oils contained

added antioxid ants. SDA soybean oil used for preparing the blends conta ined 22.9% of SDA
(%wt of total fatty acid). Oil Stability Index measurements were obtained on an Omnion OSI

instrument at 110 o C using AOCS Official Method Cd 12b

- 92

. OSI was run in duplicate for each
blend sample, and means were averaged to obtain the d ata points shown. Target blend accuracy
in the commodity/SDA soybean oil blends was confirmed by fatty acid composition determined
using capillary gas chromatographic (GC) analysis (data not shown). The “Commercial soy”
range shown in Figure 2 represents 21 unstabilized salad oils that were obtained at retail markets
in St. Louis over a 3 year period and analyzed for OSI within 2 weeks of purchase. Oils were
selected with „best by...‟ dates of 6 to 24 months from date of purchase. The OSI values of thes e

oils range d from 5.63 to 9.68 with a median value of 6.66. SDA soybean oil is estimated to have

decreased oxidative stability compared to conventional s oybean oil (Figure 2)

. Based on this
study it is reasonable to conclude that minor levels of SDA will not impact the OSI index of
commodity soybean oil . At comingling levels of 12.5% of SDA soybean oil in commodity
soybean oil, the OSI index fall s below the range observed for commercial soybean oils.Page 13 of 25

Figure 2. Oxidative Stability I ndex (OSI) of S DA Soybean Oil Blended with Commodity

S oybean O il

Heat T reatment Vegetable oils containing high levels of PUFA are known to undergo rapid

oxidation dur ing frying applications and are known to impart undesirable taste and od or to the
fried foods. SDA soybean oil contains relatively high levels of PUFA s compared to other
vegetable oils

, and therefore , is not in tended for frying applications. However, a s part of the

evaluation of the potential impact of SDA soybean oil on the functional properties of co mmodity
soybean oil, the influence of SDA soybean oil on the ability of fry oil to withstand heat treatment

was determined. From a practical standpoint, commercial fryers use the level of polar materials
formed during frying as a measure of oxidation and to determine the fry life of oil. Total polar

material values are often used to determine when the oil has deteriorated to a point where the oil
can no longer be used (Orth eo fer and List, 2007) . When the polar material reaches the threshold

level of 24%, the oil is typically discarded and fresh oil is used. Polar compounds are
by products that are generated as the oil degrades in quality. A common means of measuring
total polar material in oils is by the use of the Ebro Food O il Meter (Ebro Intern ational, Lino

Lakes, MN). As oil degrades during f rying, there is an increase in the polar materials that affect
the dielectric constant of the oil. Measurements are taken of the fry oil throughout the length of
the fry study to dete rmine when the oil has reached 24% total polar material (TPM), which
typically indicates the need to dis card the oil. Figure 3 show s the time in hours that it took for

ea ch SDA soybean oil/ commodity soybean oil blend to reach 24% TPM during frying

. ThePage 14 of 25

r esults indicate within the range of blends tested the level of SDA soybean oil had m inimal
impact on the formation of total polar materials in the oil.

Figure 3. Hours to Reach 24% Total Polar Material During Frying Application of Soybean
Oil Containing Varying Amounts of SDA Soybean Oil

Food Sensory Assessment :

A s ensory assessment of SDA soybean oil was performed to evaluate cons umer acceptability of

SDA soybean oil. Sensory assessment has concluded that the sensory attributes of SDA soybean
oil is comparable to commodity soybean oil. Even though SDA soybean oil is not intended for
frying application, sensory results for SDA soybean oil in the most challenging environ ment of
high temperature food frying application s was evaluated as a worst case scenario

. To determine
the impact SDA soybean oil on the quality of foods prepared from blends of SDA soybean oil
and commodity soybean oil, f rench fries were prepared. The f ive oil blends (ranging from 2.5 to
15% SDA soybean oil) and commodity soybean oil were heated continuously for over 40 hours.
During this time, batches of f rench fries were cooked in each of the oil samples and one batch
each day of frying was evaluated for the difference in sensory properties from a control (100%
commodity oil) using the Sensory Quality System method (King et al., 2002). Each panelist was
provided a control fry and test fry and asked to rate the overall quality score (overall difference )
along with the differences in individual French fry attributes. A rating of 5 indicates no
difference, 4 slight difference, 3 difference, 2 significant difference and 1 extremely different.
Figure 4 shows the results from this study. The outcome from the sensory e valuation showedPage 15 of 25

that up to 1 5 % of SDA soybean oil can be co mingled with commodity soybean oil without
impacting the flavor perception of fried food (Figure 4)

