USDA/APHIS/BRS Petition # 09-183-01p 

February 4, 2010 Monsanto Company 09-SY-200U 

Attachment 

Clarifications and Corrections to Petition #09-183-01p for the Determination of Non-regulated Status for M

Page, Section 

(changes) 

Existing Text or Information Revised Text or Infor

Page 108, Table VII-2 (Removed duplicated numbers following subheading on the 4th row from bottom) 

Seed Fatty Acid (% Total FA) 12.31 12 2.51 0.024 [12.24-12.39]

Seed Fatty Acid (% Total FA) 

Page 110, Table VII-2 (Subtitle inserted in between the 4th and 5th rows from the bottom)

Genistein Site OH Seed Amino Acid (% DW) Aspartic Acid Site OH Glutamic Acid Site OH Histidine Site OH 

Genistein Site OH Statistical Differences Observed in Seed Amino Acid (% DW) Aspartic Acid Site OH Glutamic Acid Site OH Histidine Site OH 
USDA/APHIS/BRS Petition # 09-183-01p 

February 4, 2010 

Monsanto Company 09-SY-200U Page 1 of 20 

Response to Review for Completeness and Acceptability of Monsanto 

Petition Number 09-183-01p for a Determination 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 Monsanto 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 extent 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 using MegAlign in the Lasergene program. As 

described on page 81 of the petition, the MON 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 9387 through 9429, corresponding 

to base pairs 934-976 of the insert) were from the Right Border region that is present in the 

transformation vector. This analysis also confirmed that the sequence of the insert in 

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

The MON 87769 insert, beginning at base 934 and 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 corresponding sequences in PV-GMPQ1972. 

USDA Question #2 (General Issue): Please supply study data or other information related to 

the stability of the fatty acids in MON 87769 soybean during storage. Please compare this to 

the stability of fatty acids in commodity soybeans (without altered fatty acid profiles). 

Monsanto Response: MON 87769 harvested seed was stored 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 analysis 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. Table 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, 1993). From the data presented in Table 1 and 

Table 2, it is concluded that the storage stability of MON 87769 soybean is comparable to the 

storage stability of conventional soybean.
USDA/APHIS/BRS Petition # 09-183-01p 

February 4, 2010 

Monsanto Company 09-SY-200U Page 2 of 2

Table 1. Fatty acid analysis of MON 87769 harvested seed stored at room temperate over 20 months 

Fatty acid composition (% 

of total fatty acids) 

0 

Month 

3 

Months 

6 

Months 

9 

Months 

14 

Months 

20 

Months 

C16:0 (Palmitic) 12.55 12.48 12.91 11.45 11.67 11.98 C18:0 (Stearic) 4.13 4.15 3.89 4.10 4.18 4.28 C18:1 (Octadecenoic) 18.45 18.19 17.64 18.70 18.34 18.66 C18:2n6 (Linoleic) 28.14 27.84 28.14 28.12 28.42 28.43 C18:3n6 (Gamma linolenic) 5.27 5.37 5.71 5.23 5.54 5.25 C18:3n3 (Alpha linolenic) 11.20 11.30 11.28 11.61 11.17 11.16 C18:4n3 (Stearidonic) 18.69 19.02 19.30 19.19 18.90 18.41 C20:0 (Arachidic) 0.32 0.33 0.28 0.36 0.36 0.49 C20:1n9 (Eicosenoic) 0.22 0.26 0.18 0.25 0.22 0.24 C22:0 (Behenic) 0.28 0.30 0.24 0.32 0.33 0.33 
USDA/APHIS/BRS Petition # 09-183-01p 

February 4, 2010 

Monsanto Company 09-SY-200U Page 3 of 2Table 2. Fatty acid analysis of conventional soybean harvested seed stored at 20 °C over 12 months1

Fatty acid composition 

(% of total fatty acids) 

0 

Month 

1 

Month 

2 

Months 

4 

Months 

8 

Months 

12 

Months 

C16:0 (Palmitic) 11.0 11.1 11.3 11.7 12.3 12.7 C18:0 (Stearic) 4.1 5.2 6.9 8.9 13.8 16.1 C18:1 (Octadecenoic) 25.2 24.9 24.7 24.3 23.0 22.3 C18:2n6 (Linoleic) 49.5 48.7 48.0 46.9 43.9 41.0 C18:3 (Linolenic) 8.9 8.3 7.1 6.5 5.7 4.9 C20:0 (Arachidic) 0.1 0.05 ND ND ND ND C20:1n9 (Eicosenoic) 0.1 ND ND ND ND ND 

ND 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 lipase and lipoxygenase activities 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 7th 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 cited in the petition. While we recognize 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, because it was cancelled and a letter of 

cancellation was sent to the USDA on April 11, 2006. If USDA/APHIS records indicate other 

field report(s) are missing, please let us know so that we can provide a copy of any missing 

field report(s). 

USDA Question #4 (General Issue): Standard deviation (or standard error) values are 

presented for some analyses in the petition, but not others. Those statistics are useful for 

reviewers to make inferences about data presented 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 presented 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, G5, 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 Petition 09-SY-200U) to show the significant 

differences among analytes between MON 87769 and the conventional soybean control 

A3525. Tables E-1 - E-12 and E15 – E18, with the 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 detailed 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 Paragraph, 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 reference varieties included 

in the study for the AOSA temperature regime (20/30 oC). The lack of significant differences 

in germination between MON 87769 and conventional soybean control demonstrate that the 

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

data are indicative of no increased 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 sentence. How is it that the lack of significant 

differences between MON 87769 and the control demonstrate that the observed values of 

MON 87769 are within the range of values expected of soybean? The sentence before states 

that both MON 87769 and the control had germination means outside of the range of reference 

varieties included in the study. 

Monsanto Response: To address the question, paragraphs 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 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 germinated seeds were detected between MON 87769 and the conventional control. 

Furthermore, the germination of both MON 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 commercial soybean variety, and were within the 

range of accepted germination values for certified soybean seed.

The biological characteristics evaluated were used to characterize MON 87769 in the context 

of a plant pest risk assessment. 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 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 

indicative of no increased plant pest or weediness potential of MON 87769 relative to 

conventional soybean. 

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

states: 

“The presence or absence of MON 87769 in the test, control, and reference seed samples was 

verified by event-specific polymerase chain reaction. 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 sites 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 samples from the isolated plots were low and 

did not negatively affect the quality of the study or interpretation of the results.” 

Please explain how MON 87769 may 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 characteristics were assessed on seed of MON 87769, the 

conventional soybean control, and the reference soybean varieties produced at the Jefferson 

County, IA; Stark County, IL; and Boone County, IN sites in the 2006 field trials 

(Appendix G). The field trial at each site was established in a randomized complete block 

design with three replications. The seed of MON 87769, the control, and reference varieties 

were harvested from all three replicated plots at each of the three field 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 

Type1 

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. The 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 absence of MON 87769 in the 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 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 

plots and from five out of a total of 36 reference plots across the three field trials contained 

between 0 and 1.84% MON 87769. In addition, seed 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 varieties were all produced from the same 

replicated field trials as MON 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 plots were low and did not negatively affect the 

quality of the study or interpretation 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 MON 87769. For the 20/30 0C 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 conducted at BioDiagnostics, Inc. in River Falls, 

WI. The principal investigator was certified to conduct seed dormancy and germination 

testing consistent with the standards established by the Association of Official Seed Analysts 

(AOSA), an internationally recognized seed testing organization (AOSA, 2000; AOSA, 2006). 

Seed materials of MON 87769, the control, and four 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 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 oC. The alternating 

temperature regimes were maintained at the 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 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, abnormal 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 periodically during the study for 

germinated, hard (viable and nonviable), dead, and firm-swollen (viable and nonviable) seed. 

H.5. Individual Site Seed Dormancy and Germination Results 

MON 87769, the control, and reference seed materials were produced at three sites to assess 

germination characteristics of seed grown under various environmental conditions. The 

individual site data in Tables 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 higher percent abnormal 
USDA/APHIS/BRS Petition # 09-183-01p 

February 4, 2010 

Monsanto Company 09-SY-200U Page 8 of 20 

germination at the 20/30 oC 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 regimes 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, 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 # 09-183-01p 

February 4, 2010 

Monsanto Company 09-SY-200U Page 9 of 2Table H-1. Germination Characteristics of MON 87769, the Conventional Control, and the Reference SoyProduced from each of Three Field Sites and Tested in the AOSA-Recommended Temperature Regime  Mean % (S.E.)1  IA IL Temp. Regime2 

Germination Category3 MON 87769 Control 

Ref. 

Range4 MON 87769 Control 

Ref. 

Range4 MON 87769 

20/30 °C (AOSA) 

Normal Germinated 81.5 (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) Abnormal Germinated 18.0 (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) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0)  Dead 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) 

Viable Firmswollen 0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0) 0.3 (0.3) 0.0-0.0 0.0 (0.0) 

Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed wchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete block and seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized deseed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the convention

sites. 

1 Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the contr

numerical rounding of the means. S.E. = Standard Error. 

2 Temperature Regime. In the alternating 20/30 °C temperature regime, the lower temperature was maintained for 16 hours and the hi

hours.

3 Germinated seed in the AOSA-recommended temperature regime were categorized as either normal germinated or abnormal germinated 

4 Minimum and maximum mean values from among four commercial reference soybean varieties produced from replicated field trials at 
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February 4, 2010 

Monsanto Company 09-SY-200U Page 10 of Table H-2. Germination Characteristics of MON 87769 and the Conventional Control Seed Produced from 

Field Sites and Tested in the Additional Temperature Regimes 

Temperature Regime Germination Category 

 Mean % (S.E.)1

 IA IL IN

 MON 87769 Control MON 87769 Control MON 87769 

10 °C Germinated 100.0 (0.0) 100.0 (0.0) 99.5 (0.3) 100.0 (0.0) 100.0 (0.0)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0)  Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

20 °C Germinated 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

30 °C Germinated 99.8 (0.3) 99.3 (0.5) 99.8 (0.3) 99.8 (0.3) 99.5 (0.3) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.3 (0.3) 0.8 (0.5) 0.3 (0.3) 0.3 (0.3) 0.5 (0.3) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed wchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete block and seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized deseed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the convention

sites. 

1 Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the contr

numerical rounding of the means. S.E. = Standard Error. 
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Monsanto Company 09-SY-200U Page 11 of Table H-2 (continued). Germination Characteristics of MON 87769 and the Conventional Control Seed Produced fField Sites and Tested in the Additional Temperature Regimes Temperature Regime1 Germination Category 

Mean % (S.E.)2

 IA IL 

 MON 87769 Control MON 87769 Control MON 87769 

10/20 °C Germinated 99.5 (0.5) 99.5 (0.3) 99.8 (0.3) 100.0 (0.0) 100.0 (0.0)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.5 (0.5) 0.5 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

10/30 °C Germinated 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed wchambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete block and seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized deseed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the convention

sites. 

1 In the alternating temperature regimes, the lower temperature was maintained for 16 hours and the higher temperature for eight hours. 2 Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the contr

numerical rounding of the means. S.E. = Standard Error.
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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-GMPQ1972 transformation vector sequence (bottom 

sequence). The MON 87769 insert sequence represents base pairs 934-8300. The 

PV-GMPQ1972 sequence represents base pairs 9387-288. Base pairs 1-933 consist of 

genomic DNA flanking the 5’ end of the insert that did not align with sequences in the 

transformation vector. 

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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 (dated 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 financial 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 available to the public by the 

provider. Critical Mass Energy Project v. NRC, 975 F.2d 871 (D.C. Cir. 1992) 

(“financial or commercial information provided to the Government on a voluntary basis 

is ‘confidential’ for the purpose of exemption 4 if it is of a kind that 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 can be said to be 

the end product of either innovation 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 substantial 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 squarely within this definition, and is the 

type of information accorded trade secret protection by the courts under Exemption 4 of 
Soybean MON 87769 

Monsanto Document #09-SY-200U 

Page 2 of 4

the FOIA request. The courts have been very 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 biotechnology submissions within the boundaries of 

these statutory and court interpretations 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 Secrets” and “Commercial or Financial Information.” “Trade 

Secrets” are, in turn, defined as: “information relating to the production process. This 

includes production data, formulas, and processes, 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 “Commercial or Financial Information will also be 

deemed confidential if review establishes that 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

BIOTECHNOLOGY USER’S GUIDE, GENERAL DOCUMENT PREPARATION GUIDELINES FOR 

SUBMISSION 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 protected 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 valuable information. 

Monsanto is at the leading edge in the development of biotechnology products in a 

rapidly growing and highly competitive industry. Monsanto faces a number of strong, 

multinational competitors in this field, including BASF, Bayer, Dow AgroSciences, 

DuPont and Syngenta. Monsanto’s competitors, 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 development 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 costs. Monsanto can document the development 

and testing costs by means of monthly summaries of the worker hours devoted to these 

projects, budgetary documents, field test agreements and project documents. 

Presently, Monsanto’s competitors cannot duplicate Monsanto’s commercially valuable 

products from information in the public domain without going through the same 

painstaking trial and error development and testing that Monsanto has undertaken. 

Although certain information regarding Monsanto 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 determine from the patent applications which 

particular combination of genes and transgenic products will prove to be commercially 

valuable. 

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

MON 87769 could provide competitors with commercially valuable knowledge regarding 

the characteristics of particular products Monsanto is planning to commercialize and the 

likely time frame for commercialization. This information would be extremely helpful 

to these companies in developing their own marketing strategies and development plans 

in a highly competitive market. 

The commercial value of the information regarding 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 protections 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 competitors after that initial ten year period. 

In 1996, Congress amended the FFDCA to provide both disclosure protections and 

compensation equivalent to that provided by FIFRA for health and safety data submitted 

to support pesticide residue tolerance and tolerance exemption applications. FFDCA § 

408(i). APHIS should recognize 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 for these types of data set forth by FIFRA and 

FFDCA, each statute contains independent provisions 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) as 
Soybean MON 87769 

Monsanto Document #09-SY-200U 

Page 4 of 4

Appendix 1 are required in order for Monsanto 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 business 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 advance of the release of the document(s), USDA 

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

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

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

expects that no information that has been identified as CBI (confidential business 

information), 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, publication or other distribution 

of this information (including website posting) without Monsanto's prior notice and 

consent. 

© 2010 Monsanto Company. All Rights Reserved. 

This document is protected under copyright law. 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 Company. Any other use of this material, 

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

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

information or intellectual property described in this document. 

Monsanto Company 09-SY-200U Page 4 of 395 

EXECUTIVE SUMMARY 

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

Agriculture (USDA) has responsibility under the Plant Protection Act (7 USC § 7701-

7772) to prevent the introduction and dissemination of plant pests 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 request to APHIS for a determination of 

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

MON 87769, any progeny derived from crosses 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-derived soybean MON 87769 that contains 

stearidonic acid (SDA; 18:4), a sustainable 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 Δ6 desaturase (Pj.D6D) and Neurospora crassa Δ15 desaturase (Nc.Fad3)

into conventional soybean, A3525. The two introduced genes encode for the PjΔ6D and 

NcΔ15D 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 acid (EPA; 20:5) and 

docosahexaenoic acid (DHA; 22:6) from alpha linolenic acid (ALA; 18:3), a common 

dietary constituent. In addition to SDA, MON 87769 contains approximately 7% γlinolenic acid (GLA; 18:3), which is an in vivo metabolite in the conversion of linoleic 

acid (LA: 18:2) to arachidonic 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 production of margarine, mayonnaise, 

shortenings, salad dressings, ready-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 rancid odors and taste) 

than fish oils, thereby expanding the potential formulation options for food companies 

and food products for consumers. Fish and plant oils rich in omega-3 fatty acids also are 

used in feed applications such as aquaculture and poultry feeds. SDA soybean oil from 

MON 87769 may eventually be used in aquaculture and feed applications 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 other 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 this petition demonstrate that MON 87769 is 

agronomically, phenotypically, and biochemically 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 overall 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 characterizations that demonstrate the identity of the 

PjΔ6D and NcΔ15D proteins produced in MON 87769, 

3. A comprehensive safety assessment of the PjΔ6D and NcΔ15D proteins, that shows no 

allergenic potential, acute toxicity or dietary risk to humans and animals, 

4. A detailed compositional and nutritional assessment demonstrating that MON 87769 is 

equivalent in composition to conventional soybean, except for the intended fatty acid 

change (i.e., the presence 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 adverse 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 demonstrate MON 87769 is not likely to have 

increased plant pest potential compared to conventional soybean, 

8. An assessment of the potential impact to non-target organisms (NTOs) and endangered 

species concludes that MON 87769 is unlikely to have adverse effects on these 

organisms under normal agricultural 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 cultivation practices or 

impact land use patterns. 

Molecular Characterization of Inserted DNA

The development of MON 87769 involved the introduction of two desaturase genes, 

Primula juliae Δ6 desaturase (Pj.D6D) and Neurospora crassa Δ15 desaturase (Nc.Fad3)

that encode for the PjΔ6D and NcΔ15D proteins. The genetic modification in 

MON 87769 was extensively characterized. 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 toxins. The methodology used to characterize the 
Monsanto 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 inserted 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 allow a comparison to the transfer DNA 

(T-DNA) sequence of the transformation vector to confirm that only the expected 

sequences were integrated; and 3) a segregation 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 the Mendelian laws of genetics. 

Characterization of the PjΔ6D and NcΔ15D Proteins

A multistep approach was used to characterize the PjΔ6D and NcΔ15D proteins 

expressed in MON 87769 resulting from the genetic modification. This detailed 

characterization confirms that both proteins are safe for human and animal consumption. 

The assessment involved: 1) characterizing the 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 fluids; 5) documenting the history of safe 

consumption of these proteins or their structural/functional homologues that lack 

documented adverse affects on human or animal health; and 6) investigating potential 

mammalian toxicity through an acute oral gavage assay. The PjΔ6D and NcΔ15D 

proteins share sequence similarity to several proteins that are ubiquitous in the human 

diet and are directly consumed in many common foods. The PjΔ6D and NcΔ15D 

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 allergens, toxins and antinutritional proteins known to have adverse effects on 

mammals. Additionally, both proteins are rapidly digested in simulated gastric and 

intestinal fluids and do not exhibit any signs of toxicity when administered to mice via 

oral gavage. The safety assessment supports the conclusion that dietary exposure to the 

PjΔ6D and NcΔ15D 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 available soybean varieties were conducted. 

These compositional comparisons were made by analyzing the seed and forage harvested 

from five replicated field sites across the 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 fatty 
Monsanto Company 09-SY-200U Page 7 of 395 

acid composition, while the other components analyzed in MON 87769 were 

compositionally equivalent to conventional 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 evidence of biologically-relevant changes. 

Statistical analysis was conducted 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 the 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 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 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 differences between MON 87769 and conventional 

soybean control were seen in amino acids (17 out of the 22 significant differences) and 

the magnitude of these differences were small (<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 glycitein, were lower in MON 87769 harvested seed compared to 

the values in the conventional soybean control. However, the mean and range of values 

for these three analytes in MON 87769 were within 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 influenced by many factors, ranging from 

environmental conditions, variety, and agronomic practices. The remaining two 

statistical differences observed were for carbohydrates and protein, and their magnitude 

of differences between MON 87769 and conventional soybean control were small (<6%). 

Furthermore, the mean and range values of carbohydrates and protein 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. Therefore, these differences were not considered to be biologically 

meaningful from a food and feed safety or nutritional perspective. 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, lecithin, 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 acid change, the composition of the soybean 

processed fractions from MON 87769 is equivalent to the composition of soybean 

processed fractions from the conventional soybean 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 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. 

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 ecological 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 concentrations greater than 1% of their total 

fatty acid composition. Similarly, GLA is present in a number of plant species at levels 

greater than 1% of their total fatty acid composition. As natural components of the plant 

and animal world, SDA, GLA and other PUFAs are not expected 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 scientific literature, nor is it present on the lists of noxious weed 

species compiled by the Federal government. Soybean does not possess any of the 

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

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

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

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

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

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

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

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

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

does not exist for MON 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 conventional 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 environmental impact of MON 87769 is to compare MON 87769 to 

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

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

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

plant variety whose biological properties and plant pest potential are well known. 

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

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

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

counterpart. The MON 87769 characteristics assessed included: seed dormancy and 

germination, pollen morphology, symbiont interactions conducted in the laboratory and 

greenhouse, and plant phenotypic and environmental interaction 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 seed, a well-accepted characteristic 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 assessment of pollen characteristics, there were no 

significant differences (p>0.05) observed for any 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, shoot 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 potential compared to conventional soybean. 

The field evaluation of phenotypic, agronomic, 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 conventional soybean. These evaluations were 

conducted at 21 field locations across U.S. soybean production regions 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 phenotypic characteristics of 

MON 87769 were not different from the conventional soybean control. No significant 

differences (p<0.05) were observed in either year in a combined-site analysis of the data 

for any of the phenotypic characteristics measured. The results from the 2006 and 2007 

field trials demonstrate that the assessed phenotypic and agronomic characteristics of 

MON 87769 were not altered, compared to the conventional control, as a result of the 

introduced genes (Pj.D6D and Nc.Fad3), the proteins produced from the introduced 

genes (PjΔ6D and NcΔ15D) and the intended fatty acid change. These results are 
Monsanto Company 09-SY-200U Page 10 of 395 

indicative of no increased plant pest or weediness potential 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, and arthropod damage, indicates no increased 

plant pest potential or adverse ecological interactions of MON 87769 relative to 

conventional soybean. No biologically meaningful differences were detected between 

MON 87769 and the conventional control for the 703 comparative observations of plant 

response to abiotic stressors, disease damage, and arthropod damage and 260 

comparative observations of the abundance of various pest and beneficial 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 and conventional soybean, including the 

presence of the inserted genes, the expression of the PjΔ6D and NcΔ15D proteins, and 

the production of the expected fatty acids (SDA and GLA) in MON 87769 seed. Since 

MON 87769 is a source of omega-3 enriched soybean oil with no pesticidal activity, all 

exposed organisms are considered to be NTOs. The assessment considered pertinent 

information from product characterization and 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 species under normal agricultural practices. 