.Page 16 of 25

Figure 4

. Sensory Results from Frying Application of Soybean oil Containing Varying Amounts of SDA Soybean Oil

.Page 17 of 25

The market and trade assessment has concluded that i n the unlikely ev ent comingling did occur,
the impact to labeling and functionality o f commodity soybean oil would be minimal. Monsanto

will have an appropria te stewardship plan in place to direct MON 87769 and oil to its intended
markets, both in its initial commercial phase when produced and used under CLSS as well as
longer

- term

.



### 4.3 Risk Mitigation Plan

4.3 .1 Stewardship Plan for MON 87769

- Closed Loop Stewardship System

To mitigate potential impacts to commodity soybean , a Closed Loop Stewardship System
(CLSS ) has been developed to support the comm ercialization of SDA soybean prior to obtaining
approvals in key soybean export markets with functioning regulatory systems . The CLSS
defines procedures and processes relevant to the production, handling, and processing of all

stewarded ma terials generated within the closed loop . S tewarded materials inclu de viable
soybean seed and grain , meal, hulls, oil, and downstream co

- products derived from the crude oil
that are in direct control of Monsanto, its licensees or partners, and their customers. Monsanto

w ill continue to utilize th e CLSS through the initial produ ct introduction phase until necessary global approvals are received from key soybean export countries with functioning regulatory

systems . After the approvals are received, Monsanto will refine and update the stewardship

sys tem for long

- term implementation based on the experience developed during the introduction
phase and in consultation with key stakeholders.

Under the CLSS, Monsa nto will develop and implement best p ract ices and system s consistent

with the Excellence Thro ugh Stewardship 14 initiative. Monsanto will carefully evaluate
capabilities of partners and downstream entities to contain downstream products to their intended
market(s) and include terms and conditions in agreements with those partners or entities. These
agreements wil

l require that appropriate quality management systems (QMS), including both
quality assurance (QA) and quality control (QC), are in place to manage and verify containment
of all stewarded materials in countries of production and use.

Monsanto will only wor k with downstream partners that have proven traceability, identity

prese rvation, and CLS S production capabilities. Monsanto

, its licensees, partners and p rocessors will implement an annual auditing process for material under their control . Commercial

pro duction of soybean seed for planting and harvested soybean and the subsequent processing or
manufacturing of stewarded end use products requires strict adherence to established processes

under the CLSS to ensure each material is handled and used appropriat ely

. If any part of the
process is contracted out to a third party, Monsanto will require a Stewardship Management Plan

that meets CLSS standards. Monsanto reserves the right to audit these plans dire ctly or through
a third party.

14 Excellence Through Stewardship (ETS) is an initiative to promote the global adoption of stewardship programs and quality management systems for the full life cycle of biotechnology

- derived plant products

.Page 18 of 25

Activities under the CL SS are organized into five relevant sections identifying critical control
points: (1) Production of SDA Soybean S eed for Planting

, (2) Production of SDA Soybean for
Processing , (3) SDA Soybean Processing and Oil Refining, (4) SDA Soybean Oil Packaging and
Storage, and (5) SDA Soybean Oil Distribution to Food Company Supply Chain. The
information presented in each section describes the purpose, scope, and procedures necessary to
conduct and verify the proper handling and use of all relevant materials under the CLSS

.
Figure 5. Parts of the Closed Loop Stewardship System and Critical Control Points

The requirements and processes for activities at each of these control points under a CLSS are consistent with Monsanto stewardship guidelines. These guidelines include processes to prevent
commingling with commodity soybean seed, soybean , and oil (e.g. isolation, equipment cleaning
and segregated storage). All Monsanto and non

- Monsanto perso nnel involved with pr oducing

material under the CLSS are required to follow these guidelines and requirements

.

Detection Methods Monsanto has developed event

- specific detection methods to detect the

MON 87769 event in article s of commerce including soybean seed, harvested soybean, soybean

meal and oil . More importantly, Monsanto has developed analytical methods to assess the fatty
acid profile for SDA soybean oil as well as detect the presence of SDA in vegetable oil

. Monsanto will provide these methods t o the industry upon request. Oil processors and
downstream distributors are accustomed to using oil analysis methods during their normal course
of business. Fatty acid profile analyses of soybean oil are considered routine within the i ndustry.Page 19 of 25



### Production of SDA Soybean Seed for Planting :

Monsanto is a leader in crop biotechnology having successfully introduced numerous

biotech nology

- derived crops to the marketplace globally. Monsanto has developed and
implemented seed quality s tandards and prac tices to validate soybean seed meets the standards
established for purity of a trait. These standards apply to all soybean seed sold by Monsanto and
are based upon measures that seed producers put in place to assure the genetic purity of
improved planting seed. This system is used to assure that farmers receive seed of known
quality with a m

inimum level of off type seeds.

The first step in production of SDA soybean is the production, processing and delivery of high
quality parent seed to the grower. Mons anto‟s seed manufacturing organization uses specific
SOPs and documentation forms to ensure compliance with Monsanto stewardship and
compliance standards. The entire seed production process at the majority of the seed companies
and tollers operates using International Organization for Standardization 15 (ISO) certification
standards and include internal and external audits (ISO, 2009). By following ISO qu ality
standards it is possible to validate that the processes are followed which have been designed to

g enerate the desirable characteristics of seeds and services such as quality, safety, reliability, and
efficiency. The ISO standards represent an international consensus on good management
practices with the aim of ensuring that the organization can consis tently deliver excellent product or services. The standards not only must meet the customer‟s requirements and applicable seed
regulatory requirements, but also must aim to enhance customer satisfaction and achieve
continual improvement of its performance in pursuit of these objectives (ISO, 2009).

Commercially certified soybean seed must meet state and federal seed standards and labeling
requirements. The Association of Official Seed Certifying Agencies (AOSCA) standards for
certified soybean seed are as follows: 98% pure seed (minimum), 2% inert matter (maximum).
The inert matter can contain up to 0.05% weed seed (maximum, not to exceed 10 per lb.), 0.60%
total of other crop seeds (maximum), 0.5% other varieties (maximum, includes off

- colored beans

and off

- type seeds), 0.10% other crop seeds (maximum, not to exceed three per lb.), and have
80% germination and hard seed (minimum) (AOSCA, 2009). Seed that meets or exceeds these
standards are provided in appropriately labeled seed bags to growers. Monsan to‟s seed
manufacturing practices for MON 87769 will be designed to contain MON 87769 within the
boundaries of the production system thereby assuring that levels of the trait and the subsequent
SDA and GLA fatty acids do not cause impacts to conven tional s oybeans or other commodity
soybean varieties produced at seed manufacturing sites.



### Production of SDA Soybean for Processing :

SDA soybean will be produced under contracts similar to those currently issued by processors or
elevators for commercial special ty soybean such as f

ood grade, organic, Vistive low linolenic or other specialty soybeans.

15 http://www.iso.org/iso/ims_2009.h tmPage 20 of 25

The income opportunity is achieved b y the farmer only when the soybean is delivered to the
processor within specifications. Therefore, the motivation is a financial i ncent ive for the farmer

to avoid com

ingling with commodity soybean, keeping SDA soybean i dentity preserved within
system of seed

– farmer

– processor. Many farmers may willingly choose to plant all their
acreage to specialty soybeans because it eliminates any risk of com ingling that may occur during

the harvest. If a farmer choose s to produce the specialty soybean for the processor, the farmer
will arrange to store the soybean on farm or at their local elevator, if the elevator is participating
in the specialty program with the processor.