The potential for MON 87769 to outcross with sexually compatible species in the U.S., 

including threatened or endangered plant species, is unlikely, since no known wild 

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

In those world areas where sexually compatible species do exist, the potential for 

outcrossing is low because soybean is a highly self-pollinated 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 progeny 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 (PjΔ6D and NcΔ15D), 

and the intended fatty acid change is not likely to enhance the weediness or plant pest 

potential of MON 87769. Therefore, the environmental consequence 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 planted 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 states within the next decade. 

Soybean fields are typically highly managed agricultural areas that 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 current cultivation and management 

practices for soybean. MON 87769 has been shown to be no different from conventional 

soybean in its agronomic, phenotypic, and ecological characteristics 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 apparent 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, Monsanto 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 DEVELOPMENT OF MON 87769 .................................... 29

I.A. Basis for the Request for a Determination 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 the 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/7Sα' Promotor ..................................................................... 63

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

V.D.3. tml 3′ Nontranslated Sequence .................................................................. 65

V.D.4. 7Sα 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 Introduced Traits .......................................................... 81

V.H. Conclusions of Molecular Characterization .............................................. 85

VI. CHACTERIZATION OF THE INTRODUCED PjΔ6D AND NcΔ15D 

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

VI.A. Identity and Function of the PjΔ6D and NcΔ15D Proteins ...................... 86

VI.A.1. PjΔ6D Protein ........................................................................................... 87

VI.A.2. NcΔ15D Protein ........................................................................................ 89

VI.B. Characterization of the PjΔ6D and NcΔ15D Proteins Produced in 

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

VI.C. Expression of PjΔ6D and NcΔ15D 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 PjΔ6D and 

NcΔ15D Proteins Produced in MON 87769 ............................................. 97

VI.E. Assessment of the Potential for Toxicity of the PjΔ6D and NcΔ15D 

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

VII. COMPOSITIONAL AND NUTRITIONAL 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 Fatty 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, AND ENVIRONMENTAL 

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

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

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

VIII.C. Interpretation of Detected Differences Criteria ...................................... 117
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VIII.D. Phenotypic, Agronomic and Environmental Interactions 

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

VIII.D.1. Seed Dormancy and Germination Characteristics .................................. 119

VIII.D.2. Field Phenotypic, Agronomic 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 Phenotypic, 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 Management 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 Practices 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 PjΔ6D and NcΔ15D Proteins ................. 167

X.A.3. Environmental Safety of Fatty Acids Produced in MON 87769 ............ 168

X.A.3.1. Biological Role and Metabolism 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 Fatty 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. Hybridization with Wild Perennial Species of Subgenus Glycine .......... 179
Monsanto 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 Plant 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 PjΔ6D and NcΔ15D 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 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 ..................................................................................................... 240

Appendix F. Safety and Nutritional Impact Resulting from the Intended 

Fatty Acid Change in MON 87769 ......................................................... 316

Appendix G. Materials, Methods, and Individual-Site Results of the Plant 

Phenotypic and Agronomic Assessment of MON 87769 

from the 2006 and 2007 Field Trials....................................................... 321

Appendix H. Materials, Methods, and Individual-Site Results from the Seed 

Dormancy and Germination Assessment of MON 87769 ...................... 338

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 ......................................................... 344

Appendix J. Materials and Methods from 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 Environmental Assessment ................................................. 373
Monsanto 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, and Distribution .......................................................... 35

Table IV-1. Summary of Genetic Elements in Plasmid Vector PV-GMPQ1972 ....... 46

Table V-1. Summary of Genetic Elements in MON 87769 ...................................... 50

Table V-2. Summary Chart of the Expected 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 PjΔ6D Protein Levels in Tissues Collected from 

MON 87769 Produced in the 2006 U.S. Growing Season ...................... 95

Table VI-2. Summary of NcΔ15D 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 Analysis of MON 87769 vs. the 

Conventional Control (A3525) and Commercial Reference Varieties . 106

Table VIII-1. Phenotypic, Agronomic and Environmental Interaction 

Characteristics Evaluated in U.S. Field Trials ...................................... 116

Table VIII-2. Seed Dormancy and Germination Characteristics Evaluated ................ 121

Table VIII-3. Germination Characteristics of MON 87769 and A3525 ...................... 122

Table VIII-4. Field Phenotypic Evaluation Sites for MON 87769 during 2006 

and 2007 ................................................................................................ 125

Table VIII-5. Plant Growth and Development Data across 17 Locations during 

2006 ....................................................................................................... 126

Table VIII-6. Plant Growth and Development Data across Four Locations during 

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

Table VIII-7. Pollen Grain Diameter 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 Production by Region and State in 2008 ........................ 142

Table IX-3. U.S. Soybean Production Costs and Returns in 2006 ........................... 143

Table IX-4. Ratings for Control of Volunteer Glyphosate-Tolerant Soybean in 

Labeled Rotational Crops ...................................................................... 162

Table X-1. Plants that Contain Stearidonic Acid (SDA), Gamma-Linolenic 

Acid (GLA), and Other Polyunsaturated Fatty Acids (PUFAs) ............ 172

Table X-2. Fish that Contain Stearidonic Acid (SDA) ............................................ 173
Monsanto Company 09-SY-200U Page 17 of 395 

Table X-3. Summary of Published Literature on Soybean Cross-Pollination ......... 178

Table A-1. USDA Notification Approved for MON 87769 and Status of 

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

Table C-1. N-terminal Amino Acid Sequence Analysis of the MON 87769- 

Produced PjΔ6D Protein ........................................................................ 224

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

Produced NcΔ15D Protein .................................................................... 230

Table E-1. Summary of the Compositional Comparison of MON 87769 vs. 

the Conventional Control Forage from IA-1 Site for Fiber and 

Proximates ............................................................................................. 250

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. 

the Conventional Control Soybean Seed from IA-1 Site for 

Amino Acids, Fatty Acids, Fiber, Proximates, 

Vitamin E, Antinutrients and Isoflavones ............................................. 251

Table E-3. Summary of the Compositional Comparison of MON 87769 vs. 

the Conventional Control Forage from IA-2 Site for 

Fiber and Proximates ............................................................................. 257

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. 

the Conventional Control Soybean Seed from IA-2 Site for 

Amino Acids, Fatty Acids, Fiber, Proximates, 

Vitamin E, Antinutrients and Isoflavones ............................................. 258

Table E-5. Summary of the Compositional Comparison of MON 87769 vs. 

the Conventional Control Forage from IL Site for Fiber and 

Proximates ............................................................................................. 264

Table E-6. Summary of the Compositional 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 Compositional Comparison of MON 87769 vs. 

the Conventional Control Forage from MI Site for Fiber and 

Proximates ............................................................................................. 271

Table E-8. Summary of the Compositional 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 Compositional Comparison of MON 87769 vs. 

the Conventional Control Forage from OH Site for Fiber and 

Proximates ............................................................................................. 278

Table E-10. Summary of the Compositional 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 the Combined-Site Compositional 

Analysis of MON 87769 vs. the Conventional Control (A3525) 

Forage for Fiber and Proximates ........................................................... 285
Monsanto Company 09-SY-200U Page 18 of 395 

Table E-12. Statistical Summary from the Combined-Site Compositional 

Analysis of MON 87769 vs. the Conventional 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 the 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 the 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 the 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 the Compositional Comparison of 

MON 87769 vs. the Conventional Control 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 Agronomic Comparison of MON 87769 to the 

Conventional Control in the Individual-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 Agronomic Comparison of MON 87769 to the 

Conventional Control in the Individual-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 Information, Soil Description, and Cropping 

History ................................................................................................... 336

Table H-1. Germination Characteristics of MON 87769, the Conventional 

Control, and the Reference Soybean Variety Seed Produced from 

each of Three Field Sites and Tested in the AOSA-Recommended 

Temperature Regime ............................................................................. 341

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

Control Seed Produced from each of Three Field Sites and Tested 

in the Additional Temperature Regimes ............................................... 342
Monsanto Company 09-SY-200U Page 19 of 395 

Table I-1. Abiotic Stressor Evaluations Using an Observational Severity 

Scale for MON 87769 and the Conventional Control in the 2006 

Field Trials ............................................................................................ 347

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

Scale for MON 87769 and the Conventional Control in the 2007 

Field Trials ............................................................................................ 348

Table I-3. Disease Damage Evaluations Using an Observational Severity 

Scale for MON 87769 and the Conventional Control in the 2006 

Field Trials ............................................................................................ 349

Table I-4. Disease Damage Evaluations Using an Observational Severity 

Scale for MON 87769 and the Conventional Control in the 2007 

Field Trials ............................................................................................ 350

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

Scale for MON 87769 and the Conventional Control in the 2006 

Field Trials ............................................................................................ 351

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

Scale for MON 87769 and the Conventional Control in the 2007 

Field Trials ............................................................................................ 352

Table I-7. 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 2006 .................................................... 353

Table 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 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 Arthropods 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 Uninoculated Conventional Control (A3525-UN) 

to the Inoculated Conventional Control (A3525) for Nodule 

Number and Shoot Total Nitrogen ........................................................ 370

Table L-1. Organic and Conventional Soybean Seed Sources ................................ 383

Table L-2. Deregulated Biotechnology-derived Soybean Products ........................ 391
Monsanto 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 Δ6 Desaturase 

from PV-GMPQ1972 ............................................................................. 48

Figure IV-3. Deduced Amino Acid Sequence of Neurospora crassa Δ15 

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-7sα′ 

Probe ...................................................................................................... 68

Figure V-8. Southern Blot Analysis 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-7sα 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 Stability 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 Stability of MON 87769 with T-DNA II Probes.............. 80

Figure V-17. Breeding Path for Generating Segregation Data on MON 87769 ......... 83

Figure VI-1. Deduced Amino Acid Sequence of PjΔ6D from PV-GMPQ1972 ........ 88

Figure VI-2. Hydropathy Plot of the Deduced Amino Acid Sequence of PjΔ6D ...... 88

Figure VI-3. Deduced Amino Acid Sequence of NcΔ15D from PV GMPQ1972 ..... 89

Figure VI-4. Hydropathy Plot of the Deduced Amino Acid Sequence of 

NcΔ15D ................................................................................................. 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 PjΔ6D Protein .................. 222

Figure C-2. Western Blot Analysis of MON 87769-Produced PjΔ6D Protein ....... 223

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87769-Produced 

PjΔ6D Protein ...................................................................................... 224

Figure C-4. Glycosylation Analysis of the MON 87769-Produced PjΔ6D 

Protein .................................................................................................. 227

Figure C-5. SDS-PAGE of the MON 87769-Produced NcΔ15D Protein ............... 229
Monsanto Company 09-SY-200U Page 21 of 395 

Figure C-6. MALDI-TOF MS Coverage Map of the MON 87769-Produced 

NcΔ15D Protein ................................................................................... 231

Figure C-7. Western Blot Analysis of MON 87769-Produced NcΔ15D Protein.... 232

Figure C-8. Glycosylation Analysis of the MON 87769-Produced NcΔ15D 

Protein .................................................................................................. 234

 
Monsanto 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 borders 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-nucleotidyltransferase from the 

transposon Tn7 

AA Amino Acid 

AAA Amino Acid Analysis 

ADF Acid Detergent Fiber 

AD8 Allergen Database; www.allergenonline.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 Official 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-optimized) of the gene from Neurospora 

crassa encoding Δ15 desaturase 

CS-Pj.D6D Coding sequence for the fatty acid Δ6 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 Evaluation 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 Acid 
Monsanto 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 Recognized 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 Database and Information Service 

ILSI International Life Science Institute

ILSI-CCD International Life Sciences Institute Crop Composition Database 

IRM Insect resistance Management 

ISO International Organization 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 Quantitation 
Monsanto 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 Desorption/ionization Time-of-Flight Mass 

Spectrometry 

MEEC Maximum Expected Environmental 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 

NcΔ15D 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 the broad host range plasmid RK2 

necessary for maintenance of plasmid in Agrobacterium

OSL Overseason Leaf 

PjΔ6D Primula juliae delta-6 desaturase 

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

P-7Sα Promoter and leader from the Sphas2 gene from soybean 
Monsanto 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 Deodorized (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 acid - 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 Center 
Monsanto 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 Threatened 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 Volume 
Monsanto Company 09-SY-200U Page 28 of 395 

w/v Weight Per Volume 

 
Monsanto Company 09-SY-200U Page 29 of 395 

I. RATIONALE FOR THE DEVELOPMENT OF MON 87769 

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

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

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

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

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

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

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

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

introduction of the article. 

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

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

MON 87769, any progeny derived from crosses 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-derived soybean MON 87769 that contains 

stearidonic acid (SDA; 18:4), a sustainable 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 Δ6 desaturase (Pj.D6D) and Neurospora crassa Δ15 desaturase (Nc.Fad3)

into conventional soybean, A3525. The two introduced genes encode for the PjΔ6D and 

NcΔ15D proteins, respectively. SDA is an eighteen-carbon fatty acid with four double 

bonds (18:4) and as such is found in plants, fish and fish/algal oil products. In mammals, 

SDA is a metabolic intermediate in the production of eicosapentaenoic acid (EPA; 20:5) 

and docosahexaenoic acid (DHA; 22:6) from 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 little fish, and it is not practical to expect the general 

population to take fish oil supplements on a regular basis. 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 habits. 

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 (James 

et al., 2003; Yamazaki et al., 1992). Thus, MON 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 approximately 20 to 30% SDA (wt% of total fatty 

acids). SDA soybean oil can be used for the production of margarine, mayonnaise, 

shortenings, salad dressings, ready-to-eat foods, and other food categories. The use of 

SDA soybean oil in selected food categories 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 oxidation (e.g., less prone 

to fishy or rancid odors and taste), thereby expanding the potential food formulation 

options for food companies and food products for consumers, and serving as an 

alternative to fish oils where they currently are used in food production. 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 food 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 (IDP) in the Northern U.S. soybean 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 conventional soybean meal. 

I.C. Submissions to Other Regulatory Agencies 

Under the Coordinated Framework for Regulation of Biotechnology, the responsibility 

for regulatory oversight of biotechnology-derived crops falls on two federal agencies: 

FDA and USDA (USDA, 1986). Deregulation of MON 87769 by USDA constitutes only 

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

practical matter, MON 87769 cannot be released and marketed until FDA and USDA 

have completed their reviews and assessments under their respective jurisdictions. 

Submission to FDA 

MON 87769 falls within the scope of the 1992 U.S. Food and Drug Administration 

policy statement concerning regulation of products derived from new plant varieties, 

including those developed through biotechnology (FDA, 1992). In compliance with this 

policy, on March 23, 2009, Monsanto initiated consultation with the FDA on the food 

and feed safety and nutritional assessment for MON 87769 (FDA BNF 00117). 

Submissions to Foreign Government Agencies 

To support commercial introduction of MON 87769 in the U.S., regulatory submissions 

will be made to countries that import significant quantities of soybean or its processed 

fractions from the U.S. and have established regulatory approval processes in place. 

These will include submissions to a number of foreign government regulatory authorities, 
Monsanto Company 09-SY-200U Page 31 of 395 

including: Ministry of Agriculture, People’s Republic of China; Japan’s Ministry of 

Agriculture, Forestry, and Fisheries (MAFF) and the Ministry of Health, Labor, and 

Welfare (MHLW); the Canadian Food Inspection Agency (CFIA) and Health Canada; the 

Intersectoral Commission for Biosafety of Genetically Modified Organisms 

(CIBIOGEM), Mexico; the European Food Safety Authority (EFSA); and the regulatory 

authorities in other soybean importing countries with functioning regulatory systems. As 

appropriate, notifications of importation will be made to importing countries that do not 

have a formal approval process. 
Monsanto Company 09-SY-200U Page 32 of 395 

 II. THE SOYBEAN FAMILY 

This section summarizes the taxonomy, biology, and use of soybean based on: 1) the 

consensus document for Glycine max (L.) Merr. prepared by the Organization for 

Economic Co-operation and Development (OECD, 2000; OECD, 2001), and 2) a 

summary prepared by USDA-APHIS (2006) and a biology document published by 

Canadian Food Inspection Agency-Plant Biosafety Office (CFIA, 1996), 

II.A. Soybean as a Crop 

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

million metric tons 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 countries. The major producers of soybean are 

the U.S., Brazil, Argentina, China, and India, which accounted for approximately 91% of 

the global soybean production in 2007 (Soyatech, 2008); also see Table II-1. 

Approximately one-third of the 2007 world soybean production was produced in the U.S. 

(Soyatech, 2008). The soybean produced in China and India are primarily for domestic 

use, while a significant portion of 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 seed supply was crushed to produce soybean 

meal and oil in 2007 (ASA, 2008; Soyatech, 2008), and the majority was used to supply 

the feed industry for livestock use or the food industry for edible vegetable oil and 

soybean protein isolates. Another 34% of the world soybean seed supply was traded to 

other geographies, with China, EU, Japan, and Mexico being the top soybean seed import 

geographies (ASA, 2008). The remainder of the soybean seed produced was used as 

certified seed, feed, or stocks. 
Monsanto Company 09-SY-200U Page 33 of 395 

Soybean is used in various food products, including tofu, soybean sauce, soymilk, energy 

bars, and meat products. A major food use for soybean is purified oil, for use in 

margarines, shortenings, cooking, and salad oils. Soybean oil generally has a smaller 

contribution to soybean’s overall value compared to soybean meal because the oil 

constitutes just 18 to 19% of the soybean's weight. Nonetheless, soybean oil accounted 

for approximately 30% of all the vegetable oils consumed globally, and was the second 

largest source of vegetable oil worldwide, slightly behind palm oil at approximately 32% 

share (Soyatech, 2008). 

Soybean meal is used as a supplement in feed rations for livestock. Soybean meal is the 

most valuable component obtained from processing the soybean, accounting for roughly 

50-75% of its overall value. By far, soybean meal is the world's most important protein 

feed, accounting for nearly 69% of world protein meal supplies (ASA, 2008). Industrial 

uses of soybean range from a carbon/nitrogen source in the production of yeasts via 

fermentation to the manufacture of soaps, inks, paints, disinfectants, and biodiesel. 

Industrial uses of soybean have been summarized by Cahoon (Cahoon, 2003) and the 

American Soybean Association (ASA, 2008). 

Global soybean plantings reached 90.8 million hectares in 2007/08, an 8.9% increase 

over the previous four years with an average of 82.3 million hectares planted from 

2002/03 – 2007/08 (Soyatech, 2008). Soybean production has realized, on average, a 

6.2% annual growth between 1995/96 to 2006/07. Increased planting flexibility, 

increased yield from narrow-row seeding practices, a higher rate of corn-soybean 

rotations, and low production costs favored expansion of soybean areas in the mid-1990s, 

and the expanded areas tended to be concentrated where soybean yields were highest. 

II.B. History of Soybean 

Domestication of soybean is thought to have taken place in China during the Shang 

dynasty (approximately 1500 to 1027 B.C.) or earlier (Hymowitz, 1970). However, 

historical and geographical evidence only could be traced back to the Zhou dynasty (1027 

to 221 B.C.) where the soybean was utilized as a domesticated crop in the northeastern 

part of China. By the first century A.D., the soybean probably reached Central and 

Southern China as well as peninsular Korea. The movement of soybean germplasms was 

probably associated with the development and consolidation of territories and the 

degeneration of Chinese dynasties (Ho, 1969; Hymowitz, 1970). 

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

several countries, with land races eventually developing in Japan, Indonesia, Philippines, 

Vietnam, Thailand, Malaysia, Myanmar, Nepal, and Northern India. The movement of 

soybean throughout this period was due to the establishment of sea and land trade routes, 

the migration of certain tribes from China, and the rapid acceptance of seeds as a staple 

food by other cultures (Hymowitz and Newell, 1981; Hymowitz et al., 1990). 

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

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

East to the West. 
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 subsequently throughout the Corn Belt. In 1853, soybean seed 

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

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

Commissioner of Patents sent soybean seed to dozens of growers throughout the U.S. 

Soybean has been cultivated extensively 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 diploidized tetraploid (2n=40), which 

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

the genus Glycine Willd., and the subgenus Soja (Moench) F.J. Herm. 

Family: Leguminosae 

Subfamily: Papilionoideae 

Tribe: Phaseoleae 

Genus: Glycine

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

Species: max

The genus Glycine Willd. is of Asian and Australian origin and is divided into two 

subgenera, Glycine and Soja (Moench) F.J. Herm. The subgenus Glycine consists of 22 

wild perennial species, which are indigenous to Australia, West, Central and South 

Pacific Islands, China, Russia, Japan, Indonesia, Korea, Papua New Guinea, the 

Philippines, and Taiwan (Hymowitz, 2004). The subgenus Soja includes the cultivated 

soybean, G. max (L.) Merr. and its wild annual relatives from Asia, G. soja Sieb. and 

Zucc. The list of species in the genus Glycine Willd. is presented in Table II-2. 
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 Genome1 Distribution

Subgenus Glycine

 

1. G. albicans Tind. & Craven 40 I1 Australia

2. G. aphyonota B. Pfeil 40 --2 Australia

3. G. arenaria Tind. 40 HH Australia

4. G. argyrea Tind. 40 A2A2 Australia

5. G. canescens F.J. Herm. 40 AA Australia

6. G. clandestina Wendl. 40 A1A1 Australia

7. G. curvata Tind. 40 C1C1 Australia

8. G. cyrtoloba Tind. 40 CC Australia

9. G. dolichocarpa Tateishi and Ohashi 80 -- (Taiwan)

10. G. falcate Benth. 40 FF Australia

11. G. hirticaulis Tind. & Craven 40 H1H1 Australia

80 -- Australia

12. G. lactovirens Tind. & Craven. 40 I1I1 Australia

13. G. latifolia (Benth.) Newell & 

Hymowitz

40 B1B1 Australia

14. G. latrobeana (meissn.) Benth. 40 A3A3 Australia

15. G. microphylla (Benth.) Tind. 40 BB Australia

16. G. peratosa B. Pfeil & Tind. 40 -- Australia

17. G. pindanica Tind. & Craven 40 H3H2 Australia

18. G. pullenii B. Pfeil, Tind. & Craven 40 -- Australia

19. G. rubiginosa Tind. & B. Pfeil 40 -- Australia

20. G. stenophita B. Pfeil & Tind. 40 B3B3 Australia

21. G. tabacina (Labill.) Benth. 40 B2B2 Australia

80 Complex3 Australia, West Central and 

South Pacific Islands

22. G. tomentella Hayata 38 EE Australia

40 DD Australia, Papua New Guinea

78 Complex4 Australia, Papua New Guinea

80 Complex5 Australia, Papua New Guinea, 

Indonesia, Philippines, Taiwan

Subgenus Soja (Moench) F.J. Herm. 