The steps involved in securing soybean production are:

1. Monsanto, its licensees, partners or p rocessors will be responsible to issue productio n

contracts. These contracts will be either to an elevator or directly to the farmer. If issued

to an elevator, the elevator will in turn issue contracts with farmer customers in the
amount of acreage established in its processor contract. The contract will include number

of acres and the timeline for the delivery of harvested soybeans

. 2. Monsanto, its licensees or partners will distribute SDA soybean seed to the farmer after
verifying that the farmer has a valid contract that has b een issued for SDA soybe an production and stewardship in their operation. This will include an amount of seed
needed to plant the number of acres established on the production contract by the
processor or elevator.

3. Monsanto, its licensees or partners will work with all parties i nvolved to confirm and
reconcile acres contracted with seed sales. Processors can plan processing schedules
according to demand of the oil and supply of SDA soybean anticipated from contracted

farmers.

4. After harvest, t he farmer will deliver the harvested SDA soybean to the location specified
in his contract, either a participating elevator or processor. If delivered to the elevator,
the elevator will keep the SDA soybean segregated from commodity soybean and pay the
farmer any premiums as applicable, prov ided it passes the analytical testing. The
elevator will deliver the SDA soybean to the processor as delivery windows and crush
schedules have been established. Upon delivery of SDA soybean by the farmer, samples
will be analyzed from every truckload. T his is to confirm th e soybean contains the SDA

trait as required by the production contr act. Upon confirmation that the SDA soybean
meets specification, the processor or elevator will approve the premium payment to the

fa rmer. SDA soybean that does not meet specifications at the elevator or processor will
be segregated, processed and the oil will be directed to appropriate uses such a s for the
production of biofuel

.

Soybean Grain E levator Grain elevators play an important role in specialt y programs with thei r long term storage of the soybeans

. Because processing facilities crush soybeans throughout the
year, soybeans used to supply these crush plants need to be stored year round. Farmers typicallyPage 21 of 25

prefer to empty their storage prior to pl anting of the new crop and prior to temperature warm up
in the spring. The warm spring and summer weather present s challenges as condensation can
build up in the bins creating m oisture

- r elated issues that make soybean

- eating insects more

active. To avoid this, c ommercial elevators have expert grain ma nagers on staff to monitor
soybean quality and keep soybean in good condition in all weather situations throughout the
year. Commercial grain elevators are al so better equipped to ship soybeans to processors du ring

times of severe weather even when f armers cannot get to their bins

.
Processors will therefore enter into supply contracts with commercial grain elevators and work

with farmers to establish inventories and to as sure proper routing of SDA soybean through the
supply chain. The elevator pay s the farmer the specialty soybean premium upon successful
analytical testing performed at the elevator location. Testing equipment will be provided as
needed by Monsanto to participating elevators

.
In order for the elevator to be reimbursed for the premiums paid out to farmers that have

delivered SDA soybeans to them, the elevator must in turn pr eserve the identity of the soybean
as it is delivered to the processor. Every load delivered to the proc essor by the elevator will be
analyzed to make this determination. Processors will approve the premium payment to the
elevator after analysis of th e soybean to confirm the SDA trait

. SDA soybean that does not meet
specifications at the elevator or proces sor will be segregated, processed and the oil will be
directed to appropriate uses such a s for the production of biofuel

. 

### SDA Soybean Processing and Oil Refining :

All SDA soybean processors must be approved in advance by Monsanto. In order for the
processor to be approved by Monsanto to process SDA soybean , certain requirements must be
met. They must posses the ability to store, segregate and ide ntity preserve SDA soybeans . They
must demonstrate the ability to clean the equipment associated with t he receiving and subsequent
handling of SDA soybeans . The processo r‟s facility must have procedures in place and the
ability to assure that SDA soybean processing, extracting, and oil refining equipments can be

flushed to commercially acceptable stan dards after processing SDA soybeans

. Monsanto and the

processor will enter into an agreement to establish the terms and conditions of SDA soybean

pro cessing services. These agreement s will also include terms for distribution of SDA soybean

co

- products resulti

ng from processing as stewarded products within the Unite d States prior to
receiving necessary regulatory approvals in soybean export markets . The resulting oil from
processing SDA soybean is generally recognized as safe in the United States