23. G. soja Sieb. & Zucc. 40 GG China, Russia, Taiwan, Japan, 

Korea (Wild Soybean)

24. G. max (L.) Merr. 40 GG Cultigen (Soybean)

1 Genomically similar species carry the same letter symbols. 

2

 Genome designation has not been assigned to the species.

3

 Allopolyploids (A and B genomes) and segmental allopolyploids (B genomes). 

4

 Allopolyploids (D and E, A and E, or any other unknown combination). 

5

 Allopolyploids (A and D genomes, or any other unknown combination). 

Note: Table is adapted from Hymowitz (2004).
Monsanto Company 09-SY-200U Page 36 of 395 

Glycine soja grows wild in China, Japan, Korea, the Russian Far East, and Taiwan and is 

commonly found in fields, hedgerows, roadsides, and riverbanks (Lu, 2004). The plant is 

an annual, slender in build with narrow trifoliolate leaves. The purple or very rarely 

white flowers are inserted on short, slender racemes. The pods are short and tawny with 

hirsute pubescence, producing oval-oblong seeds (Hermann, 1962). 

Glycine max (L.) Merr., the cultivated soybean, is an annual that generally exhibits an 

erect, sparsely branched, bush-type growth habit with trifoliolate leaves. The leaflets are 

broadly ovate, and the purple, pink, or white flowers are borne on short axillary racemes 

or reduced peduncles. The pods are either straight or slightly curved, and one to three 

ovoid to 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 intermediate in morphology between G. max and G. soja, and is 

sometimes considered a variant of G. max. The three species in the Soja subgenus can 

cross-pollinate, and the hybrid seed can germinate normally and subsequently produce 

fertile pollen and seed (Singh and Hymowitz, 1989). The taxonomic position of 

G. gracilis has been an area of debate, and neither ILDIS (International Legume Database 

and Information Service) nor USDA-GRIN (USDA Germplasm Resources Information 

Network) recognizes G. gracilis as a distinct species. The wild and weedy relatives 

(G. soja and G. gracilis) of soybean do not occur in the U.S., and, therefore, are not likely 

to contribute to the potential for outcrossing (USDA-APHIS, 2006). 

II.D. The Genetics of Soybean 

Glycine is the only genus in the tribe Phaseoleae where species have diploid chromosome 

numbers of 40 and 80, but not 20 (Lackey, 1981). The unique chromosome number of 

Glycine is probably derived from diploid ancestors with a base number of 11. The 

ancestral species have undergone aneuploid reduction (loss of a specific chromosome), 

which is prevalent throughout the Papilionoideae, to a base number of 10 chromosomes 

(Lackey, 1981). Tetraploidization (2n = 2x = 40) through autopolyploidy or 

allopolyploidy of the progenitor species occurred either prior to or after dissemination 

from the ancestral region. The path of migration from a common progenitor is assumed 

by Singh et al. (2001) as: wild perennial (2n = 4x = 40, unknown or extinct) to wild 

annual (2n = 4x = 40; G. soja) to soybean (2n = 4x = 40; G. max). Soybean should be 

regarded as a stable tetraploid with diploidized genome (Gurley, 1979; Lee and Verma, 

1984; Skorupska, 1989). 

II.E. Pollination of Cultivated Soybean 

Soybean is largely a self-pollinated species, propagated by seed (OECD, 2000). The 

papilionaceous flower consists of a tubular calyx of five sepals, a corolla of five petals, 

one pistil, and nine fused stamens with a single separate posterior stamen. The stamens 

form a ring at the base of the stigma and elongate one day before pollination, at which 

time the elevated anthers form a ring around the stigma (OECD, 2000). The soybean 

flower stigma is receptive to pollen approximately 24 hours before anthesis and remains 
Monsanto Company 09-SY-200U Page 37 of 395 

receptive for 48 hours after anthesis. The anthers mature in the bud and directly pollinate 

the stigma of the same flower. As a result, soybean is considered to be a highly selfpollinated species, with cross-pollination to adjacent plants of other soybean varieties 

occurring at very low frequency (0.3 to 3.62%) (Beard and Knowles, 1971). Pollination 

typically takes place on the day the flower opens. The pollen naturally comes in contact 

with the stigma during the process of anthesis. Anthesis normally occurs in late morning, 

depending on the environmental conditions. The pollen usually remains viable for two to 

four hours, and no viable pollen can be detected by late afternoon. Natural or artificial 

cross-pollination can only take place during the short time when the pollen is viable. 

II.F. Cultivated Soybean as a Volunteer

Cultivated soybean plants are annuals, and they reproduce solely by means of seeds. 

Volunteer soybean in rotational crops is typically not a concern in most environments 

where soybean is cultivated (CFIA, 1996; OECD, 2000). Soybean seed rarely exhibit 

any dormancy characteristics, and seed remaining in the field after harvest likely will 

readily imbibe water (Lersten and Carlson, 2004), germinate, and will be killed by frost 

or field cultivation. If they did become established, volunteer plants would not compete 

well with the succeeding crop, and could be controlled readily by either mechanical or 

chemical means (OECD, 2000). 

II.G. Characteristics of the Recipient Plant 

The soybean variety used as the recipient for the DNA insertion to create MON 87769 

was A3525, a nontransgenic conventional variety developed by Asgrow Seed Company. 

A3525 is a mid-maturity group III soybean variety with very high yield potential. It has 

superior yields relative to lines of similar maturity and has excellent agronomic 

characteristics (Steffen, 2004). 

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

In developing the data to support the safety assessment of MON 87769, A3525 was used 

as the nontransgenic conventional 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 PjΔ6D and NcΔ15D proteins could be assessed 

in an unbiased manner. Since MON 87769 was derived from the A3525 conventional 

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

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

assessments. In addition, commercial conventional varieties were used as reference 

materials to establish ranges of responses or values representative of commercial soybean 

varieties. Reference varieties were selected based on prevalence and performance of the 

soybean varieties at each trial location. As a general principle, varieties that were well 

adapted to the local environments and could 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 protein (5-enolpyruvylshikimate-3-phosphate synthase protein 

from Agrobacterium sp. strain CP4) that provides tolerance to the 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 the 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 within the genome of the plant. Following selection of the 

transformants that contain both T-DNAs, the inserted T-DNA encoding the selectable 

marker (e.g., T-DNA II) can be segregated in progeny through subsequent selfing of 

plants (self-pollination) and genetic selection, 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 successfully used 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 transformation to produce MON 87769 was based 

on published methods that generate transformed plants without utilization of callus 

(Armstrong et al., 2007; Martinell et al., 2002). Agrobacterium tumefaciens strain ABI 

contains a disarmed Ti plasmid that is incapable of inducing tumor formation due to the 

deletion of the phytohormone genes originally present in the Agrobacterium plasmid 

(Koncz and Schell, 1986). The transformation vector, PV-GMPQ1972, contains both the 

left and right border sequences flanking the transfer DNA (T-DNA) to facilitate 

transformation. 

Embryos were excised from germinated A3525 seed and meristematic 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 untransformed plant cells, and antibiotics carbenicilin 

and claforan to inhibit the growth of excess Agrobacterium, respectively. The meristems 

then were placed on media conducive to shoot and root development, and only rooted 

 

® Roundup is a registered trademark 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 aforementioned 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 quantify 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 (Southern blot) and phenotypic assessments. 

MON 87769 was selected as the lead event based on superior phenotypic characteristics 

and molecular profile. Regulatory studies on MON 87769 were initiated to further 

characterize the genetic insertion and the expressed proteins, and to confirm the food, 

feed, and environmental safety relative to conventional soybean. The major steps 

involved in the development of MON 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 

tumefaciens, 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 plants for insert integrity and 

SDA production, and selected homozygous plants 
Monsanto 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 PjΔ6D and 

NcΔ15D proteins produced in MON 87769, and a description of the CP4 EPSPS protein 

selectable marker employed to produce MON 87769. In this section, T-DNA refers to 

DNA that is transferred to the plant during transformation. An expression cassette is 

comprised of a coding sequence and the regulatory elements necessary for the expression 

of the coding sequence. 

IV.A. Plasmid PV-GMPQ1972 

This section describes the donor genes and regulatory sequences used in the development 

of MON 87769 and the deduced amino acid sequences of the Primula juliae Δ6 

desaturase (Pj∆6D) and Neurospora crassa Δ15 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 vector is an approximately 16.5 kb plasmid 

containing two T-DNA regions, each delineated by a left and right border region. The 

first T-DNA (T-DNA I) region 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 was used for 

early event selection and was segregated away from T-DNA I by conventional breeding 

(self-pollination). The T-DNA II elements are 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 incorporated 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 7Sα′ promoter and the tml 3' nontranslated sequence. The Nc.Fad3

expression cassette consists of the Nc.Fad3 coding sequence under the regulation of the 

7Sα 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 contains 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 provided in Table IV-1. 

IV.B. T-DNA I 

This section describes the elements contained 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 PjΔ6D, of 446 amino acids (Ursin et al., 2008). The deduced 

full-length amino acid sequence is shown in Figure IV-2. The PjΔ6D protein is a Δ6 

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

acid. 

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

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

cassette. The Pj.D6D coding sequence is under the regulatory control of the P-7Sα' 

transcriptional promoter. P-7Sα' 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 octopine-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 NcΔ15D, of 429 amino acids (Ursin et al., 2006). The deduced 

full-length amino acid sequence is shown in Figure IV-3. The NcΔ15D protein is a Δ15 

desaturase which creates a double bond at the 15th 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 regulatory control of the P-7Sα promoter. P-7Sα 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 directs transcriptional termination and 

polyadenylation of the Nc.Fad3 mRNA. 

IV.B.5. The T-DNA Borders 

Plasmid vector PV-GMPQ1972 contains right border and left border regions that 

delineate the T-DNAs and are involved in their efficient transfer into soybean genome. 

These border regions (Figure IV-1; Table IV-1) were derived from Agrobacterium 

tumefaciens plasmids (Barker et al., 1983; Depicker et al., 1982). 
Monsanto Company 09-SY-200U Page 43 of 395 

IV.C. T-DNA II 

This section describes the elements contained 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 protein 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 chloroplast transit peptide sequence, 

(TS-CTP2), derived from the Arabidopsis thaliana epsps gene (Klee et al., 1987). This 

transit peptide directs the transport of the CP4 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). Transit peptides are typically cleaved from the translated 

polypeptide following delivery to the plastid (Della-Cioppa et al., 1986). 

IV.C.3. The cp4 epsps Regulatory Sequences

The CTP2/cp4 epsps gene cassette that produces the CTP/CP4 EPSPS protein consists of 

the promoter (P-FMV) for the 35S RNA from figwort 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 carboxylase, and the small subunit (rbcS2) E9 gene 

(Coruzzi et al., 1984) that directs transcriptional termination and polyadenylation of the 

CTP2/cp4 epsps mRNA. 

The unlinked T-DNA II insertion was eliminated as a result of segregation during 

breeding. T-DNA II elements are not present 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 Outside of the T-DNA Borders 

Four genetic elements exist outside of the 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 repressor of primer (ROP) protein for 
Monsanto 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 border regions, they are not expected to be 

transferred into the soybean genome. The 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 Map of Vector PV-GMPQ1972 

Plasmid PV-GMPQ1972 was used in Agrobacterium-mediated transformation to develop 

MON 87769. Approximate locations of the genetic elements (with approximate positions 

relative to the plasmid vector) are shown on the exterior of the map. PV-GMPQ1972

contains two T-DNA regions designated as T-DNA I and T-DNA II. 

 
Monsanto Company 09-SY-200U Page 46 of 395 

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

B1-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

CS2-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

OR3-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 an 

aminoglycoside-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

P4-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

L5-ShkG 5492–5558 5' nontranslated leader sequence from the 

Arabidopsis ShkG gene encoding EPSPS (Klee et 

al., 1987) that helps regulate gene expression

TS6–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; 2CS – Coding Sequence; 3OR – Origin of Replication; 4P – Promoter; 5L-Leader; 6

TS – Targeting Sequence.
Monsanto Company 09-SY-200U Page 47 of 395 

Table IV-1 (continued). Summary of Genetic Elements in Plasmid Vector PV-GMPQ1972

T-DNA II (cont.) 

T7-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-7Sα′ 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 Δ6 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-7Sα 12738-14417 Promoter and leader from the Sphas2 gene from 

soybean 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 Δ15 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.
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 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 Δ6 Desaturase from 

PV-GMPQ1972 

The amino acid sequence of the PjΔ6D 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 Δ15 Desaturase 

from PV-GMPQ1972 

The amino acid sequence of the NcΔ15D 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 used to characterize the integrated DNA insert 

in MON 87769. Southern blot analysis confirmed 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 segregation 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 restriction enzymes and 

subjected to Southern blot analyses to characterize the T-DNA that was integrated into 

the soybean genome. Genomic DNA from conventional soybean (A3525) was used as a 

negative control to determine potential nonspecific hybridization signals and to establish 

hybridization signals to endogenous soybean DNA sequences since several genetic 

elements in T-DNA I were derived from soybean. There were two types of positive 

controls used. For all Southern blots, PV-GMPQ1972 plasmid DNA was digested with a 

restriction enzyme or enzyme combination 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 lanes were obtained using the molecular weight 

markers on the left side. The genetic elements present in MON 87769 are listed in Table 

V-1 starting at partial Right Border 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 shown 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 MON 87769. This linear map depicts restriction 

sites identified in the insert and the soybean genomic DNA flanking the insert, and 

provides information on the expected banding 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 plasmid map, summarizing the expected DNA 

fragments for Southern analyses, is presented in Table V-2. The materials and methods 

used in the analyses are presented in Appendix B. 

 
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Table V-1. Summary of Genetic Elements in MON 87769 

Genetic Element1

Location in 

Sequence1 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-7Sα′ 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 Δ6 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-7Sα 4285-5964 Promoter and leader from the Sphas2 gene from 

soybean 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 includes the insert in MON 87769 and adjacent genomic 

DNA starting with 933 bp flanking the 5’ of the sequence and ending with 831 bp flanking 3’ end 

of the insert. 

2

B – Border; 3P – Promoter; 4CS – Coding Sequence; 5T – Transcript Termination Sequence and 

polyadenylation signal sequences. 

 
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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 regions 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 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 the accompanying Table V-2. PV-GMPQ1972 

contains two T-DNA regions designated as T-DNA I and T-DNA II. 
Monsanto Company 09-SY-200U 

Figure V-3. Schematic Representation of the Insert and Genomic Flanking Sequences in MON 877

A linear map of the insert and genomic DNA flanking the insert in MON 87769 is shown. The upper pdisplays genetic elements within the insert (rectangular bar), as well as all restriction sites used in SouThe numbered designation of the restriction sites are consistent with the information presented in Tunderneath the designated insert indicate the direction of transcription. Shown on the lower portion 

expected sizes of the DNA fragments after digestions with respective restriction enzyme or combinatio

two large arrows above the designated insert indicate the end of the insert and the beginning of soybea

sequence. 

BsrG I/PshA I

> 9.2 kb

6.8 kb 1.6 k

2.0 kb 1.9 kb 4.2 kb

Lgu I 121

BsrG I 304

1

5' Genomic Flank DNA

P-7Sα'

CS-Pj.D6D

T-tml

P-7Sα

T-E9

B-Right Border

BstX I 6912

PshA I 2282

PshA I 4167

Lgu I

Lgu I/BstX I

CS-Nc.Fad3

BsrG I/PshA I

> 9.2 kb

6.8 kb 1.6 k

2.0 kb 1.9 kb 4.2 kb

Lgu I 121

BsrG I 304

1

5' Genomic Flank DNA

P-7Sα'

CS-Pj.D6D

T-tml

P-7Sα

T-E9

B-Right Border

BstX I 6912

PshA I 2282

PshA I 4167

Lgu I

Lgu I/BstX I

CS-Nc.Fad3
Monsanto Company 09-SY-200U 

Table V-2. Summary Chart of the Expected Sizes of DNA Fragments Using Combinations of RestrictProbes 

Probes used 13 - 15 16 – 18

7

8

9 10 11 12 

Southern blotin Figure

V-4a and 

V-14a*

V-4b and 

V-14b* V-7 V-8 V-9 V-10 V-11 V-12 

PlasmidBamHI

1.3, 1.9, 2.3, 3.1, 

and 7.9 kb

2.3, 3.1, 

and 7.9 kb

1.3, 2.3, 

and 7.9 

kb

1.3 and 

1.9 kb 1.9 kb 3.1 kb 3.1 kb 2.3 and 7.9 kb 

Probe templates 1.6, 1.7, and 2.0 kb 0.8, 1.0, and 1.3 kb --1 -- -- -- -- -- MON 87769Lgu I > 9.2 kb > 9.2 kb -- -- -- -- -- -- Lgu I and BstX I 6.8 kb 6.8 and 1.6 kb 6.8 kb 6.8 kb 6.8 kb 6.8 kb 6.8 and 1.6 kb 1.6 kb BsrG I and PshA I -- -- 2.0 kb 1.9 and 2.0 kb 1.9 and 4.2 kb 4.2 kb 4.2 kb 4.2 kb 

1 ‘--’ indicates that the particular restriction enzyme or combination 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 with BsrG I and PshA I.
Monsanto Company 09-SY-200U Page 55 of 395 

Southern blot analysis confirmed 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 stability across generations. In addition, DNA 

sequencing analyses confirmed the expected organization and nucleotide sequence of the 

insert in MON 87769. Furthermore, insert segregation analysis confirmed 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 the T-DNA I in 

MON 87769. 

V.A. Insert and Copy Number Determination 

The number of T-DNA inserts (insert 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, releases 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 number) integrated at a single locus was 

determined by digesting the MON 87769 DNA with the combination of restriction 

enzymes Lgu I and BstX I, which cleave once within the 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 portion of the T-DNA I, 

along with adjacent plant genomic DNA. The samples were subjected to Southern blot 

analysis using six overlapping 32P-labeled T-DNA I probes (probes 13-18, Figure V-2) 

spanning the entire T-DNA I insert. This analysis was divided between two Southern 

blots. Each blot was probed with a combination 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 hybridization 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 BstX I (lanes 1 and 5) 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 DNA sequences 

residing in the conventional soybean genome and are not specific to the inserted DNA. 

These signals were produced in all lanes, including those containing the conventional 

soybean DNA material and therefore, are considered to be endogenous background 

hybridization. 

To ensure that each of the T-DNA probes hybridized 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 BamH I, mixed with conventional 

soybean DNA predigested with Lgu I and BstX I, and analyzed using probes 13-15 

(lane 9, Figure V-4a) produced the expected 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 
Monsanto 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 conventional DNA and hybridized with probes 13-15 

(lanes 10 and 11) produced the expected bands 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 BamH I, mixed with predigested 

conventional soybean DNA and analyzed with 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, and 1.3 kb, in addition to the endogenous 

background hybridization. The 0.1 genomic equivalent 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 hybridization 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 BstX I (lanes 2 and 6, Figure V-4a) and 

hybridized with probes 13-15 produced a single unique band of approximately 6.8 kb in 

addition to the endogenous background hybridization observed in the conventional 

soybean controls (lanes 1 and 5). MON 87769 DNA digested with Lgu I and BstX 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 (lanes 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 simultaneously with three overlapping 32P-labeled T-DNA 

probes (probes 13-18, Figure V-2). Figure V-4a was hybridized 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (Lgu I) 

4: MON 87769 (Lgu I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (Lgu I)

8: MON 87769 (Lgu I)

9: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

10: Conventional soybean (Lgu I and BstX I) spiked with probe template [~1 

genomic equivalent] 

11: Conventional soybean (Lgu I and BstX 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 BstX I or restriction enzymes BsrG I and PshA 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 BstX I (lanes 1 and 5) or BsrG I and PshA I (lanes 3 and 

7) and hybridized with the backbone probes (probes 1-3 and 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 BamH I and probe template 

spikes were mixed with conventional soybean DNA (predigested with Lgu I and BstX I) 

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

BamH I, mixed with predigested conventional 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 probe template controls, mixed with digested 

conventional soybean DNA, and hybridized with 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 exists between lanes 5 and 6. There is no DNA present at 

the location of the hybridization spot since lanes 5 and 6 contain the “short run” 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 their intended targets. 

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

and BstX I (lanes 2 and 6) or BsrGI and PshA 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 32P-labeled probes that 

span the entire backbone sequence (probes 1-3 and 6, Figure V-1) of plasmid 

PV-GMPQ1972. 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

10: Conventional soybean (Lgu I and BstXI) spiked with probe templates [~1 

genomic equivalent] 

11: Conventional soybean (Lgu I and BstX 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 segregated away from MON 87769 by traditional breeding after 

the initial transformation. 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 BstX I or BsrG I and PshA I and analyzed by Southern blot (Figure 

V-6) using probes 4 and 5 (Figure V-1). The results are summarized in Table V-2. 

Conventional soybean DNA was used as a control in order to determine the endogenous 

background of MON 87769 on the Southern blot used to confirm the absence of 

T-DNA II sequences (Figure V-6). Conventional soybean DNA digested with a 

combination of Lgu I and BstX I (lanes 1 and 5) or BsrG I and PshA 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 plasmid PV-GMPQ1972 DNA digested with BamH I and 

probe template spikes were mixed with conventional soybean DNA (predigested with 

Lgu I and BstX I) and used on the Southern blot (Figure V-6). PV-GMPQ1972 DNA 

digested with BamH I and analyzed using the two overlapping T-DNA II probes 

produced the expected size bands of approximately 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 templates resulted in a fairly faint hybridization 

signal (lane 11), but is detectable on the actual autoradiography 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 sequences not associated with T-DNA I, 

MON 87769 DNA 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 contain the right border sequence, the E9 3′ nontranslated region sequence, and 

the left border sequence that are also contained on T-DNA I. Therefore, given the 

common genetic elements shared between T-DNA I and T-DNA II, the T-DNA II probes 

are expected to hybridize to fragments derived from T-DNA I. Southern blot analysis of 

MON 87769 DNA digested with Lgu I and BstX I (lanes 2 and 6) produced a single band 

of approximately 1.6 kb. This band is consistent with the 1.6 kb band detected with the 

E9 3′ nontranslated region + left border sequence (lanes 2 and 6, Figure V-12). Although 

a band at 6.8 kb, that represents the right border sequence in the insert, was also expected 

in MON 87769, this band was not visible in 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 right border sequence in the MON 87769 insert. There were no additional 

fragments observed. Southern blot analysis of MON 87769 DNA digested with BsrG I 

and PshA 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 E9 3′ nontranslated region + left border 

sequence (lanes 4 and 8, Figure V-12). Although a band at 2.0 kb that represents the 
Monsanto Company 09-SY-200U Page 61 of 395 

right border sequence in the insert 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 right border sequence in the 

MON 87769 insert. No additional fragments were observed. These results indicate that 

MON 87769 does not contain any additional detectable 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 Analysis of MON 87769 with T-DNA II Probes

The blot was hybridized simultaneously with two overlapping 32P-labeled probes that 

span the entire T-DNA II sequence (probes 4 and 5, Figure V-1) of plasmid 

PV-GMPQ1972. 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Conventional soybean (BsrG I and PshA I) spiked with PV-GMPQ1972 (BamH 

I) [~1 genomic equivalent] 

10: Conventional soybean (BsrG I and PshA I) spiked with probe templates [~1 

genomic equivalent] 

11: Conventional soybean (BsrG I and PshA 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 assessed by digesting MON 87769 DNA with a 

combination of Lgu I and BstX I or a combination of BsrG I and PshA I and hybridizing 

the Southern blots with individual genetic 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 BstX I was expected to generate 

two border fragments of approximately 6.8 and 1.6 kb (Figure V-3). The 6.8 kb fragment 

contains genomic DNA flanking the 5′ end of the insert, right border sequence, 7Sα′

promoter sequence, Pj.D6D coding sequence, tml 3′ nontranslated region, 7Sα 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 BsrG I and PshA I was expected to 

generate one internal restriction fragment of approximately 1.9 kb and two border 

fragments of approximately 2.0 and 4.2 kb (Figure V-3). The 2.0 kb fragment contains 

genomic DNA flanking the 5′ end of the insert, right border sequence, 7Sα′ 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 contains the remaining portion of the tml 3′

nontranslated region, 7Sα 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 with the following probes: the right border + 

7Sα′ promoter probe, Pj.D6D coding region probe, tml 3′ nontranslated region probe, 7Sα

promoter probe, Nc.Fad3 coding sequence probe, or E9 3’ nontranslated region + left 

border probe (probes 7-12, Figure V-1). The expected DNA fragments identified by 

probes 7-12 are summarized in Table V-2. To ensure that each of the probes was capable 

of hybridizing to the respective target, plasmid PV-GMPQ1972 DNA digested with 

BamH I and combined with predigested conventional soybean DNA was used as a 

positive hybridization control. 

V.D.1. Right Border/7Sα' Promotor 

Conventional soybean DNA was used as a control to determine the endogenous 

background hybridization of MON 87769 on the Southern blot used to assess the copy 

number of the right border and 7Sα′ promoter (Figure V-7). Conventional soybean DNA 

digested with Lgu I and BstX I (lanes 1 and 5) or BsrG I and PshA I (lanes 3 and 7) and 

hybridized with the right border + 7Sα′ promoter probe (probe 7, Figure V-1) produced a 

single hybridization signal. This hybridization 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 control, plasmid PV-GMPQ1972 DNA was digested with 

BamH I, mixed with conventional soybean DNA, and included on the Southern blot 

analysis that was hybridized with probe 7 (Figure V-7). The results of this experiment 

showed the expected size hybridization bands at approximately 1.3, 2.3 and 7.9 kb in 

addition to the endogenous hybridization band observed in the conventional soybean 

control (lanes 10 and 11). 

Southern blot analysis of MON 87769 DNA digested with Lgu I and BstX I (lanes 2 and 

6) using probe 7 produced the expected single unique band of approximately 6.8 kb in 

addition to the endogenous hybridization band observed in the conventional soybean 

control (lanes 1 and 5; Figure V-7). Southern blot analysis of MON 87769 DNA digested 

with BsrG I and PshA I (lanes 4 and 8) produced the single expected unique band of 

approximately 2.0 kb in addition to the endogenous hybridization band observed in the 

conventional soybean control (lanes 3 and 7; Figure V-7). There were no unexpected 

bands present in the MON 87769 DNA samples. This indicates that MON 87769 

contains no additional detectable Right Border or 7Sα′ promoter elements other than 

those endogenous to the soybean genome 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 Southern 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 BstX I (lanes 1 and 5) or BsrG I and 

PshA I (lanes 3 and 7) and hybridized with the Pj.D6D coding sequence probe (probe 8, 

Figure V-1) showed no detectable hybridization 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, plasmid PV-GMPQ1972 DNA was digested with 

BamH I, mixed with conventional soybean DNA (predigested with Lgu I and BstX 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 BstX I (lanes 2 and 

6) using probe 8 produced the expected single unique band of approximately 6.8 kb. 

Southern blot analysis of MON 87769 DNA digested with BsrG I and PshA 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 short 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 Southern 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 BstX I (lanes 1 and 5) or BsrG I and PshA 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 indicates that there are no endogenous soybean 

DNA sequences that hybridized to the tml 3' nontranslated region probe. 

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

BamH I, mixed with conventional soybean DNA (predigested with Lgu I and BstX I) and 

included on the tml 3' nontranslated region Southern blot. The results for this probe 

showed the expected size band at approximately 1.9 kb (lanes 10 and 11). 

Southern blot analysis of MON 87769 DNA digested with Lgu I and BstX I (lanes 2 and 

6) using probe 9 produced the expected single unique band of approximately 6.8 kb. 

Southern blot analysis of MON 87769 DNA digested with BsrG I and PshA I (lanes 4 

and 8) produced two expected unique bands of approximately 1.9 and 4.2 kb. There were 

no additional bands detected using the tml 3′ nontranslated region probe. 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. 7Sα Promoter 

Conventional soybean DNA was used as a control to determine the endogenous 

background hybridization of MON 87769 on the Southern blot used to confirm the copy 

number of the 7Sα Promoter (Figure V-10). The conventional soybean DNA, digested 

with Lgu I and BstX I (lanes 1 and 5) or BsrG I and PshA I (lanes 3 and 7) and hybridized 

with the 7Sα promoter probe (probe 10, Figure V-1), produced two hybridization signals. 

These hybridization signals result from the 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 control, plasmid PV-GMPQ1972 DNA was digested with 

BamH I, mixed with conventional soybean DNA (predigested with Lgu I and BstX I), and 

included in the 7Sα promoter Southern blot analysis. The results of this experiment 

showed the expected size band at approximately 3.1 kb in addition to the endogenous 

hybridization bands observed in the conventional soybean control (lanes 10 and 11). 

Southern blot analysis of MON 87769 DNA digested with Lgu I and BstX I ( lanes 2 and 

6) produced the expected single unique band of approximately 6.8 kb in addition to the 

endogenous hybridization bands observed in the conventional soybean control (lanes 1 

and 5). Southern blot analysis of MON 87769 DNA digested with BsrG I and PshA I 

(lanes 4 and 8) produced the expected single unique band of approximately 4.2 kb in 

addition to the endogenous hybridization bands observed in the conventional soybean 
Monsanto 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 molecular 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 87769 DNA samples. This indicates that 

MON 87769 contains no additional detectable 7Sα 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 Southern blot used to confirm the copy 

number of the Nc.Fad3 coding sequence (Figure V-11). Conventional soybean DNA 

digested with Lgu I and BstX I (lanes 1 and 5) or BsrG I and PshA 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 indicates that there are no endogenous soybean 

DNA sequences that hybridized to the Nc.Fad3 coding sequence probe. 

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

BamH I, mixed with conventional soybean DNA (digested with Lgu I and BstX I), and 

included on the Nc.Fad3 coding sequence Southern blot analysis using probe 11. The 

results of this experiment showed the expected size hybridization band at approximately 

3.1 kb (lanes 10 and 11). 

Southern blot analysis of MON 87769 DNA digested with Lgu I and BstX I (lanes 2 and 

6) using probe 11 produced the two expected 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 band 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 BsrG I and PshA 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 Southern blot used to confirm the copy 

number of the E9 3′ nontranslated region and left border sequence (Figure V-12). 

Conventional soybean DNA digested with Lgu I and BstX I (lanes 1 and 5) or BsrG I and 

PshA I (lanes 3 and 7) and hybridized with the E9 3′ nontranslated region + left border 

probe (probe 12, Figure V-1) produced no detectable hybridization bands. This indicates 

that there are no endogenous soybean DNA sequences that hybridized to the E9 3′

nontranslated region and left border sequence. 
Monsanto Company 09-SY-200U Page 67 of 395 

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

BamH I, mixed with conventional soybean DNA (digested with Lgu I and BstX I), and 

included in the E9 3′ nontranslated region 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 BstX I (lanes 2 and 

6) using probe 12 produced the expected single unique band of approximately 1.6 kb. 

Southern blot analysis of MON 87769 DNA digested with BsrG I and PshA I (lanes 4 

and 8) produced the expected single unique band of approximately 4.2 kb. There were no 

additional bands detected using the E9 3′ nontranslated region + left border probe. This 

indicates that MON 87769 contains 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-7sα′ Probe

The blot was hybridized with a 32P-labeled probe that spanned the right border and 7sα′

promoter sequence (probe 7, 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8 MON 87769 (BsrG I and PshA I) 

9: Empty 

10: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

11: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH 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 Analysis of MON 87769 with CS-Pj.D6D Probe 

The blot was hybridized with a 32P-labeled probe that spanned the Pj.D6D coding 

sequence (probe 8, Figure V-1). Each lane contains approximately 10μg of digested 

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

Lane 1: Conventional soybean (Lgu I and BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Empty 

10: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

11: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH 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 32P-labeled probe that spanned the tml nontranslated 

region (probe 9, 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Empty 

10: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

11: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH 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-7sα Probe 

The blot was hybridized with a 32P-labeled probe that spanned the 7sα 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Empty 

10: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

11: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH 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 Analysis of MON 87769 with CS-Nc.Fad3 Probe

The blot was hybridized with a 32P-labeled probe that spanned the Nc.Fad3 coding 

sequence (probe 11, 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 BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshAI) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Empty 

10: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

11: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH 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 32P-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 tissue. Lane designations are as follows:

Lane 1: Conventional soybean (Lgu I and BstX I) 

2: MON 87769 (Lgu I and BstX I) 

3: Conventional soybean (BsrG I and PshA I) 

4: MON 87769 (BsrG I and PshA I) 

5: Conventional soybean (Lgu I and BstX I) 

6: MON 87769 (Lgu I and BstX I) 

7: Conventional soybean (BsrG I and PshA I) 

8: MON 87769 (BsrG I and PshA I) 

9: Empty 

10: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

11: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH 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

0.5
Monsanto 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 using DNA obtained from multiple generations of 

MON 87769. DNA from four generations was analyzed by Southern blot using six 

overlapping probes that span the entire T-DNA I region of plasmid PV-GMPQ1972 

(probes 13-18, Figure V-2). For reference, the breeding history of MON 87769 is 

presented in Figure V-13. The expected Southern hybridization 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 used 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 BstX 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 lanes, including those 

containing the conventional soybean DNA and, 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 BamH I, mixed with 

conventional soybean DNA (digested with Lgu I and BstX I), and analyzed using probes 

13-15 (lane 1, Figure V-14a) produced the expected 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 hybridization produced by the conventional 

soybean DNA. 

Plasmid PV-GMPQ1972 DNA digested with BamH I, mixed with conventional soybean 

DNA (digested with Lgu I and BstX I) and analyzed with probes 16-18 (lane 1, Figure 

V-14b) 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 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 BstX I (lanes 5-9, Figure V-14a) and hybridized simultaneously with probes 13-15 

produced the expected band of approximately 6.8 kb in addition to the endogenous 

background hybridization observed in the conventional soybean DNA controls (lanes 4). 

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

and BstX I (lanes 5-9, Figure V-14b) using probes 16-18 produced the two expected 
Monsanto 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 soybean control DNA (lane 1). Also, several 

nonspecific hybridization spots exist 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 stably maintained across multiple generations 

of its breeding history. 

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

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

examined for the absence of backbone sequence by Southern blot. MON 87769 and 

control DNA samples were digested with Lgu I and BstX I and the blot was hybridized 

simultaneously with four overlapping probes, which taken together, span the backbone 

sequence of plasmid PV-GMPQ1972 (probes 1-3 and 6, Figure V-1). 

Conventional soybean DNA was used as a control in order to determine 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 BstX I (lane 4) and hybridized 

with the backbone probes (probes 1-3 and 6, Figure 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 BamH 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, and 1.8 kb (lanes 2 and 3). 

These results indicate that all the backbone probes labelled properly and hybridized to 

their intended targets. 

To confirm the absence 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. Southern blot analysis of MON 87769 DNA 

digested with a combination of Lgu I and BstX I (lanes 5-9) showed no detectable 

hybridization signal. These results are consistent with the data depicted in Figure V-5. 

This indicates that MON 87769 does not contain 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 assess generational stability 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 BstX 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 control in order to determine the endogenous 

background of MON 87769 on the Southern blot used to confirm the absence of T-DNA 

II sequences (Figure V-16). Conventional soybean DNA digested with a combination of 

Lgu I and BstX I (lanes 4) and hybridized with the T-DNA II probes produced no 

detectable hybridization bands. This indicates 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 controls on the Southern blot (Figure V-16). PV-GMPQ1972 

DNA digested with BamH 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 hybridization. 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 sequences not associated with T-DNA I, 

MON 87769 DNA from four generations 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 contain the right border sequence, the E9 3′

nontranslated region sequence, and the left border sequence that also are contained on 

T DNA I. Therefore, the T-DNA II probes are expected to hybridize to fragments 

derived from T-DNA I. Southern blot analysis of MON 87769 DNA from all four 

generations digested with Lgu I and BstX I (lanes 5-9) produced two bands of 

approximately 1.6 and 6.8 kb. The 1.6 kb band is consistent with the 1.6 kb band 

detected with the E9 3′ nontranslated region + left border 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 because 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 the selection of plants homozygous for the T-DNA I insert 

(Section III). R5 seed material was used for commercial variety development. 

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

R5a

, R6b, and R6c 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 32P-labeled probes that spanned the T-DNA 

(probes 13-18, Figure V-2). Each blot was hybridized simultaneously with three of the 

overlapping probes. Figure V-14a was hybridized with probes 13-15 and Figure V-14b 

was hybridized with probes 16-18. Each lane contains 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 BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent]

2: Conventional soybean (Lgu I and BstX I) spiked with probe templates [~1 

genomic equivalent] 

3: Conventional soybean (Lgu I and BstX I) spiked with probe templates [~0.1 

genomic equivalent] 

4: Conventional soybean (Lgu I and BstX I) 

5: R3: MON 87769 (Lgu I and BstX I) 

6: R4: MON 87769 (Lgu I and BstX I) 

7: R5a

: MON 87769 (Lgu I and BstX I) 

8: R6c

: MON 87769 (Lgu I and BstX I) 

9: R6b

: MON 87769 (Lgu I and BstX I) 

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

stained gel. 

20 15

2.0

8.1 7.16.1

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 7.16.1

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 7.16.1

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 7.16.1

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 32P-labeled probes that span the entire 

backbone sequence (probes 1, 2, 3, and 6, Figure V-1) of plasmid PV-GMPQ1972. Each 

lane contains approximately 10 μg of digested genomic DNA isolated from leaf tissue. 

The breeding history of MON 87769 is illustrated in Figure IV-17. Lane designations are 

as follows:

Lane 1: Conventional soybean (Lgu I and BstX I) spiked with PV-GMPQ1972 (BamH I) 

[~1 genomic equivalent] 

2: Conventional soybean (Lgu I and BstX I) spiked with probe templates [~1 

genomic equivalent] 

3: Conventional soybean (Lgu I and BstX I) spiked with probe templates [~0.1 

genomic equivalent] 

4: Conventional soybean (Lgu I and BstX I) 

5: R3: MON 87769 (Lgu I and BstX I) 

6: R4: MON 87769 (Lgu I and BstX I) 

7: R5a

: MON 87769 (Lgu I and BstX I) 

8: R6c: MON 87769 (Lgu I and BstX I) 

9: R6b

: MON 87769 (Lgu I and BstX 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 9
Monsanto 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 32P-labeled probes that span the entire TDNA II (probes 4 and 5, Figure V-1) of plasmid PV-GMPQ1972. Each lane contains 

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 BstX I ) spiked with PV-GMPQ1972 (BamH 

I) [~1 genomic equivalent] 

2: Conventional soybean (Lgu I and BstX I) spiked with probe templates [~1 

genomic equivalent] 

3: Conventional soybean (Lgu I and BstX I) spiked with probe templates [~0.1 

genomic equivalent] 

4: Conventional soybean (Lgu I and BstX I) 

5: R3: MON 87769 (Lgu I and BstX I) 

6: R4: MON 87769 (Lgu I and BstX I) 

7: R5a

: MON 87769 (Lgu I and BstX I) 

8: R6c

: MON 87769 (Lgu I and BstX I) 

9: R6b: MON 87769 (Lgu I and BstX 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 within the insert of MON 87769 was confirmed 

by PCR and DNA sequence analyses. Overlapping 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 right border region, and ending at base 288 in the left 

border region of PV-GMPQ1972. In addition, 933 base pairs of soybean genomic DNA 

flanking the 5' end of the insert and 831 base pairs of soybean genomic DNA flanking the 

3' end of the insert were also determined. 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. Results confirm that the DNA sequence of the insert in MON 87769 is 

identical to that of plasmid PV-GMPQ1972, and the organization of the insert genetic 

elements is as depicted in Figure V-3. 

V.G. Inheritance of the Introduced Traits 

The genotypic stability and segregation 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 based on testing the observed segregation ratio to 

the expected segregation ratio according to Mendelian principles. The breeding path for 

these segregation data on MON 87769 is described in Figure V-17. The R0 plant was 

self-pollinated to produce R1 seed, which is expected to segregate 1:2:1 

(1 homozygote:2 hemizygote:1 null segregant) for the insert. A homozygous selection 

(R1 plant) was identified from the segregating population by Invader and Southern blot 

analyses. The selected R1 plant was self-pollinated to produce R2 seed, which was 

expected to be fixed for the trait, meaning 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 plants 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 the 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. Hemizygous F3 plants were 

selected and self-pollinated to produce F4 seed. Likewise, the F4 plants 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 predicted 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: 
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 χ 2 = ∑ [( | o – e | )2 / e] 

where o = observed frequency of the genotype and e = expected frequency of the 

genotype. The level of statistical significance was predetermined to be 5% (p≤0.05). 

Table V-3 shows the segregation data obtained with Invader-based assay. The 

Chi-square value for the F2, F3, and F4 generations indicated no significant difference 

between the observed and expected segregation ratios. These results are consistent with 

molecular characterization data, indicating a single insertion site of the insert within 

MON 87769 and show the insert follows the expected Mendelian pattern of segregation. 
Monsanto Company 09-SY-200U Page 83 of 395 

Figure V-17. Breeding Path for Generating Segregation Data on MON 87769 
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Table V-3. Chi-square (

χ2) analysis of MON 87769 Progeny 

 1:2:1 Segregation

Generation1

Total 

Plants 

Tested2

Observed # 

Plants 

Homozygous 

Positive

Observed # 

Plants 

Hemizygous 

Positive

Observed # 

Plants 

Homozygous 

Negative

Expected # 

Plants 

Homozygous 

Positive

Expected # 

Plants 

Hemizygous 

Positive

Expected # 

Plants 

Homozygous 

Negative

F2 47 13 23 11 11.75 23.5 11.75 

F3 174 45 81 48 43.5 87 43.5 

F4 222 60 102 60 55.5 111 55.5 

1 F2 progeny were from the cross of homozygous MON 87769 with a soybean variety that did not contain the MON 87769 

progeny were from self-pollinated F2 and F3 plants hemizygous positive for the MON 87769 insert. 

2 Plants were tested for the presence of the tml 3′ 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 characterize the integrated DNA insert in 

MON 87769. Southern blot analyses were used to determine the DNA insert number 

(number of integration sites within the soybean genome), copy number (the number of 

copies within one insert), 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 sequencing analyses were 

performed to confirm the organization of the elements within the MON 87769 DNA 

insert, and to confirm the sequence identity 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-GMPQ1972 was not detected and it also was 

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

elements other than those associated with T-DNA I. Generational stability analysis 

demonstrated that the expected Southern blot fingerprint of MON 87769 has been 

maintained across four generations of breeding, thereby confirming the stability of the 

DNA insert over multiple generations. These generations were also shown not to contain 

any detectable T-DNA II or backbone sequence from plasmid PV-GMPQ1972. In 

addition, DNA sequence analyses confirmed 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 results 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. Finally, heritability 

and stability of the insert within MON 87769 were as expected across multiple 

generations, which corroborates the molecular insert stability analysis and establishes the 

genetic behavior of the DNA insert at a single chromosomal locus. 
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VI. CHACTERIZATION OF THE INTRODUCED PjΔ6D AND NcΔ15D 

PROTEINS 

This section summarizes the safety assessment of the PjΔ6D and NcΔ15D proteins 

produced in MON 87769 based on the following: 

1. Characterization of their identity and function, 

2. Isolation from immature soybean seed for use in subsequent laboratory and regulatory 

safety evaluations, 

3. Quatitation of expression levels in key soybean tissues, 

4. An assessment of the potential allergenicity, and 

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

Results show that the PjΔ6D and NcΔ15D proteins share sequence 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 toxins. Additionally, both proteins are rapidly 

digested in simulated gastric and intestinal fluids 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 PjΔ6D and the NcΔ15D proteins derived from 

MON 87769 poses no meaningful risk to human or animal health. 

These results, all of which have been submitted to FDA as part of the pre-market 

consultation, support a conclusion of safe dietary consumption for humans and animals. 