.
Upon the t ransition from commodity soybeans to the SDA soybeans, a sample of crude oil is
required to confirm the presence of the appropriate fatty acid composition (FAC) unique to SDA
soybean oil. Crude oil will be collected and handled in a way that ensures that any SDA soybean

oil as well as SDA soybean oil com ingled with commodity soybean oil are handled in an
appropriate manner and segregated from other oils. This will include all co mingled oil before

and after the collection of SDA soybean oil. Testing metho ds will be used to aid in identifyingPage 22 of 25

the appropriate time to start and stop collecting crude SDA soybean oil . Storage t anks approved
for SDA soybean oil will be assigned for the storage of crude and refined SDA soybean oil.
Upon the completion of SDA so ybean processing, a commodity soybean flush will be initiated.

After all SDA soybean and commodity soybean used to flush the channels have been processed,
crude oil exiting the extractor will be sampled and analyzed to confirm the equipment has been

flush ed to an acceptable level, yielding commercially acceptable commodity soybean oil. Off

spec, commingled or flush SDA soybean oil will be segregated from commodity soybean oil and
directed to appropriate us e such a s for the production of biofuel

.



### SDA Soybean Oil Packaging and Storage :

Soybean oil produced under the CLSS will be distributed as a stewarded product w ithin the

United States until receiving approv als in all key soybean export markets. An agreement will be

executed between Monsanto, its licensees, partners, the processor and/or the packager to
establish the terms and conditions for packaging stewarded SDA soybean oil at a contract
packaging facility. A separate agreement will be executed with a long

- term storage facility

, if

used

, to establish the terms and conditions for SDA soybean oil receipt, storage, and distribution.

Bulk liquid semi

- trailers (tankers) will be used to transport packaged and/or bulk SDA soybean
oil. Refined, bleached, and deodorized (RBD) SDA soybean oil packa ging will be conducted in
an area that is clean, secure, and segre gated to eliminate potential co mingling with commodity
soybean and/or other vegetable oil s . The containers will be properly labeled using
predetermined methods that will include chain of cu stody documentation. Periodic visual
inspections will occur in and around the packaging area for the collection and disposal of any
spilled oil or damaged containers. All semi

- trailers used in the transportation of bulk SDA
soybean oil will be verified t o be clean at the processing plant or other Monsanto approved
facility prior to being released . After packaging is complete, the facility will be flushed with
adequate amounts of commodity soybean oil to remove SDA soybean oil and return the oil

stream to commodity soybean oil specifications. Flush oil containing SDA soybean oil will be
segregated and directed to appropriate us e such as for the produ ction of biofuel

.


### SDA Soybean Oil Distribution to Food Company Supply Chain :

Monsanto, its licensees and/or partners will execute agreement s with food companies to estab lish

the terms and conditions for the use of stewarded SDA soybean oil. During the development of
these contracts, it will be necessary to verify that the food companies have the capabilities in
place to control and limit distribution of stewarded products and track all products that use
specific ba tches of the SDA soybean oil as ingredient. These contracts will include stewardship
obligations and will require compliance including auditing. T hird party consultan ts will be used
if necessary under the CLSS. These third party consultants will have ex pertise in supply and demand chain procedures, to enable verification of a food company‟s capabilities.

SDA soybean oil will be used by the food industry and will be supplied to specific customers by
Monsanto, its licensees or partners and suppliers afte r verifying that it meets specific customerPage 23 of 25

requirements including quality factors. From the time the oil is packaged until it is utilized at the
speci fic customer‟s facility there will be proper identification of the oil through labeling and

manufacturin g codes allowing for sufficient product traceability. Individual facilities will utilize
proprietary inventory and ordering systems that are in place to insure that the appropriate oil is

ordered, delivered and utilized.

Food companies will use SDA soybea n oil as a food ingredient consistent with the G RAS

notification 16 . Each individual food company has in place systems for ingredient (oil) ordering,
receipt, storage, access and lot identification at spe cific manufacturing locations, as well as
finished pr oduct (the food which incorporates SDA soybean oil) batch identification,
manufacturing facility, storage, shipment to distribution centers, customer order picking,
customer order shipment and receipt. Appropriate proced ures are currently in place to e nsu re traceability from receipt of the ingredient through distribution to a specific retailer‟s facility

. Food manufacturing facilities also comply with federal and state requirements for good
manufacturing practices and product traceability. Supply chain c onsultants can be employed to
confirm appropriate systems have been established that meet ingredient and product traceability
requirements.