The following sections provide further details regarding the safety assessment of the 

PjΔ6D and NcΔ15D proteins produced in MON 87769. 

VI.A. Identity and Function of the PjΔ6D and NcΔ15D Proteins 

Fatty acid desaturases are enzymes that convert a single bond between two carbon atoms 

(C-C) to a double bond (C=C) in a fatty acyl chain (Stumpf, 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 membranes that consist of a 

bilayer of phospholipids (Aguilar and Mendoza, 2006). All organisms synthesize 

unsaturated fatty acids de novo using a common metabolic pathway (Somerville and 

Browse, 2000; Voet and Voet, 1995). Hence, enzymes involved in this metabolic 

pathway, including fatty acid desaturases, are conserved across all kingdoms (Los and 

Murata, 1998). 

The PjΔ6D and NcΔ15D proteins are members of a family of integral membrane fatty 

acid desaturases found in all eukaryotic organisms (plants, animals, fungi) and some 

prokaryotes, e.g. cyanobacteria (Hashimoto et al., 2008; Los and Murata, 1998). Fatty 
Monsanto Company 09-SY-200U Page 87 of 395 

acid desaturases are non-heme iron-containing enzymes that introduce a double bond 

between defined carbons of fatty acyl chains with strict 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, the 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 membrane desaturase proteins is not known. 

VI.A.1. PjΔ6D Protein 

The PjΔ6D 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 isoelectric point of 8.8. Analysis of the 

PjΔ6D amino acid sequence demonstrates three histidine motifs that are characteristic of 

integral membrane desaturases (Figure VI-1). The active site of integral membrane 

desaturases consists of three histidine motifs 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 catalytic center of the 

desaturases (Los and Murata, 1998). Analysis of PjΔ6D deduced amino acid sequence 

has shown that, similar to other Δ6 desaturases (Nakamura and Nara, 2004) PjΔ6D 

contains the amino-terminal cytochrome b5 domain carrying the heme-binding motifs 

(Figures VI-1 and VI-2). As in other Δ6 desaturases, the first His residue of the third 

His-box in PjΔ6D is substituted with glutamine (QXXHH instead of HXXHH) and, 

therefore, is called Q-box. The cytochrome b5 domain and glutamine residue are 

essential for the activity of Δ6 desaturases because mutations abolish the enzyme activity 

(Sayanova et al., 2001; Sayanova et al., 2000). 

The PjΔ6D protein also contains multiple membrane-spanning regions that are 

characteristic of the integral membrane desaturase proteins (Figure VI-2) based on the 

patterns observed from hydropathy plots. 

 
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Figure VI-1. Deduced Amino Acid Sequence of PjΔ6D from PV-GMPQ1972 

Three histidine-motifs are represented by black shaded blocks while the heme binding motif is 

shaded in light gray. 

Figure VI-2. Hydropathy Plot of the Deduced Amino Acid Sequence of PjΔ6D 

The deduced amino acid sequence was analyzed by the DNAStar-Protean software 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 PjΔ6D 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 Δ6 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 when 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 the 
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requisite electron transfer proteins. In the case of PjΔ6D, 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 substrate. Enzymatic activity specific to the 

PjΔ6D protein was observed in the crude homogenate obtained from very young (stage 

0-1, green immature seed, up to 4 mm) fresh 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 14C-ALACoA was incubated with the immature seed homogenate of MON 87769, 14C-SDA was 

detected in the assay mixture. The formation of SDA is a unique reaction attributed to 

the presence of the PjΔ6D protein in MON 87769. 

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

extracts demonstrate the Δ6 desaturase function of the PjΔ6D protein encoded by the 

Pj.D6D gene. 

VI.A.2. NcΔ15D Protein 

The NcΔ15D 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 that was introduced to facilitate the insertion of the gene 

into the plant transformation vector. The MON 87769-expressed NcΔ15D protein 

consists of 429 amino acids, has a calculated molecular mass of 49,195 Da, and a 

predicted isoelectric point of 7.1. Analysis of the NcΔ15D amino acid sequence 

demonstrates the presence of three histidine motifs that are characteristic of integral 

membrane desaturases (Figure VI-3). The active site of integral membrane desaturases is 

thought to be comprised of three histidine-rich motifs with a total of nine essential 

histidine residues (Shanklin et al., 1994). Similar to PjΔ6D, the NcΔ15D 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 NcΔ15D 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 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 
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Figure VI-4. Hydropathy Plot of the Deduced Amino Acid Sequence of NcΔ15D 

The deduced amino acid sequence was analyzed in the DNAStar-Protean software 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 three histidine-rich motifs are shown below the 

hydropathy plot. 

The functional activity and specificity of the NcΔ15D protein was confirmed in vivo via 

the expression of the yeast expression vector containing Nc.Fad3 (NcΔ15D-encoding 

gene) in S. cerevisiae as described for PjΔ6D protein in Section VI.A.1. S. cerevisiae

was grown in the absence or presence of various 18 and 20 carbon fatty acids. In the 

absence of the exogenous fatty acids, the yeast cells produced ALA from oleic acid. The 

Δ15D protein is the only known enzyme capable of catalyzing this reaction. Results also 

demonstrated that other 18 ( e.g., LA and GLA) or 20 carbon fatty acids (e.g., di-homo-γlinolenic acid and arachidonic acid tested) were substrates for NcΔ15D and all of the 

exogenous fatty acid substrates were desaturated three carbons from the methyl terminus 

(omega-3 desaturation). 

A demonstration of the functional activity of the NcΔ15D 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 Δ15 desaturase; therefore, this 

Δ15 desaturation reaction occurs in the conventional 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 NcΔ15D protein encoded by Nc.Fad3 gene. 

VI.B. Characterization of the PjΔ6D and NcΔ15D 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 proteins 
Monsanto Company 09-SY-200U Page 91 of 395 

as surrogates for the plant-expressed proteins. In such cases, the physicochemical and 

functional properties of the plant-produced and heterologously produced proteins must be 

established to justify the use of the surrogate protein for safety testing. In the case of the 

two integral membrane desaturase proteins, PjΔ6D and NcΔ15D, safety assessment 

studies were conducted with proteins isolated directly from MON 87769 immature seed. 

Therefore, no equivalence study was required. 

Expression of desaturase proteins is higher during seed development, therefore, the level 

of PjΔ6D and NcΔ15D is higher in immature seeds than mature seeds (Tables VI-1 and 

VI-2). Purification of integral membrane desaturases is a challenging task that requires 

removal of membranes and replacement of the lipids surrounding the protein’s 

hydrophobic membrane-spanning regions with an appropriate detergent that will keep the 

protein in solution (Wiener, 2004). To purify the integral membrane PjΔ6D and NcΔ15D 

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 their solubility. As a result, 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 adopted for use in NMR studies of membrane 

proteins due to its structure-stabilizing properties (Choowongkomon 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 integral membrane protein diacylglycerol kinase 

(Gorzelle et al., 1999) and had less deleterious effects than other tested detergents on 

functional activities of some integral membrane proteins (Li et al., 2001; Narayanan et 

al., 2007). After the PjΔ6D and NcΔ15D proteins were solubilized from the membranes, 

multiple chromatographic steps were applied to further purify these proteins. Standard 

precautions to retain the integrity of the protein during purification were undertaken, 

including conducting all chromatographic 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 characterization of PjΔ6D and NcΔ15D proteins. 

Although the activity of integral membrane desaturases 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 PjΔ6D and NcΔ15D enzymatic activities 

following their solubilization from the membranes. This most likely is due to the 

desaturases’ requirement for the requisite electron transfer proteins co-localized in the 

membrane. 

To characterize the purified PjΔ6D and NcΔ15D proteins produced in MON 87769, a 

number of analyses were performed. The analyses employed for the characterization of 

MON 87769-produced PjΔ6D and NcΔ15D proteins included: 

1. N-terminal sequence analysis, 
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2. Matrix-assisted laser desorption ionization - time of flight (MALDI-TOF) mass 

spectrometry to generate a tryptic peptide map, 

3. Immunoblot analysis to establish protein identity through immunoreactivity with 

PjΔ6D and NcΔ15D-specific antibodies, 

4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to 

assess the apparent molecular weight of the protein, and 

5. Glycosylation analysis to evaluate potential post-translational modification of the 

protein. 

Results show that the identity of the MON 87769-produced PjΔ6D and NcΔ15D proteins 

was confirmed by: 1) their recognition by anti-PjΔ6D and anti-NcΔ15D antibodies, 

respectively; 2) the identification of the first 15 amino acids of the expected N-terminus 

sequence of each protein; 3) the identification of tryptic peptide masses of each protein 

that yielded >40% overall coverage of the expected 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 

PjΔ6D and NcΔ15D proteins isolated from MON 87769. The PjΔ6D and NcΔ15D 

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 PjΔ6D and NcΔ15D Proteins in MON 87769 

PjΔ6D and NcΔ15D 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 detergent, cell membranes tend to form vesicles of 

different orientation depending on the charge and hydrophobicity of the surface 

(Rosenberg, 1996). Hydrophilic portions of the integral membrane proteins are often 

trapped inside membrane vesicles and not available for interaction with antibody. Due to 

their membrane localization, the PjΔ6D and NcΔ15D 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 interact with antibody, extracts containing 

solubilized PjΔ6D and NcΔ15D 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 (or immunoblot), protein extracts are separated on SDS-PAGE 

performed in the presence of a denaturing detergent. The subsequent electrotransfer of 

proteins from the gel to a blotting membrane yields an immunoblot that can be probed in 

buffer conditions that facilitate specific protein-antibody interactions. For PjΔ6D and 

NcΔ15D protein detection in MON 87769, optimized tissue extraction conditions were 

combined with standardized electrophoretic, blotting, and immunodetection 

methodologies to utilize PjΔ6D and NcΔ15D peptide antibodies specific to a fragment 

(peptide) of soluble portion of the respective proteins. This method allowed for the 

reproducible and accurate estimation of the PjΔ6D and NcΔ15D protein levels in plant 

tissue samples. Densitometric analysis of the PjΔ6D- and NcΔ15D-specific immunoblots 

yielded the reported quantitative values of PjΔ6D and NcΔ15D proteins by interpolation 

from standard curves prepared using purified PjΔ6D and NcΔ15D protein standards, 
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respectively. Limit of quantitation (LOQ) for PjΔ6D and NcΔ15D in each plant tissue 

type was determined from the corresponding immunoblot and was defined as the lowest 

amount of the standard that could be reliably determined. Limit of detection (LOD) was 

defined as the lowest amount of PjΔ6D and NcΔ15D protein standards visually observed 

on X-ray films. The levels of the PjΔ6D and NcΔ15D 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 PjΔ6D- and NcΔ15Dspecific antibodies and visualized using chemiluminescent 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 represent major soybean-growing regions of the U.S. 

and provide a range of environmental conditions that would be encountered in the 

commercial production of soybean. At each site, three replicated plots of MON 87769 

and conventional soybean control (A3525) were grown using a randomized complete 

block field design. Over-season leaf (OSL), forage, root, mature seed, and immature seed 

were collected from each replicated plot from all field sites. A description of the tissues 

collected is provided below. 

Twenty sets of the youngest expanded trifoliate 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: 
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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 receipt. Tissue samples were extracted and 

analyzed using an immunoblot according to applicable standard operating procedures 

(SOP). Appendix D describes the materials and methods used for the protein expression 

analysis. 

The levels of PjΔ6D and NcΔ15D proteins in OSL-1 through 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 all tissue types were 

calculated on a microgram (μg) per gram (g) fresh weight (FW) basis. For tissues 

displaying measurable quantities of PjΔ6D and NcΔ6D proteins, moisture content also 

was measured and protein levels were also expressed on a microgram (μg) per gram (g) 

dry weight of tissue (DW) basis. 

The expression of the PjΔ6D and NcΔ15D proteins is driven by a 7Sα' and 7Sα seed 

specific promoter, respectively. As expected, both PjΔ6D and NcΔ15D 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 signal specific to each protein at or above 

the tissue-specific LOD of the immmunoblot (Tables 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 PjΔ6D protein levels 

across all sites for immature seed, mature seed and forage were 100, 1.8, and 16 μg/g 

DW, respectively (Table VI-1). The mean NcΔ15D protein levels across all sites for 

immature seed, mature harvested seed and forage were 200, 10, and 14 μg/g DW, 

respectively (Table VI-2). The PjΔ6D and NcΔ15D proteins were not detected in the 

conventional soybean control, A3525. 

 
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Table VI-1. Summary of PjΔ6D Protein Levels in Tissues Collected from 

MON 87769 Produced in the 2006 U.S. Growing Season 

Tissue 

Type

PjΔ6D Mean

 (μg/g FW) 

(SD)1

Range2

(μg/g FW)

PjΔ6D Mean

 (μg/g DW) 

(SD)3

Range

(μg/g DW)

LOD

(μg/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 

 

Mature4 

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 expressed as micrograms (μg) of protein per gram (g) of tissue on a fresh weight 

(FW) basis. When applicable, an arithmetic mean and a standard deviation (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 “μg/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 PjΔ6D 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 μg/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 PjΔ6 desaturase western blot are as follows: 

Limit of Quantitation (μg/g FW)1 

Tissue Type PjΔ6D 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 NcΔ15D Protein Levels in Tissues Collected from 

MON 87769 Produced in the 2006 U.S. Growing Season

Tissue 

Type

NcΔ15D Mean

 (μg/g FW) 

(SD)1

Range2

(μg/g FW)

NcΔ15D Mean

 (μg/g DW) 

(SD)3

Range

(μg/g DW)

LOD

(μg/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 (μg) of protein per gram (g) of tissue on a fresh weight 

(FW) basis. When applicable, an arithmetic mean and a standard deviation (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 “μg/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 NcΔ15 desaturase western blot are as follows: 

Limit of Quantitation (μg/g FW)1 

Tissue Type NcΔ15D 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 Potential for Allergenicity of the PjΔ6D and NcΔ15D 

Proteins Produced in MON 87769 

The NcΔ15D and PjΔ6D proteins have been assessed for their potential allergenicity 

according to the recommendations of the Codex Alimentarius Commission (Codex, 

2003). Both proteins are from nonallergenic sources, 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 PjΔ6D protein is from Primula juliae, an organism that is not known to be allergenic. 

Bioinformatics analyses demonstrated that the PjΔ6D protein does not share structurally 

or immunologically relevant amino acid sequence similarities with known allergens and, 

therefore, is highly unlikely to contain immunologically cross-reactive allergenic epitopes. 

Digestive fate experiments conducted with the PjΔ6D protein demonstrated that the fulllength protein is rapidly digested in SGF, a characteristic shared among many proteins 

with a history of safe consumption. The transiently stable protein fragments in the SGF 

assay were quickly degraded during a short exposure to SIF. Rapid digestion of the fulllength PjΔ6D protein in SGF and SIF, together with rapid degradation of the transiently 

stable fragment from the SGF assay by SIF, indicates that it is highly unlikely that the 

PjΔ6D protein and its fragment will reach absorptive cells of the intestinal mucosa. 

Finally, the PjΔ6D protein represents no more than 0.00043% of the total protein in 

MON 87769 mature seed. 

The NcΔ15D 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 analyses demonstrated that the NcΔ15D protein does 

not share structurally or immunologically relevant amino acid sequence similarities with 

known allergens and, therefore, is highly unlikely to contain immunologically crossreactive allergenic epitopes. Digestive fate experiments conducted with the NcΔ15D 

protein demonstrated that the full-length protein is rapidly digested in SGF, a 

characteristic shared among many proteins with a history of safe consumption. The 

transiently stable protein fragments from the SGF assay of NcΔ15D were very quickly 

degraded during short exposure to SIF. Rapid digestion of the full-length NcΔ15D 

protein in SGF and SIF, together with rapid degradation of the transiently stable fragment 

from the SGF assay by SIF, indicates that it is highly unlikely that the NcΔ15D protein 

and its fragment will reach absorptive cells of the intestinal mucosa. Finally, the 

NcΔ15D 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 PjΔ6D and NcΔ15D proteins do 

not pose a significant allergenic risk. 

VI.E. Assessment of the Potential for Toxicity of the PjΔ6D and NcΔ15D Proteins 

The PjΔ6D and NcΔ15D proteins have been assessed for their potential toxicity. 

Homologues of NcΔ15D and PjΔ6D are present in all kingdoms, from prokaryotes to 
Monsanto Company 09-SY-200U Page 98 of 395 

humans. Many organisms that either are consumed as food or used to produce food 

constituents contain homologous desaturases, supporting the general familiarity of these 

proteins in human and animal diets. The proteins lack structural similarity to known 

toxins or biologically active proteins known to have adverse effects to mammals. The 

PjΔ6D and NcΔ15D proteins are present at a very low level in the harvested seed of 

MON 87769 and, therefore, constitute a negligible portion of the total protein present in 

food and feed derived from MON 87769. The PjΔ6D and NcΔ15D proteins were readily 

digestible in simulated gastric and intestinal fluids. 

Acute toxicology studies were conducted with PjΔ6D and NcΔ15D purified from 

MON 87769 immature seed. The PjΔ6D and NcΔ15D 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 PjΔ6D and 47,000 for 

NcΔ15D. Estimated dietary intake of the PjΔ6D and NcΔ15D proteins by animals was 

at extremely low levels compared to the consumption of total proteins present in animal 

feed. These data indicate that the PjΔ6D and NcΔ15D proteins are safe for human and 

animal consumption. 

 
Monsanto Company 09-SY-200U Page 99 of 395 

VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENT OF MON 87769 

Compositional analyses were conducted to assess whether the nutrients and antinutrient 

levels in harvested seed and forage derived from MON 87769 are comparable to those in 

the conventional soybean control, A3525. In addition, 10 commercial conventional 

soybean varieties were included in the analysis as references to establish a range of 

natural variability for each analyte, where the range of variability is defined by a 99% 

tolerance interval for that particular analyte. Compositional analysis included the 

significant nutrients, antinutrients, 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 polyunsaturated fatty acids, small amounts of 

the trans-isomers of SDA and ALA that are formed 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 and 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 conventional soybean control were harvested 

from soybean grown in three replicated plots at each of five sites across the U.S., during 

the 2006 growing season. Samples were 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 varieties were analyzed for compositional 

components. Appendix E describes the materials 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 for 49 components (42 in seed and seven in 

forage). The overall data set was examined for evidence of biologically relevant changes 

using a mixed model of variance. Six sets of statistical analyses were conducted, five 

based on the data from each of the replicated field sites (individual-site) and the sixth 

analysis based on data from a combination of all five field sites (combined-site). The 

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% level 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 intentionally changed. This interval is expected to 

contain, with 95% confidence, 99% of the values obtained from the population of 

commercial reference varieties. It is important to establish the 99% tolerance interval 

from representative conventional soybean varieties for each of the analytes, because such 
Monsanto Company 09-SY-200U Page 100 of 395 

data illustrate the compositional variability that occurs naturally in commercially grown 

varieties. By comparison to the 99% tolerance interval, any significant difference 

(p<0.05) between MON 87769 and the conventional control can be assessed for 

biological relevance in the context 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 Sciences Institute Crop Composition Database 

(ILSI-CCD, 2008).2

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

MON 87769 was developed to contain SDA at levels 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 Δ6 desaturation of linoleic acid (LA) by 

PjΔ6D (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%. Lower levels of two other fatty acids, trans-SDA 

(mean = 0.18%, range = 0.058 - 0.26% of total 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 extraction and processing. Trans 

isomerization of unsaturated fatty acids are known 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 analysis of MON 87769. 

Since SDA, GLA, and the two trans-isomer analytes were not detected in the 

conventional soybean control seed, statistical comparisons between MON 87769 and the 

conventional soybean control were not possible 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-site analysis between harvested seed from 

MON 87769 and the conventional soybean control, a total of 28 statistically significant 

differences, representing six fatty 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 the conventional soybean control in the 

combined-site analysis. A summary of the significant differences (p<0.05) from the 

combined-site statistical evaluation of MON 87769 versus conventional soybean control 

can be found in Table VII-2. Of the 28 detected differences in seed, 27 (except LA) were 

small in magnitude and/or their mean component values were all within the 99% 

tolerance interval for the population of conventional references grown at the same time 

and location as MON 87769 and also within the range of values found in the published 

literature and ILSI-CCD values. Therefore, these 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 analysis and outside of 
Monsanto Company 09-SY-200U Page 102 of 395 

the 99% tolerance interval for the population of conventional references. These results 

were expected, because LA is the starting material from which SDA and GLA are 

produced and, therefore, its levels were expected to be significantly different in 

MON 87769 seed compared to conventional soybean seed. 

Further assessment of the statistically significant differences observed between 

MON 87769 and the conventional soybean control are discussed in the sections below. 

The analytes that are significantly different between MON 87769 and the conventional 

soybean control in the combined-site analysis are presented in Table VII-2. The 

statistical summaries of the combined-site analysis data, individual-site 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 between 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 VII-2). In MON 87769 seed, oleic acid, LA, and 

ALA were found to be significantly different from the conventional soybean control in 

the combined-site and in the five individual-site analyses. Since oleic acid, LA, and ALA 

are directly involved in the pathway to SDA (Figure VII-1), their concentrations are 

interdependent with that of other fatty acids and, therefore, were expected to be different 

in MON 87769 seed. Arachidic acid was found to be significantly different from the 

control in the combined-site analysis and in four of the five individual-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 differences 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 varieties. Therefore the differences in fatty acid levels with 

the exception of the intended change are not considered to be biologically 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 levels 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. Nineteen nutrient analytes in the seed were 

statistically different (p<0.05) between MON 87769 and the conventional control in the 

combined-site analysis. For five of these 19 nutrient analytes (proline, arginine, cystine, 

glycine, and phenylalanine), statistically significant differences were observed in the 

combined-site analysis and in more than one individual-site analyses (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 analyses. The magnitude of the differences 

between MON 87769 and conventional soybean control for arginine, cystine, glycine, 

and phenylalanine also were very small (≤ 10%). The mean and range values for proline, 

arginine, cystine, glycine, and phenylalanine 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 different between MON 87769 

and conventional control (aspartic acid, glutamic 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 

Δ9 Δ12 

Δ6 

18:3 

γ-linolenic 

acid 

18:4 

stearidonic 

acid 

Δ15 

Δ6 

Δ15 
Monsanto 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, methionine, serine, threonine, tyrosine and 

carbohydrates). The magnitude of these differences 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 the 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 Antinutrient Levels in Soybean Seed 

Soybean seed contains several well-described antinutritional factors according to OECD 

(2001), which include: trypsin inhibitors, lectins, isoflavones (genistein, daidzein and 

glycitein), stachyose, raffinose, and phytic acid. The analytes that are significantly 

different between MON 87769 and the conventional soybean control are presented in 

Table VII-2. 

Trypsin inhibitors are heat-labile antinutrients that interfere with the digestion of proteins 

and result in decreased animal growth (Liener, 1994). Lectins are also heat labile, and 

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 processed appropriately, the final edible soybean 

fractions contain minimal levels of these antinutrients. No statistical differences were 

observed for all comparisons of trypsin inhibitors 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 have been reported to possess biochemical 

activities, including estrogenic, anti-estrogenic, and hypocholesterolemic effects, it is not 

universally accepted that the isoflavones are antinutrients as they have also been reported 

to have beneficial anti-carcinogenic effects (OECD, 2001). Genistein 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 analyses were lower than the 

values observed in the conventional soybean control (Table VII-2). Glycitein levels in 

MON 87669 were found to be significantly lower 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 influenced 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 established from conventional soybean varieties, and 

within the literature and ILSI-CCD (Tables VII-2 and E-14). Therefore, these differences 
Monsanto 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 weight carbohydrates present in soybean seed 

that are considered to be antinutrients due to the gas production and resulting flatulence 

caused by consumption. There were no significant differences (p>0.05) observed for 

stachyose and raffinose between MON 87769 and the conventional soybean control 

(Table VII-2). 

Phytic acid is present in soybean seed, where 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 significantly 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 calculated 99% tolerance 

interval for the population of conventional references grown at the same time and 

locations, therefore, the two differences found in one individual-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 Section 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 isomerization of polyunsaturated fatty acids during 

the seed extraction and processing, small amounts of the trans-isomers of SDA and ALA 

are also seen in MON 87769 compostion analysis. In addition, the forage from 

MON 87769 is compositionally equivalent to conventional soybean forage. 
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositiona

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties 

Mean Difference 

(Test minus Control) 

Component (Units)1 

MON 87769 

Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Test 

Range 

Statistical Differences Observed in Combined-Site Analysis Seed Amino Acid (% DW) Alanine 1.78 1.74 2.51 0.001 [1.76 - 1.84Arginine 3.23 2.95 9.35 <0.001 [3.00 - 3.61Aspartic Acid 4.54 4.36 4.04 0.007 [4.41 - 4.73Cystine 0.62 0.6 3.23 <0.001 [0.56 - 0.65Glutamic Acid 7.63 7.29 4.7 <0.001 [7.42 - 7.90Glycine 1.79 1.73 3.6 0.003 [1.76 - 1.87Histidine 1.09 1.05 3.42 <0.001 [1.06 - 1.14Isoleucine 1.87 1.78 4.95 <0.001 [1.75 - 1.97Leucine 3.19 3.09 3.28 <0.001 [3.13 - 3.32Lysine 2.67 2.6 2.69 <0.001 [2.61 - 2.75Methionine 0.6 0.58 2.99 0.038 [0.54 - 0.62Phenylalanine 2.14 2.06 3.63 0.002 [2.08 - 2.24Proline 2.09 1.99 5.13 <0.001 [2.03 - 2.19Serine 2.2 2.14 2.55 0.043 [2.08 - 2.25
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositio

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (C

Mean Difference 

(Test minus Control) 

Component (Units)1 MON 87769 Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Te

Ran

Seed Amino Acid (% DW) 

Threonine 1.6 1.57 1.9 0.035 [1.54 - Tyrosine 1.4 1.34 4.43 0.013 [1.27 - Valine 1.98 1.88 5.08 <0.001 [1.84 - Seed Fatty Acid (% Total FA)16:0 Palmitic 12.06 11.77 2.5 <0.001 [11.53 - 18:1 Oleic 15.18 19.19 -20.92 0.001 [12.66 - 18:2 Linoleic 22.78 54.93 -58.53 <0.001 [16.46 - 18:3 Linolenic 11.18 9.2 21.51 0.016 [10.20 - 20:0 Arachidic 0.34 0.31 9.88 <0.001 [0.31 - 22:0 Behenic 0.29 0.32 -8.3 0.023 [0.26 - Seed Proximate (%DW) Carbohydrates 36.45 38.68 -5.78 <0.001 [33.23 - Protein 41.92 39.75 5.47 <0.001 [40.92 - Seed Isoflavone (μg/g DW) Daidzein 1187.81 1807.36 -34.28 0.006 [957.23 - Genistein 733.64 1136.52 -35.45 0.007 [576.70 - 
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositio

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (C

Mean Difference 

(Test minus Control) 

Component (Units)1 MON 87769 Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Te

Ran

Glycitein (μg/g DW) 82.73 102.18 -19.04 0.004 [65.37 - Statistical Differences Observed in More than One Individual Site Seed Amino Acid (% DW)Arginine Site IA-2 3.42 3.11 10.03 0.003 [3.36 - Arginine Site OH 3.11 2.86 8.67 0.012 [3.07 - Cystine Site IA-2 0.62 0.6 4.05 0.005 [0.62 - Cystine Site IL 0.63 0.61 3.21 0.01 [0.62 - Glycine Site IA-1 1.8 1.72 4.38 0.046 [1.79 - Glycine Site OH 1.76 1.71 2.87 0.024 [1.76 - Phenylalanine Site IA-1 2.13 2.03 5.18 0.024 [2.08 - Phenylalanine Site OH 2.12 2.05 3.69 0.015 [2.12 - Proline Site IA-1 2.12 2.0 5.77 0.039 [2.11 - Proline Site IA-2 2.12 2.06 2.94 0.014 [2.09 - Proline Site OH 2.05 1.95 4.98 0.011 [2.03 - Seed Fatty Acid (% Total FA) 16:0 Palmitic Site IL 12.31 12.0 2.51 0.024 [12.24 – 16:0 Palmitic Site MI 12.11 11.79 2.66 0.019 [12.03 - 18:1 Oleic Site IA-1 13.42 18.45 -27.29 0.001 [13.14 - 
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositio

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (C

Mean Difference 

(Test minus Control) 

Component (Units)1 MON 87769 Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Te

Ran

18:1 Oleic Site IA-2 13.56 18.53 -26.84 0.003 [12.93 - 18:1 Oleic Site IL 17.89 20.89 -14.35 0.01 [17.52 - 18:1 Oleic Site MI 12.92 17.44 -25.95 <0.001 [12.66 - 18:1 Oleic Site OH 18.1 20.65 -12.31 0.046 [16.73 - 18:2 Linoleic Site IA-1 18.46 54.9 -66.39 <0.001 [18.24 - 18:2 Linoleic Site IA-2 21.19 55.33 -61.7 <0.001 [20.36 - 18:2 Linoleic Site IL 30.48 54.33 -43.9 <0.001 [30.26 - 18:2 Linoleic Site MI 18.4 55.6 -66.92 <0.001 [16.46 - 18:2 Linoleic Site OH 25.37 54.5 -53.45 <0.001 [25.06 - 18:3 Linolenic Site IA-1 11.11 9.85 12.85 <0.001 [11.08 - 18:3 Linolenic Site IA-2 11.14 9.96 11.84 0.018 [11.10 - 18:3 Linolenic Site IL 10.27 7.59 35.25 0.002 [10.20 - 18:3 Linolenic Site MI 11.76 10.59 10.99 0.001 [11.72 - 18:3 Linolenic Site OH 11.63 8.02 45.05 0.001 [11.41 - 20:0 Arachidic Site IA-1 0.33 0.3 10.66 0.005 [0.32 - 20:0 Arachidic Site IL 0.36 0.34 8.18 0.025 [0.36 - 
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositio

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (C

Mean Difference 

(Test minus Control) 

Component (Units)1 MON 87769 Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Te

Ran

20:0 Arachidic Site MI 0.32 0.29 10.39 0.029 [0.31 - 20:0 Arachidic Site OH 0.36 0.33 8.57 0.011 [0.35 - 22:0 Behenic Site IA-1 0.29 0.31 -8.9 0.018 [0.26 - 22:0 Behenic Site IL 0.31 0.33 -6.71 0.046 [0.30 - Seed Isoflavone (μg/g DW) Daidzein Site IA-1 995.39 1550.96 -35.82 0.001 [978.37 - Daidzein Site IA-2 1076.04 1583 -32.03 0.002 [999.02 - Daidzein Site MI 1662.22 2750.13 -39.56 0.016 [1389.19 Daidzein Site OH 1125.54 1668.07 -32.52 0.002 [1094.38 Genistein Site IA-1 594.53 973.04 -38.9 <0.001 [584.75 - Genistein Site IA-2 656.8 1044.68 -37.13 0.001 [612.27 - Genistein Site MI 1000.9 1683.74 -40.55 0.01 [841.05 - Genistein Site OH 760.07 1143.19 -33.51 0.003 [750.00 - Statistical Differences Observed in One Site Seed Amino Acid (% DW) Aspartic Acid Site OH 4.43 4.27 3.72 0.021 [4.41 - Glutamic Acid Site OH 7.51 7.17 4.67 0.02 [7.42 - Histidine Site OH 1.07 1.03 3.2 0.003 [1.06 - 
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Compositio

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (C

Mean Difference 

(Test minus Control) 

Component (Units)1 MON 87769 Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Te

Ran

Isoleucine Site OH 1.87 1.75 6.53 0.025 [1.84 - Leucine Site OH 3.16 3.05 3.42 0.016 [3.14 - Lysine Site OH 2.62 2.56 2.46 0.02 [2.61 - Valine Site OH 1.96 1.84 6.62 0.027 [1.94 - Seed Fiber (% DW) Acid Detergent Fiber Site IL 16.16 17.76 -9 0.005 [15.91 - Neutral Detergent Fiber Site IL 17.06 17.87 -4.52 0.043 [16.72 - Seed Proximate (% DW) Ash Site OH 5.83 5.71 2.12 0.022 [5.78 - Protein Site OH 41.37 39.54 4.63 0.008 [40.92 - Seed Antinutrient (% DW, unless noted) Lectin (H.U./mg DW) Site MI 1.49 3.44 -56.79 0.019 [0.55 - Phytic Acid Site IA-1 1.28 1.17 9.49 0.041 [1.22 - Seed Isoflavone (μg/g DW) Glycitein Site IL 84.86 120.49 -29.57 0.044 [83.23 - 
Monsanto Company 09-SY-200U 

Table VII-2. Summary of the Soybean Seed and Forage Analyte Differences (p<0.05) in the Composition

MON 87769 vs. the Conventional Control (A3525) and Commercial Reference Varieties (C

Mean Difference 

(Test minus Control) 

Component (Units)1 

MON 87769 

Mean 

A3525 

Mean 

Mean Difference 

(% of A3525) 

Significance 

(p-Value) 

Test 

Range 

Forage - Statistical Differences Observed in One Site Forage Fiber (% DW) Acid Detergent Fiber Site OH 32.29 28.31 14.09 0.009 [30.74 - 33.95] Forage Proximate (% DW) Total Fat Site IA-2 4.87 6.27 -22.39 0.037 [4.40 - 5.28] 

1DW = dry weight; FA = fatty acid. 2With 95% confidence, interval contains 99% of the values expressed in the population of commercial soybean varieties. Negative lim
Monsanto Company 09-SY-200U Page 113 of 395 

VII.C. Compositional Comparisons of Processed 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 soybean 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 processed into defatted toasted soybean meal (DT 

soybean meal); refined, bleached, and deodorized soybean oil (RBD oil); protein isolate; and 

crude lecithin fractions. The processed fractions were analyzed according to the principles 

outlined in the OECD consensus document for soybean composition (OECD, 2001). The DT 

soybean meal was analyzed for proximates (moisture, 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 protein 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 the processed fractions are summarized in 

Appendix E, Section E.6. The statistical 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 from the seed of MON 87769 and the conventional 

control are included in Appendix E, Section E.6. 

As expected, apart from the intended fatty acid changes, the composition of the soybean 

processed fractions from MON 87769 is equivalent to the composition of conventional 

soybean control (OECD, 2001). Thus, the 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 processed 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 Assessment 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 also decreased the levels of LA in MON 87769. 

The safety of SDA soybean oil is supported by the results of a published 90-day/onegeneration reproductive rat toxicity study in which a no observable 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 corroborate 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 derived, 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, AGRONOMIC, 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 variety 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 MON 87769 is no more likely to pose a plant 

pest risk or to have a significant environmental impact compared to conventional 

soybean. The conclusions are based on the results of the multiple evaluations reported 

herein. 

Phenotypic and agronomic characteristics of MON 87769 were evaluated in a 

comparative manner to assess plant pest potential (OECD, 1993). These assessments 

included 14 plant growth and development characteristics, five seed germination 

parameters, two pollen characteristics, six plant-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 environmental impact compared to conventional soybean. 

Data on environmental interactions also indicate that MON 87769 does not confer any 

increased susceptibility or tolerance to specific diseases, insects, or abiotic stressors. 

VIII.A. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental 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 encompasses 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) plant interactions with insect, disease, and abiotic stressors. 

An overview of the characteristics assessed 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 conventional control, A3525, which had a genetic 

background similar to MON 87769 but did not possess the genes introduced in 

MON 87769 (Pj.D6D and Nc.Fad3), the proteins produced from the introduced genes 

(PjΔ6D and NcΔ15D), 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 commercial 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 reference varieties reflect a range of selection and 

breeding for desirable characteristics and therefore 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 

timing1 

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 

assessment1 

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 Pubescence on plants in each plot 

categorized as hairy, hairless, or mixed 

Plant height Maturity; R8 Distance from the soil surface to the 

uppermost node on the main stem of five 

representative plants per plot 

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 using 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 biotechnology-derived crops are, by OECD (1993) 

standards, comparative assessments. Familiarity provides a basis from which the 

potential environmental impact of a biotechnology-derived plant can be evaluated. The 

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

developed from a well-characterized conventional plant variety. Familiarity considers 

the biology of the crop, the introduced trait, the receiving environment and the interaction 

of these factors, and provides a basis for comparative environmental risk assessment 

between a biotechnology-derived plant and its conventional counterpart. 

Expert knowledge and experience with conventionally bred soybean were the basis for 

selecting appropriate endpoints and estimating the range of responses that would be 

considered typical for soybean. Thus, assessment of phenotypic and agronomic 

characteristics and environmental interactions was essential to compare the 

biotechnology-derived plant to the conventional counterpart. An overview of the 

characteristics assessed is presented in Table VIII-1. A subset of the data relating to well 

understood weediness criteria (e.g., 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 determination. Based on all of the data collected, an assessment 

was made whether the biotechnology-derived plant is likely to pose an increased plant 

pest risk compared to the conventional counterpart. 

During the processes of data collection, summarization, and analysis, experienced 

scientists familiar with each experimental design and evaluation criteria were involved in 

all steps. This level of oversight ensured that the evaluation system was functioning 

appropriately, measurements were taken properly, and data were consistent with 

expectations based on experience with the crop. In addition, the overall dataset was 

evaluated for evidence of biologically relevant changes, and for possible evidence of an 

unexpected plant response. Data then were submitted to statistical analysis. 

VIII.C. Interpretation of Detected Differences Criteria 

Comparative plant characterization data between a biotechnology-derived crop and the 

control are interpreted in the context of contributions to increased plant pest potential as 

assessed by APHIS. Under the framework of familiarity, characteristics for which no 

differences are detected support a conclusion of no increased plant pest potential of the 

biotechnology-derived crop compared to the conventional crop. Characteristics for 

which differences are detected are considered in the step-wise method (Figure VIII-1 or a 

similar method). All detected differences for a characteristic are considered in the 

context of whether the difference would increase the plant pest potential of the 

biotechnology-derived crop. Ultimately, a weight of evidence approach considering all 

characteristics and studies was used for the overall risk assessment of differences and 

their significance. In detail, Figure VIII-1 illustrates the stepwise assessment process 

employed: 
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 terms 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 evaluated on each measured 

characteristic. Differences detected in the individual-site analysis must be observed in 

the combined-site analysis to be considered further for plant pest potential. Any 

difference detected in the combined-site analysis is further assessed. 

▪ Step 3. Evaluate Differences Relative to Reference Range. If a difference is 

detected in the combined-site analysis across multiple environments, then the test 

substance mean value is assessed relative to the reference substances. 

▪ Step 4. Evaluate Differences in the Context of the Crop. If the test substance mean 

is outside the variation of the reference substances (e.g., reference range), the test 

substance mean is considered in the context of known values common for the crop. 

▪ Step 5. Plant Pest Potential. If the test substance mean is outside the range of values 

common for the crop, the detected difference is then assessed for whether or not it is 

adverse in terms of plant pest potential. 

▪ Step 6. Conduct Risk Assessment on Identified Hazard. If an adverse effect 

(hazard) is identified, risk assessment on the difference is conducted. The risk 

assessment considers contributions to enhanced plant pest potential of the crop itself, 

the impact of differences detected in other measured characteristics, and potential for 

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 analysis 
Monsanto Company 09-SY-200U Page 119 of 395 

and effects of trait transfer to feral populations of the crop or a sexually compatible 

species. 

VIII.D. Phenotypic, Agronomic and Environmental 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 characteristics, and symbiont interactions were 

evaluated. The phenotypic, agronomic, and environmental interaction evaluations are 

based on replicated laboratory, greenhouse, and/or multi-site field trials and experiments. 

In evaluating the phenotypic and agronomic characteristics of MON 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. However, 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 germination assays are available and routinely 

used to measure the germination characteristics of soybean seed. The Association of 

Official Seed Analysts, an internationally recognized seed testing organization, 

recommends a temperature range of 20/30 °C as optimal for germination of soybean 

(AOSA, 2007) (Table VIII-2). 

Comparative assessments of seed dormancy and germination characteristics were 

conducted on MON 87769 and A3525, where A3525 served 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 commercial 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 environmentally 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 other temperature regimes of 10, 20, 30, 10/20, and 

10/30 °C to assess seed germination characteristics. 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 regimes and types of 

observation are listed in Table VIII-2. The details of the materials, experimental 

methods, and data from all individual production sites are presented in Appendix H. 

A total of 25 comparisons were made between MON 87769 and the conventional control 

for seed germination characteristics across the three seed production sites (Table VIII-3). 

No statistically or biologically significant differences were detected between MON 87769 
Monsanto Company 09-SY-200U Page 120 of 395 

and the conventional control for percent normal 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 present in the data. For 

these comparisons, the values for MON 87769 and the control were all the same, 

indicating no biological differences. 

One viable hard seed was observed at each 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 germinated seeds were detected between 

MON 87769 and the conventional control. Furthermore, the germination of both 

MON 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 commercial soybean variety, and were within the range of 

accepted germination values for certified soybean seed.

The biological characteristics evaluated were used to characterize MON 87769 in the 

context of a plant pest risk assessment. 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 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 indicative of no increased plant pest or 

weediness potential of MON 87769 relative to conventional soybean. 
Monsanto Company 09-SY-200U 

Table VIII-2. Seed Dormancy and Germination Characteristics Evaluated 

Evaluation Timing1

Temperature Regimes and Seed Characteristics Evaluated 

AOSA2 Additional Temperatures 

20/30 oC 10 oC, 20 oC, 30 oC, 10/20 oC and 10/30 o

C

Day 5 Normal germinated Germinated

Dead Dead

Day 8 Normal germinated Germinated

Abnormal germinated DeadDeadHard viable and non-viable3Firm swollen viable and non-viable3

Day 13 No data collected Germinated

DeadHard viable and non-viable3Firm swollen viable and non-viable3

1 Seed in the 20/30 oC temperature regime were evaluated in Days 5 and 8 (according to AOSA guidelines), while seed in the additiona

regimes were evaluated on Days 5, 8, and 13. 

2 Association of Official Seed Analysts (AOSA, 2007). 3 Hard 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 Characteristics of MON 87769 and A3525 

Temperature 

Regime

Germination 

Characteristic1 

Mean % (S.E.) 2 Reference Range

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 results 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 individual 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 categorized as either normal 

germinated or abnormal 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 characteristics were assessed under field conditions 

as part of the plant characterization assessment of MON 87769. These data were 

developed to provide APHIS with a detailed description of any unintended phenotypic 

differences in MON 87769 relative to the conventional soybean control, A3525, and 

commercial soybean. Environmental interactions also were assessed as an indirect 

indicator of phenotypic changes to MON 87769 relative to the same comparators 

described above. The purpose of these field evaluations was to assess whether the 

introduced genes (Pj.D6D and Nc.Fad3), the proteins produced from the introduced 

genes (PjΔ6D and NcΔ15D), 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 control. Certain growth, 

reproduction, and pre-harvest seed loss characteristics (such as lodging and pod 

shattering) can be used in the assessment of whether MON 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 locations 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. Fourteen phenotypic and agronomic 

characteristics were evaluated: 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. In addition, 

observational data were collected on plant response to abiotic stressors, disease damage, 

and arthropod damage. These data were collected four times during the growing season 

at all 21 sites over the two years to assess the susceptibility and tolerance of MON 87769 

to specific abiotic stressors, diseases, or arthropod pests compared 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. The categories and timing of phenotypic 

characteristic and environmental interaction evaluations are included in Table VIII-1. 

The methods and detailed results of these individual site data comparisons are presented 

and discussed in Appendix G, while the combined-site analyses are summarized below. 

The results of this assessment showed the introduced trait did not unexpectedly alter the 

phenotypic or agronomic characteristics of MON 87769 compared 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 (PjΔ6D and NcΔ15D), and the intended fatty acid change are 

unlikely to increase plant 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 characteristics were evaluated. In the combinedsite analysis of the data from the 2006 and 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). Furthermore, no differences in plant 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 differences between MON 87769 compared to the 

nontransformed control (A3525). A subset of these characteristics were useful to assess 

the weediness potential of MON 87769 compared to the conventional soybean control. 

The results from the 2006 and 2007 field trials demonstrate that the assessed phenotypic 

and agronomic characteristics of MON 87769 were not altered 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 (PjΔ6D and NcΔ15D), and the intended 

fatty acid change. The observed values of phenotypic and agronomic characteristics of 

MON 87769 were within the range expected for soybean. These results are indicative of 

no increased plant pest or weediness potential of MON 87769 compared to conventional 

soybean. 