### 4.3 .2 Incident Response

All activities conducted by Monsanto and non

- Monsanto pers onnel under the scope of the CLSS

must be consistent with Monsanto ‟s corporate expectations for regulatory and stewardship
compliance prior to obtaining all relevant global regulatory approvals. Monsanto has an incident
response policy in place. If an unforeseen process failure or a bre ach of containment of

st ewarded material occurs, the CLSS incident response would be managed according to
Monsanto‟s Global Product Stewardship and Quality process and procedures document. The
Incident Response Process engages a multi

- disciplinary team for immediate response
investigation, assessment and mitigation of any potential adverse impacts that could result from
the process failure. T he incident investigation and analysis process will determine the reason for
the occurrence and develop r ecommenda ti ons for corrective action including process
improvement to prevent similar occurrences in the future.



### 4

. 3 .3 Development of Long Term Stewardship Processes

The CLSS described above to support the commercialization of SDA soybean oil prior to
obtaining al

l relevant regulatory approvals will be utilized through the initial product
introduction phase until all relevant global ap provals in key soybean export countries with
functioning regulatory systems are received

. Monsanto will refine and update the stewa rdship
system for long

- term implementation based upon the experience an d learning developed during

the CLS S production phase and through continued dialogue with stakeholders. This dialog will

16 http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000283.pdfPage 24 of 25

aid in the assessment and development processes while enabling transparent communication
regarding policy implementation. The long

- term stewardship plan for MON 87769 der ived
through these actions will be designed to ensure that the SDA soybean oil is directed to its

intended specialty market.



### 5

. Summary

SDA soybean offers U

. S

. soybean growers the opportunity to supply a value added and

sustainable omega

- 3 alternativ e that will provide food industry with choices to satisfy a growing

omega

- 3 mar ket

. The closed loop stewardship system for the production and handling of

MON 87769 and derived product s is designed to ensure that SDA soybean and oil will be
isolated from commodity soybean and oil as well as other vegetable oils in the market place and
are directed to their intended specialty uses

. The market and trade assessment has concluded that

i n the unlikely ev ent comingling did occur

, the impact to labeling and functionality would be

minimal. Because SDA soybean is generally recognized as safe in the United States 17 and the

defatted meal fro m SDA soybean is similar to commodity soybean meal, there would be no
negative impact to human heath from the use of any of the other processed fractions produced
from soybean due to comingling of SDA soybean with commodity soybean. G iven the

abundance of vegetable oils on the market and demonstrated ability of the system to adapt to consumer preferences incorporating new oils into existing food manufacturing processes, the

introduction of SDA soybean is expected to be easily managed by existing industry m echanisms
and identity preservation systems . Monsanto continues to engage stakeholders and educate them

on the benefits of SDA soybean s and proper stewardship pra c tices

.

17 http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm185688.htmPage 25 of 25



### References

James, M.J., V.M. Ursin, and L.G. Cleland. 2003. Metabolism of stearidonic acid in human
subjects: comparison with the metabolism of other n

- 3 fatty acids. American Journal of Clinical

Nutrition 77:1140

- 1145.

Lemke, S.L., Vicini, J.L., Su, H., Goldstein, D.A., Nemeth, M.A., Krul, E.A., and Harris, W.S.

2010. Dietary intake of stearidonic acid

– enriched soybean oil increases the omega

- 3 index:

randomized, double

- blind clinical study of efficacy and safety

. Am J Clin Nutr 92:766

– 75.

NOPA. 2006. Trading Rules for the Purchase and Sale of Soybean Meal. National Oil seed
Processors Association, Washington, D.C.

Orthoefer, F. T. and List, G. R. 2007. Evaluation of Used Frying Oil in Deep Frying: Chemistry,
Nutrition and Practical Applications, 2 nd Edition. Erickson, M.D. Editor. p 329

- 342.

King, S. et a l. 2002. The sensory quality system: a global quality control solution. Food
Quality and Preference (13) 385

- 395.