 
Monsanto Company 09-SY-200U Page 125 of 395 

Table VIII-4. Field Phenotypic Evaluation 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 07-052-104n 
Monsanto Company 09-SY-200U 

Table VIII-5. Plant Growth and Development Data across 17 Locations during 2006 

MON 87769 Control Reference Range1

Phenotypic Characteristic (units) Mean (S.E.) Mean (S.E.) Minimum Maximum Early stand count (#/plot) 217.0 (8.1) 219.7 (9.1) 150.5 298.6 Seedling vigor (1-9 scale) 3.2 (0.2) 3.1 (0.2) 2.3 5.0 Days to 50% flowering2 195.4 (1.1) 195.6 (1.1) 184.7 206.5 

Flower color3 Purple Purple Purple Purple Plant pubescence3 Hairy Hairy Hairy Hairy 

Plant height (in) 36.2 (1.0) 36.1 (0.9) 30.4 43.9 

Lodging (0-9 scale) 1.1 (0.2) 1.3 (0.2) 0.0 4.3 Pod shattering (0-9 scale) 0.3 (0.1) 0.3 (0.1) 0.0 1.5 Final stand count (#/plot) 199.6 (8.0) 203.4 (8.2) 154.3 271.3 Seed moisture (%) 12.2 (0.3) 12.2 (0.3) 11.0 16.0 100 seed weight (g) 15.1 (0.2) 15.4 (0.2) 14.0 18.0 Test weight (lb/bu)4 56.5 (0.5) † 56.1 (0.4) † 49.7 57.7 

Yield (bu/ac)4 52.7 (1.7) † 55.7 (1.9) † 28.8 66.9 Note: The experimental design was a randomized complete block. S.E. = Standard Error. Means based on n = 51, except w

which means are based on n = 48. No statistically significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525). 1 Reference range = Minimum and maximum mean values among 18 commercial reference soybean varieties. 2 Calendar day number when approximately 50% of the plants in each plot were flowering. 3 Flower color and plant pubescence data were categorical and not statistically analyzed (see Appendix G, Table G-3). 4 Test weight and yield data from the IA4 site were excluded from the statistical analysis because an inaccurate measuring 

collect the data. 
Monsanto Company 09-SY-200U 

Table VIII-6. Plant Growth and Development Data across Four Locations during 2007 

MON 87769 Control Reference Range1

Phenotypic Characteristic (units) Mean (S.E.) Mean (S.E.) Minimum Maximum Early stand count (#/plot) 357.8 (28.1) 354.2 (31.6) 180.0 492.7 Seedling vigor (1-9 scale) 2.8 (0.4) 2.5 (0.3) 1.0 6.0 Days to 50% flowering2 205.4 (1.5) 205.3 (1.5) 201.5 211.7 

Flower color3 Purple Purple Purple Purple 

Plant pubescence3 Hairy Hairy Hairy Hairy Plant height (in) 38.2 (2.4) 39.3 (1.6) 30.6 49.4 

Lodging (0-9 scale) 1.7 (0.6) 1.8 (0.6) 0.0 3.7 Pod shattering (0-9 scale) 0.2 (0.1) 0.0 (0.0) 0.0 0.3 Final stand count (#/plot) 331.6 (21.8) 339.6 (21.3) 158.7 455.7 Seed moisture (%) 11.8 (0.3) 11.7 (0.3) 10.6 12.2 100 seed weight (g)4 17.3 (0.9)† 17.0 (0.8)† 13.9 20.0 

Test weight (lb/bu) 54.2 (0.9) 54.5 (0.7) 51.2 57.0 Yield (bu/ac) 46.4 (1.6) 50.4 (2.0) 39.1 53.5 Note: The experimental design was a randomized complete block. S.E. = Standard Error. Means based on n = 12, except w

which means are based on n = 9. No statistically significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525). 1 Reference range = Minimum and maximum mean values among 12 commercial reference soybean varieties. 2 Calendar day number when approximately 50% of the plants in each plot were flowering. 3 Flower color and plant pubescence data were categorical and not statistically analyzed (see Appendix G, Table G-5). 4 100 seed weight data from the WI site were excluded from the statistical analysis because the balance used to weigh the s

sufficient 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 characteristics of MON 87769, data were 

collected on plant response to abiotic stressors (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 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 (Appendix I; Tables I-1 

through I-10). These data are used as part of the environmental risk assessment to 

evaluate plant pest potential 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 differences in plant response to abiotic stressors, 

disease damage, arthropod damage, or the abundance 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 (PjΔ6D and NcΔ15D), and the intended fatty acid 

change are unlikely to be biologically meaningful in terms 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, and arthropod pests were “natural” (i.e., 

no artificial infestation or interference was used) and, therefore, 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 replicatons at each site. MON 87769 and the 

control were considered qualitatively different in response to a stressor on a particular 

observation date if the range of ratings across 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 collected quantitatively from plots three times 

during the growing season at four sites each in 2006 and 2007 to assess the interactions 

between MON 87769 and these arthropods. 

A total of 703 comparative observations of plant response to abiotic stressors, disease 

damage, and arthropod damage and 260 comparative observations of the abundance of 

various pest and beneficial arthropods were conducted in field trials over two consecutive 

years to assess environmental interactions of MON 87769 compared to the conventional 

control. No biologically meaningful differences were observed 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, diseases, or arthropod pests or in 

their interactions with specific pest and beneficial arthropods, as indicated by the 
Monsanto 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 abiotic stressors, diseases, 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 (PjΔ6D and NcΔ15D), and the intended 

fatty acid change, compared to the conventional control and were within the range 

expected for soybean. These results are indicative of no increased plant pest potential or 

adverse ecological interactions of MON 87769 relative to conventional soybean. 

VIII.D.3. Pollen Characteristics 

In determining the potential for a biotechnology-derived plant to increase weedy or 

invasive characteristics in other plants, APHIS considers the potential for gene flow and 

introgression of the biotechnology-derived trait into other plant varieties or wild 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 conventional 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 assess the morphology and viability of pollen 

collected from MON 87769 compared to a conventional 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 block design with three replications. A 

minimum of 20 flowers were collected from each plot. Pollen was extracted, combined 

among flowers collected from the same plot, and stained with Alexander’s stain 

(Alexander, 1980). Pollen viability was evaluated for each sample and pollen grain 

diameter was measured for ten representative viable pollen grains per replication. 

General morphology of the pollen was observed for each of the three replications of 

MON 87769, the control, and the reference soybean varieties (see Appendix J). 

No statistically significant differences 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 general pollen morphology were observed between 

MON 87769 and the control. These results demonstrate that the introduction of the genes 

(Pj.D6D and Nc.Fad3), the proteins produced from the introduced genes (PjΔ6D and 

NcΔ15D), 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 differences between the pollen collected from 

MON 87769 compared to the conventional soybean control for the assessed 

characteristics demonstrate that the observed 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 Range1 

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 maximum 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 the formation of root nodules, 

providing an environment in which differentiated bacteria called bacteroids are capable 

of reducing or “fixing” atmospheric nitrogen. The product of nitrogen fixation, 

ammonia, can then be utilized by the plant. In soybean, atmospheric nitrogen is fixed 

into organic nitrogen through a symbiotic association with the bacterium Bradyrhizobium 

japonicum. As a result of this relationship, no nitrogen inputs are needed for agricultural 

production of soybean. 

As part of the plant pest risk assessment, APHIS considers the impact of the 

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., the 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 been altered as a result of the 

introduction of the genes (Pj.D6D and Nc.Fad3), the proteins produced from the 

introduced genes (PjΔ6D and NcΔ15D), and the intended fatty acid change, compared to 

the conventional soybean control. 

The relative effectiveness of the symbiotic association between a leguminous plant and 

its rhizobial symbiont can be assessed by various methods. Assessment of nodule 

number and mass along with plant growth and nitrogen status are commonly used to 

assess differences in the symbiotic association between a legume and its associated 

rhizobia (Israel et al., 1986). It should be noted, however, that nodule number relative to 

nodule dry weight may be variable in soybean experiments because some nodules may be 

larger in diameter and less numerous while others are not as developed (smaller) but 

more abundant (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 planted 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 randomized complete block design with eight 

replicates. At four and/or six weeks after emergence, plants 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 enumeration and determination of dry 

weight. Detailed information on materials and 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, results of the study were variable and did not provide 

conclusive evidence to assess the objectives of the study. Therefore, a subsequent study 

was conducted using a refined methodology in which a negative isoline control was 

included to determine whether differences detected between MON 87769 and the 

conventional control in the initial study were associated with the trait or the plant 

transformation process. Additionally, reference soybean varieties representing more 

diverse soybean germplasm were used to determine whether the measured endpoint 

values of MON 87769 are within the range of responses familiar to what is expected for 

soybean. The results from both studies were considered in assessing the impact from the 

introduction of the genes (Pj.D6D and Nc.Fad3), the proteins produced from the 

introduced genes (PjΔ6D and NcΔ15D), 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 results from both studies are summarized below 

and in Appendix K. 

In the initial study, four out of the ten measurement endpoints assessed among the four 

and six week sampling times were significantly different between MON 87769 and the 

control, although the detected differences were not consistent among the two sampling 

times (Table VIII-8). Soybean varieties 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 conventional control variety A3525, it was 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 (PjΔ6D and NcΔ15D), and the intended fatty acid change or were due 

to inherent variability within the A3525 variety. In addition, the mean values for both 

MON 87769 and the control were greater than the 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 reference 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 whether the measured endpoint 

values of MON 87769 are within the range of responses 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 included as a control, in addition 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 trait or the plant transformation 

process. Additionally, six conventional reference soybean varieties representing more 

diverse soybean germplasm from maturity groups 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 plants were at approximately the R4 (full 

pod) growth stage and not at four weeks after 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 strip-plot design used in the 

initial study. In addition, smaller pots and a single B. japonicum inoculation, instead of 

two inoculations (as was performed in the initial study), were used to further reduce 

variability within the test system. The concentration of inoculum was reduced from 

approximately 1 × 108 cells to approximately 1 × 107 cells of B. japonicum due to the use 

of smaller pots and to reduce the potential for pseudo-nodulation. MON 87769 was 

compared to the conventional control as well as to the negative isoline for nodule number, 

nodule dry weight, shoot dry weight, root dry weight, and shoot nitrogen. A summary of 

the results is provided below, while the details of the materials and methods are provided 

in Appendix K. 

No statistically significant differences were detected between MON 87769 and either the 

conventional or negative isoline control for any measured parameter, including nodule 

number, shoot nitrogen (percent and total nitrogen), and biomass (dry weight) of nodules, 

shoot material, or root material (Table VIII-9). Apart from shoot total nitrogen (% dry 

weight), each measurement endpoint evaluated 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 less than or equivalent to the percent 

differences between MON 87769 and the conventional control for five of the six 

measurement endpoints (Table VIII-10). 

The results from the refined assessment support the hypothesis that differences between 

MON 87769 and the conventional control observed in the first study were not attributed 

to the introduction of the genes (Pj.D6D and Nc.Fad3), the proteins produced from the 

introduced genes (PjΔ6D and NcΔ15D), and intended fatty acid change or plant 

transformation process. The results also demonstrate 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 (PjΔ6D and NcΔ15D), and 

intended fatty acid change does not alter the symbiotic relationship between B. japonicum 
Monsanto Company 09-SY-200U Page 133 of 395 

and MON 87769 compared to that of conventional soybean. Thus, there is no expected 

impact to cultivation practices relative to nitrogen inputs and no increased plant pest 

potential of MON 87769 compared to conventional soybean. 
Monsanto Company 09-SY-200U 

Table VIII-8. Symbiont Interaction Assessment of MON 87769 and the Conventional Control at 4- anafter Emergence in an Initial Assessment 

Measurement 

Endpoint 

 Mean (S.E.) Reference Range1

Samplin

g

Time 

MON 87769 Control Minimum Maxim

Nodule Number (per plant) 

4 week 77.63 (2.91) 68.29 (4.26) 44.25 75.8

6 week 180.13 (11.39)* 140.50 (14.56) 60.00 128.2

Nodule Dry Wt (g/plant) 

4 week 0.13 (0.01)* 0.10 (0.01) 0.07 0.6 week 0.30 (0.03) 0.25 (0.02) 0.13 0.2

Root Dry Wt (g/plant) 

4 week 0.89 (0.05) 0.82 (0.05) 0.81 0.8

6 week 1.94 (0.07) 1.71 (0.13) 1.35 1.6

Shoot Dry Wt (g/plant) 

4 week 1.81 (0.14) 1.49 (0.08) 1.33 1.5

6 week 4.57 (0.25)* 3.50 (0.30) 2.64 3.7

Shoot Total Nitrogen  (% dry wt) 

4 week 2.83 (0.10) 2.74 (0.10) 2.34 2.4

6 week 2.54 (0.05)* 2.80 (0.09) 2.11 2.2

Note: Pots were arranged in eight replicated blocks (n = 8) in a greenhouse for 4- and 6-week sampling periods using 

strip-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 
Monsanto Company 09-SY-200U 

Table VIII-9. Symbiont Interaction Assessment of MON 87769, the Conventional Control, and the Ne6-Weeks after Emergence in a Refined Assessment 

 Mean (S.E.) Referenc

Measurement 

Endpoint 

MON 

87769 A3525 Negative Isoline Minimum 

Nodule Number (per plant) 208 (24) 185 (23) 206 (16) 83 Nodule Dry Wt (g/plant) 0.56 (0.04) 0.49 (0.06) 0.53 (0.04) 0.40 Root Dry Wt (g/plant) 1.53 (0.12) 1.63 (0.08) 1.57 (0.17) 1.08 Shoot Dry Wt (g/plant) 6.21 (0.54) 6.29 (0.32) 6.36 (0.52) 5.05 Shoot Total Nitrogen  (% dry wt) 3.77 (0.06) 3.66 (0.07) 3.74 (0.13) 2.85 Shoot Total Nitrogen (g) 0.23 (0.02) 0.23 (0.01) 0.23 (0.01) 0.19 

Note: Pots were arranged in a greenhouse in a randomized complete block design with eight replications (n = 8). S.E

No significant differences were detected (p>0.05) between MON 87769 and the conventional control (A3525) or the nmeasured endpoint. 

1 Reference range is the minimum and maximum mean value observed among the six conventional reference soybean v
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 (PjΔ6D and NcΔ15D), and the intended fatty acid change altered the 

plant pest potential of MON 87769 compared to the conventional soybean control, 

A3525. Phenotypic and agronomic characteristics of MON 87769 were evaluated and 

compared to those of the conventional soybean control. These assessments included 14 

plant growth and development characteristics; five seed dormancy and germination 

parameters under six different temperature regimes; two pollen characteristics; more than 

960 observations for plant interactions with arthropods, diseases, and abiotic stressors 

under field conditions; six plant-symbiont interaction characteristics; and compositional 

evaluation (Section VII) of 75 different components (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 pest potential compared to 

conventional soybean. Data on environmental interactions indicate that MON 87769 

does not confer any biologically meaningful increased susceptibility or tolerance to 
Monsanto Company 09-SY-200U Page 137 of 395 

specific diseases, arthopods, or abiotic stressors, 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 key 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 and diseases, soybean rotational crops, and 

volunteer soybean management. Compared to traits such as herbicide tolerance or 

resistance to insects that directly benefit the growers, the production of SDA through 

cultivation of MON 87769 is neutral with respect to agronomic 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 the 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 impacts on agronomic practices used for production 

of specialty type soybean upon deregulation of MON 87769. 

Soybean is planted in over 30 states, demonstrating its wide adaptation to soils and 

climate. The soil, moisture, and temperature requirements for producing soybean are 

generally similar to those for corn and thus the two crops share a similar cultivation area. 

Proper seedbed preparation, appropriate variety selection, proper planting dates and plant 

population, and good integrated pest management practices are important to optimizing 

the yield potential and economic returns of soybean. 

MON 87769 is expected to bring added value for growers, soybean handlers, crushers 

and food processors. An identity preservation system will be used for production, postharvest handling and processing to preserve the enhanced value of MON 87769. As such, 

production practices, post-harvest handling of soybean, and processing will fall under a 

separate production system and distribution channel termed for this specialty soybean. 

MON 87769 is expected to utilize existing agricultural practices employed for production 

and identity preservation of specialty soybean. 

Annual and perennial weeds are perceived to be the greatest pest problem in soybean 

production. Economic thresholds for controlling weeds in soybean require some form of 

weed management practice on all soybean acreage. Approximately 98% of the soybean 

acreage receives an herbicide application. Soybean insects and diseases are generally 

considered less problematic, although infestations do occasionally reach economic 

thresholds requiring treatment. 

Volunteer soybean is not considered a significant concern in rotational crops, primarily 

because of climatic conditions and adequate control 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 potential, introduction of 

MON 87769 in the soybean production system would have a negligible impact on 

managing soybean volunteer plants in rotational crops such as corn, cotton, and rice 

because control measures are available for volunteer soybean when they arise. Pre-plant 

tillage is the first management tool for control of emerging volunteer soybean in the 

spring. If volunteer soybean emerges 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 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 commercialization 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 18th century (Hoeft et al., 2000). Sometime 

during the 1930s, soybean started to be processed industrially in the U.S. for edible oil 

and protein meal. Currently, the U.S. produces approximately 32% of the global soybean 

supply (ASA, 2008). In 2007, the U.S. exported 1 billion bushels (27.9 million metric 

tons) of soybean, which accounted for 37 percent of the world's soybean exports (ASA, 

2008). In total, the U.S. exported $12.9 billion USD worth of soybean and soybean 

products globally in 2007 (ASA, 2008). China is the largest export market for U.S. 

soybean with purchases totalling $4.1 billion. Japan is the second largest export market 

with sales of $1.1 billion in the same year. Other significant markets include the 

European Union and Mexico. 

The production of soybean is highly dependent upon soil and climatic conditions. In the 

U.S., the soil and climatic requirements for growing soybean are very similar to corn. 

The soils and climate in the eastern 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 structure are key components 

determining water availability in soils, where medium-textured soils hold more available 

water, allowing soybean roots to penetrate deeper in medium-textured soils than in clay 

soils. Irrigation is used on approximately 9% of the acreage to supplement the water 

supply during dry periods in the western and southern soybean growing regions (ASA, 

2008). 

Most of the soybean acreage is grown as a full-season crop. Approximately 8% of the 

soybean acres are planted in a double-crop system following winter wheat south of 35o 
Monsanto 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 decision to plant double-crop soybean is influenced 

by both agronomic and economic factors. Agronomic factors include harvest date of the 

wheat crop, which determines the double-crop soybean planting date, and available soil 

moisture. Economic factors include expected soybean price and anticipated economic 

return (Boerma and Specht, 2004). 

The vast majority of soybean grown in the U.S. is grown for animal feed and is usually 

fed as soybean meal. However, approximately 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 Herbek, 2004; Muth et al 2003; and Smyth and 

Phillips 2002) for the soybean variety. Product differentiation and market segmentation 

in the specialty soybean industry includes mechanisms to keep track of the soybean 

(traceability), methods for identity preservation (IDP), including closed-loop systems, 

and quality assurance processes (e.g., ISO9001-2000 certification), as well as contracts 

between growers and buyers that specify delivery agreements. MON 87769 is an 

alternate source of omega-3 fatty acid and will be considered a specialty soybean product 

and marketed in a manner similar to other high-value specialty crops.

The U.S. soybean acreage in the past ten years has varied from approximately 64.7 to 

75.7 million acres, with the lowest acreage recorded in 2007 and the highest in 2008 

(Table IX-1). Average soybean yields have varied from 33.9 to 43.3 bushels per acre 

over this same time period. Soybean production ranged from 2.45 to 3.19 billion bushels 

over the past ten years, with 2006 being the largest production year on record. According 

to data from USDA-NASS (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 down significantly from 2006, mainly due 

to a large increase in corn acreage. The value of soybean reached $27.4 billion in the 

U.S. in 2008 (USDA-NASS, 2009). In comparison, corn and wheat values in 2008 were 

$47.37 and $16.57 billion, respectively (USDA-NASS, 2009). 

For purposes of this agronomic practices discussion, soybean production is divided into 

three major soybean growing regions accounting for 99.1% of the 2008 U.S. soybean 

acreage: Midwest/Great Plains region (IL, IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, 

SD, and WI), Southeast region (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern 

Coastal region (DE, MD, NJ, NY, PA, and VA) (Table IX-2). The vast majority of 

soybean was grown in the Midwest region, representing 82.1% of the total U.S. acreage. 

 

3

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4

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Monsanto 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 average state yields in this region ranged from 

28.0 to 47.0 bushels per acre. The average yield in the Southeast region was 34.4 bushels 

per acre, with states within this region averaging from 30.0 to 40.0 bushels per acre. The 

average yield in the Eastern Coastal region was 34.1 bushels per acre, with individual 

state averages ranging from 27.5 to 46 bushels 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 less than 3.5 million acres (~5% of the total 

U.S. soybean acreage). MON 87769, as a speciality 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 anticipated 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 Research Service (USDA-ERS, 2006). The 

value of the production less operating cost was reported to be $161.43 per acre. A 

summary of potential production costs and returns are presented in Table IX-3. 

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

Year 

Acres 

Planted 

(×1000) 

Acres 

Harvested

(×1000) 

Average 

Yield 

(bushels/acre)

Total 

Production 

(×1000 bushels) 

Value 

(billions $) 

2008 

2007 

75,718 

64,741 

74,641 

64,146 

39.6 

41.7 

2,959,174 

2,677,117 

27.40 

26.97 

2006 75,522 74,602 42.7 3,188,247 20.42 

2005 72,142 71,361 43.3 3,086,432 16.93 

2004 75,208 73,958 42.2 3,123,686 17.89 

2003 73,404 72,476 33.9 2,453,665 18.01 

2002 73,963 72,497 38.0 2,756,147 15.25 

2001 74,075 72,975 39.6 2,890,682 12.61 

2000 74,266 72,408 38.1 2,757,810 12.47 

1999 73,730 72,446 36.6 2,653,758 12.21 

Source: USDA-NASS, 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 

Planted1

(thousands)

Acres 

Harvested1

(thousands)

Average Yield1

(bushels/acre)

Total 

Production1

(×1000 bushels)

Value1

(billions $)

Midwest Region

Illinois 9,200 9,100 47.0 427,700 4.00

Indiana 5,450 5,430 45.0 244,350 2.27

Iowa 9,750 9,670 46.0 444,820 4.29

Kansas 3,300 3,250 37.0 120,250 1.03

Kentucky 1,390 1,380 34.0 46,920 0.42

Michigan 1,900 1,890 37.0 69,930 0.64

Minnesota 7,050 6,950 38.0 264,100 2.54

Missouri 5,200 5,030 38.0 191,140 1.72

Nebraska 4,900 4,860 46.5 225,990 2.12

North Dakota 3,800 3,760 28.0 105,280 0.96

Ohio 4,500 4,480 36.0 161,280 1.55

South Dakota 4,100 4,060 34.0 138,040 1.25

Wisconsin 1,610 1,590 35.0 55,650 0.51

Region Totals 62,150 61,450 38.6 2,495,450 23.30

Southeast Region

Alabama 360 350 35.0 12,250 0.12

Arkansas 3,300 3,250 38.0 123,500 1.09

Georgia 430 415 30.0 12,450 0.11

Louisiana 1,050 950 33.0 31,350 0.29

Mississippi 2,000 1,960 40.0 78,400 0.69

North Carolina 1,690 1,670 33.0 55,110 0.47

South Carolina 540 530 32.0 16,960 0.15

Tennessee 1,490 1,460 34.0 49,640 0.43

Region Totals 10,860 10,585 34.4 379,660 3.35

Eastern Coastal Region

Delaware 195 193 27.5 5,308 0.05

Maryland 495 485 30.0 14,550 0.13

New Jersey 92 90 29.0 2,610 0.02

New York 230 226 46.0 10,396 0.09

Pennsylvania 435 430 40.0 17,200 0.15

Virginia 580 570 32.0 18,240 0.16

Region Totals 2027 1994 34.1 68,304 0.60

1 Source: USDA-NASS, 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 Category Itemized Costs

Return per Planted 

Acre ($ USD)

 

Total Gross Value of Production 254.84

 

Operating Costs: Seed 32.30

Fertilizer 13.05

Chemicals 14.46

Custom operations 6.01

Fuel, lube and electricity 13.51

Repairs 11.80

Purchased irrigation 

water

0.11

Interest on operating 

capital

2.17

Total, operating costs 93.41

Hired labor 1.78

Allocated overhead: Opportunity cost of 

unpaid grower’s labor

15.20

Capital recovery of 

machinery and equipment

60.38

Opportunity cost of land 

(rental rate)

86.17

Taxes and insurance 7.93

General farm overhead 13.22

Total, allocated overhead 184.68

 

Total cost listed 278.09

Value of production less total cost listed (23.25)

 

Value of production less operating costs 161.43

Supporting Information: Yield = 46 bushels/acre, Price = $5.54/bushel, Enterprise size = 268 

planted acres, Irrigated = 9%, Dry land = 91%. 

Source: USDA-ERS. 2006. 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/costsandreturns/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 the 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 for food. The oil produced during the crushing 

of the soybean is used for cooking or food ingredients. The goal of the commodity chain 

is to supply a homogenous product to the enduser. The grower producing soybean for 

this chain has a choice from many different varieties for production; however, harvested 

soybean is viewed to be the same for all commodity soybean varieties. At harvest, the 

grower either delivers soybean to a handler or stores them on farm for later delivery. The 

first handler is not interested in differentiating the commodity soybean for later use. 

Commodity soybean handlers typically have large volume storage capacity. Similarly, 

commodity soybean processors crush large volumes of soybean to produce homogeneous 

oil and meal products. The commodity system is designed to maximize efficiency at a 

low profit margin resulting in 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 began in earnest in the 1960s (Sonka et al., 

2004). 

In recent years, there has been an increased demand by consumers and food processers 

for soybean that has specific physical or chemical characteristics that are required by 

certain customers to meet specific food or feed needs. As a result a separate specialty 

soybean channel has developed. This production system and distribution channel is 

focused on value-added traits that involve much smaller volumes than commodity 

soybean (Sonka et al., 2004). Specialty soybean varieties are produced on approximately 

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 acreage in certain states 

within the next decade. This supply chain for 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 cost incurred from an identity preservation 

system is offset by the higher value received for the final product. 

According to the American Soybean Association (2009), specialty soybean can be 

grouped into ten broad categories: Non-biotechnology-derived, certified seed, organic 

food-grade, low saturated fat, clear hilum, tofu, natto, high sucrose, high oleic, low 

 

5 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 145 of 395 

linolenic, and high protein. The categories refer to soybean with characteristics such as 

altered seed composition (e.g., low saturated fat, high sucrose, high oleic, low linolenic, 

and high protein), varieties of soybean with unique physical characteristics suited to their 

specific uses (e.g., clear hilum for direct human consumption), or refer to a production 

process (e.g., organic, certified seed). The categories are not meant to be exclusive; for 

instance, soybean used to produce natto or tofu may employ organic production processes, 

and soybean from all of the categories is often derived from varieties produced according 

to certified seed production practices. Tofu and soymilk produced from the tofu soybean 

category represent a large segment of the specialty soybean market and are produced 

from unique soybean varieties that have clear hilum and large seed size (Lee and Herbek, 

2004). Tofu varieties also must be high in protein (40% or higher) and low in oil 

concentration compared to commodity soybean. Clear hilum and other characteristic are 

required for soybean used in the production of other soybean food products consumed 

directly by humans, such as natto, soybean sprouts, edamame (vegetable soybean), and 

soy nuts (Lee and Herbek, 2004; UK Cooperative Extension Service, 2009). Organic 

food-grade and non-biotechnology-derived soybean are identity protected and produced 

not to contain biotechnology-derived traits. Organic soybean have additional production 

restrictions requiring the soybean to be produced using no synthetic fertilizers or 

pesticides. In recent years, public and private soybean breeders have developed soybean 

varieties with improved nutritional characteristics (e.g., high sucrose, high oleic, low 

linolenic, and high protein). The characteristics or compositional modifications in this 

group include varieties with fatty acid profiles that reduce the need for hydrogenation of 

the resulting soybean oil, increased sugar concentration, increased protein concentration, 

decreased concentration of saturated and 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 varieties are offered in Group II and early Group III 

varieties which are adapted to the upper Midwest/Great Plains region (Lee and Herbek, 

2004). Maturity Groups II and III soybean varieties are grown 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 few exceptions, the agronomic or management 

practices for growing specialty soybean from 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 variety selection is dictated by the specialty 

soybean buyer or processor. Nonbiotechnology-derived and organic soybean by 

definition must be produced utilizing only conventional soybean varieties. As with 

commodity soybean, weed control is extremely 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 soybean must be grown without synthetic 

fertilizers or pesticides for three or more years prior to the current crop of soybean, 

fertilization and pest management is more difficult (Lee and Herbek, 2004). Weed 

control in organic soybean relies on a combination of crop rotation, tillage, in-crop 

cultivation, and hand-weeding. Insect and disease control is managed primarily through 
Monsanto 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 facilities for specialty soybean must be clean of seed from 

other soybean varieties or plants, dirt, pathogens, and other foreign material. Some 

soybean contracts may require a special inspection of the handling and storage facilities. 

The specialty soybean for soybean foods may require special harvesting equipment since 

some of this soybean is harvested before full maturity (e.g., edemame or vegetable 

soybean). 

IX.B.3. Identity Preservation 

Identity Preservation (IDP) refers to a system of production, handling, and marketing 

practices that maintains the integrity and purity of agricultural commodities (Sundstrom 

et al., 2002). Commodity grains, on the other hand, are marketed in mass according to 

USDA grading standards. Specialty crops require some form of segregation or full-scale 

identity preservation to keep these grains separate from conventional commodity grains 

(Elberhi, 2007). This market segmentation within the grain channeling system is driven 

by the need to preserve market value or ensure a specified purity of the product. With 

certain specialty crops, IDP is required to prevent accidental or unintended commingling 

(e.g., nonbiotechnology, organic) or to segregate products that are approved only for 

certain uses (industrial use only). 

IDP grain production has been in existence for a long time. Agricultural producers have 

over many decades developed practices that allowed for differentiation between food vs. 

feed grain, or grain vs. seed production, or organic vs. nonorganic (Massey, 2002). Seed 

certification programs such as those used by the 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 available to growers (see Section IX.B.4). Standards were 

established to ensure production of seed from known pedigree with high purity and 

quality. Similarly, commodity grain traders, marketing organizations, and food 

processors have established purity and quality tolerances for specific end-product uses. 

The need for segregation and IDP production systems has increased with the 

development of specialty crops or crops with special output traits, such as high oil corn, 

high oleic sunflower, and low-linolenic soybean (Sundstrom et al., 2002). 

The production of IDP grains requires special processes in order for growers to meet 

buyers’ criteria for variety identification, composition, and quality. Buyers of IDP 

soybean typically contract for the production of seed-variety specific soybean and work 

directly with seed suppliers, growers, independent certification agencies, intermediate 

processors, and freight companies to deliver the preferred product within specified 

tolerances.6

 Contract specifications are written to ensure the delivery of the desired 

 

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Monsanto Company 09-SY-200U Page 147 of 395 

attribute-specific product and that predetermined management practices are used (Massey, 

2002). Lack of compliance with a product specification can lead either to a price 

discount or rejection of a shipment by the buyer. Depending on the end use, contracts 

can be extensive, defining many of the production, harvest and storage activities, or less 

stringent dealing more with pricing, quality specifications and only the most critical 

production practices. Premiums are paid to growers for the additional risk and 

management only when the grain meets the contract specifications. Premium prices 

provide the impetus to maintain the specialty grain’s purity and identity separate from 

commodity grains (Massey, 2002). Premiums are affected by various factors, including 

the proximity of suppliers to buyers and the cost and availability of substitutes 

(Sundstrom et al., 2002). For many trait-specific crops, price premiums rise or fall, 

depending on the supply conditions for the generic commodity. 

Growers of IDP grains must have good managerial ability and implement certain 

management practices to produce and deliver grain possessing the desired physical and 

chemical characteristics. The production and marketing of trait-specific grains involves 

additional financial risks (Elberhi, 2007). The growers’ managerial ability can affect 

both yield performance and proficiency in meeting the contract specifications. From a 

soybean buyer’s perspective, contracts help the buyer meet the demand for specific 

product qualities, improve cost efficiencies of product processing, and reduce transaction 

costs. 

While specific IDP production practices vary depending on the characteristics of the 

product to be delivered, general elements are implemented to ensure that the enduser or 

processor receives the grain or end-product with the intended identity and desired quality. 

As mentioned above, many IDP systems were developed using the principles similar to 

those used in seed certification. IDP production begins with a system of standards, 

records, and auditing that are put in place throughout the entire crop production, 

harvesting, handling, and marketing process (Sundstrom et al., 2002). Some key 

considerations in the establishment of an IDP system include: 1) planted seed identity and 

tolerances, 2) appropriate field isolation, 3) inspection and clean-out of equipment and 

facilities, 4) end-product sampling and testing, and 5) record maintenance and identity 

labeling (Sundstrom et al., 2002). Each of these components are described in greater 

detail below: 

Planted Seed Identity and Tolerances

The purity and identity of starting seed should be tested and confirmed. The purity of the 

seed stock should equal or exceed the purity standards of the desired final product. 

Single or multiple quality tolerances may be established in specialized IDP programs 

based on market-driven standards. 

Field Isolation

Crops must be isolated either spatially or temporally from pollen sources that could 

impact the quality or purity of the harvested seed. The amount of isolation depends on 

flower characteristic, sexual compatibility with neighboring crops, pollen quantity and 
Monsanto 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 vehicles are cleaned and inspected to assure 

that segregation is maintained and no physical contamination occurs. 

Sampling and Testing

In some cases, the IDP grain is sampled and tested at various stages to confirm the 

product identification, purity, and quality. Special consideration should be given to 

sampling and testing techniques that ensure reliable results.

Record Maintenance and Identity Labeling

Records typically are maintained on field designations, harvest amounts, storage bin 

locations, and product transfers. IDP products must be identified, segregated, and labeled 

in the market chain. 

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 identity, 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 responsible for maintaining high quality seed 

stocks, an essential basis for U.S. agriculture. By the early 20th century, agronomists 

learned how to develop specific plant varieties with desirable traits. In the U.S., state 

agricultural experiment stations developed many varieties which were distributed to 

growers for use. Growers saved seed and later sold to neighbors; however, the desirable 

traits of the varieties often were lost through random genetic changes and contamination 

with other crop and weed seed (Sundstrom et al., 2002). The value of seed quality 

(including genetic purity, vigor, absence of weed 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 seed met established seed quality standards 

(Bradford, 2006). The federal government passed the U.S. Federal Seed Act of 1939 to 

recognize seed certification and official certifying agencies. Regulations first adopted in 

1969 under the Federal Seed Act recognize land history, field isolation, and varietal 

purity standards for foundation, registered, and certified seed. Under international 

agreements such as the Organization for Economic Co-Operation and Development 

scheme, the U.S. and other countries mutually recognize minimum seed quality standards 
Monsanto Company 09-SY-200U Page 149 of 395 

(Bradford, 2006). The Association of Official Seed Certifying Agencies represents state 

and private seed certification in the U.S., and includes international member countries in 

North and South America, as well as in Australia and New Zealand. 

Soybean seed is separated into four seed classes: 1) breeder, 2) foundation, 3) registered, 

and 4) certified (AOSCA, 2009). Breeder seed is seed directly controlled by the 

originating or sponsoring plant breeding organization or firm. Foundation seed is first 

generation seed increased from breeder seed and is handled to maintain specific varietal 

purity and identity. Registered seed is the progeny of foundation seed that is handled to 

maintain satisfactory variety purity and identity. Certified seed is the progeny of breeder, 

foundation or registered seed and is two generations from foundation seed. All soybean 

seed sold may not be officially certified; however, commercial soybean seed sold and 

planted for normal soybean production predominately is produced to meet or exceed 

certified seed standards. This section of the petition will provide a broad overview of the 

practices utilized in producing certified seed. 

Soybean seed breeders and producers have put in place practical measures to assure the 

quality and genetic purity of soybean seed varieties for commercial planting. The need 

for such systems arose from the recognition that the quality of improved soybean 

varieties quickly deteriorated in the absence of monitoring for quality and genetic purity 

(CAST, 2007). Seed certification programs were initiated in the early 1900s in the U.S. 

to preserve the genetic identity and variety purity of seed. There are special land 

requirements, seed stock eligibility requirements, field inspections and seed labeling 

standards for seed certification. Seed certification services are available through various 

state agencies affiliated with the Association of Official Seed Certifying Agencies. Large 

seed producers implement their own seed quality assurance programs. However, large 

seed producers often will utilize the services of state certifying agencies as a third-party 

source to perform certain field inspections and audits. 

The U.S soybean production for all purposes according to USDA-NASS statistics has 

varied from approximately 64.1 to 74.6 million acres in the past ten years, 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 allowances for seed losses due to weather, poor 

yields, and quality issues. Additional allowances are included for distribution excess, 

seed returns, replants and potential increases in soybean acreage. Assuming an average 

soybean yield of 45 bushels, or 54 units (50 lbs/unit) per acre, 1.9 to 2.3 million acres 

would be required to produce this volume of commercial certified soybean seed each 

year. 

Soybean seed is produced throughout most of the U.S. soybean growing regions. 

Soybean varieties are developed and adapted to certain geographical zones and are 

separated into ten maturity groups – Group 00 to Group VIII (see Section IX.C). Seed 

production for these maturity groups is grown in the respective geographical zone for 

each maturity group. However, the production areas generally are on the northern edge 

of the respective zone to minimize incidences of disease. 
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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 growers to produce the required amount of soybean seed. 

Production or processing plants at these seed companies identify top soybean growers to 

produce the seed and also monitor and inspect seed fields throughout the growing season. 

The production plants also clean, condition, and bag the harvested soybean seed as well 

as monitor and inspect all the processes at the plant. Production plants typically produce 

between 100,000 units to 2,000,000 units of soybean seed. Production plants will 

produce the various soybean varieties in different climates or environments to spread 

production risks. 

The entire seed production process at the majority of private seed companies and tollers 

is International Organization for Standardization (ISO) certified and, therefore, includes 

internal and external audits (ISO, 2009). The ISO standards ensure desirable 

characteristics of seeds and services such as quality, safety, reliability, and efficiency. 

ISO standards represent an international consensus on good management practices with 

the aim of ensuring that the organization can consistently deliver excellent product or 

services. The standards not only must meet the customer’s requirements and applicable 

seed regulatory requirements, but must aim to enhance customer satisfaction and achieve 

continual improvement of its performance in pursuit of these objectives. 

The field operations and management practices for producing soybean seed are similar to 

normal soybean production. However, special attention is needed in certain areas to 

produce seed with high quality, high germination rates, and high genetic purity (Helsel 

and Minor, 1993). General guidelines specific for seed production are discussed below. 

The seed production field should not have been planted with soybean the previous crop in 

order to avoid volunteer soybean plants (even though the risk of soybean volunteer plants 

is negligible) and to ensure genetic purity. 

Very early planting should be avoided because the seed produced from early planting 

often results in poorer quality seed (Helsel and Minor, 1993). Every effort must be made 

to eliminate weeds in a seed field through the use of herbicides and cultivation to prevent 

weed seed in the harvested soybean seed. Fields are scouted frequently for insect pests 

and insecticides are applied when insect pest infestations reach economic threshold 

levels. Foliar-applied fungicides should be considered when disease infestations are 

predicted in the area. Harvest should occur as soon as the mature soybean seed reaches 

13% moisture content. Harvesting soybean seed with less than 13% moisture can cause 

damage to the seed coat and result in split soybean seed that can affect germination and 

viability. Harvesting equipment must be adjusted to minimize or avoid seed damage. 

Harvesting equipment must be cleaned before entering the seed fields to minimize 

genetic contamination. Certain handling equipment such as auger elevators, should be 

avoided because they can increase seed damage. 

Field inspections are vital to ensure the soybean seed meets seed certification 

requirements, ISO certification standards, regulatory standards, and trait licensing 
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agreement standards. Field inspections are conducted on seed production fields 

throughout the soybean growing season to evaluate variety purity, ensure soybean plants 

are developing properly, and fields are maintained free of weeds, insects, and diseases. 

The fields also are mapped to ensure the seed field has the minimum federal isolation 

requirement of five feet (AOSCA, 2009). Some states and seed producers have a stricter 

isolation requirement of 10 feet. 

Production plant personnel make every effort to avoid mechanical damage to the 

harvested seed during the screening, cleaning, and bagging processes. 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 appearance, disease, discoloration, 

seed coat, mechanical damage, inert matter, and weed seed. Warm and cold germination 

tests are conducted on all seed lots to verify acceptable germination rates. Many seed 

companies will also conduct tetrazolium staining tests to assess seed viability. 

Commercially certified soybean seed must meet state and federal seed standards and 

labeling requirements. AOSCA standards for certified soybean seed are as follows: 98% 

pure seed (minimum), 2% inert matter (maximum), 0.05% weed seed (maximum, not to 

exceed 10 per lb.), 0.60% total of other crop seeds (maximum), 0.5% other varieties 

(maximum, includes off-colored beans and off-type seeds), 0.10% other crop seeds 

(maximum, not to exceed three per lb.), and 80% germination and hard seed (minimum) 

(AOSCA, 2009). State seed certification standards vary slightly from state to state and 

can be more restrictive than the seed standards of AOSCA. 

MON 87769 seed will be produced in the same manner as commercially certified 

soybean seed, such that it will meet all state and federal seed standards and labeling 

requirements. 

IX.C. Production Management Considerations 

Pre-Season 

Crop rotation, tillage system, row spacing, planting equipment, seed or variety 

selection(s), and soil fertility are areas that require production decisions well in advance 

of planting the soybean crop. Many of the decisions 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 planted in a two-year corn-soybean rotation (see 

Section IX.G). Crop rotation is generally a long-term decision, but the rotation sequence 

can be modified to take advantage of a particular economic or market opportunity. The 

decision to plant soybean in a conservation tillage or no-till system may require special 

equipment and will be made long before planting. In addition, this decision usually will 

be a long-term commitment, provided the system is successful. A decision to change row 

spacing is a similar long-term commitment that generally requires new equipment. 
Monsanto Company 09-SY-200U Page 152 of 395 

The benefits of conservation tillage or no-till systems are well documented and include 

reduced soil erosion, reduced fuel and labor costs, and conserving soil moisture. In 2004, 

approximately 29.3 million acres (38.6%) of soybean were planted in a no-till system 

(CTIC, 2004). Slow soybean emergence and growth plus lower yields have been some of 

the concerns associated with adoption of conservation tillage systems in soybean, 

especially no-till. Research in Wisconsin and Minnesota shows that soil temperatures 

can be four to five degrees colder in no-till than conventional tillage systems, which can 

slow emergence but have little effect on soybean yield (Pedersen, 2008a). Improved 

planters for establishment of good soybean populations and planting Roundup Ready 

soybean to effectively control weeds in no-till fields have made no-till a viable 

production system for soybean. Researchers still recommend some spring tillage on finetextured and poorly drained soils for proper seedbed preparation (Pedersen, 2008a). 

Most field crops, including soybean, respond very well to fertilizer when planted in soils 

with low fertility levels. Soybean requires 16 essential elements for growth and 

development. Deficiencies in any of these elements can reduce yields (Hoeft et al., 

2000). The primary or major essential nutrients are nitrogen, phosphorus and potassium. 

The soybean plant is a member of the legume family, like alfalfa and clover, and fixes a 

significant portion of its own nitrogen through the symbiotic relationship with the 

nitrogen-fixing Bradyrhizobia bacteria (Bradyrhizobium japonicum) that live in the 

nodules on its roots. Bradyrhizobia are unicellar, microscopic bacteria that invade the 

soybean plant through its root hairs (Hoeft et al., 2000). The plant responds to this 

invasion by forming nodules which contain colonies of bacteria. After being established 

on the soybean root, bacteria in the nodule take gaseous nitrogen from the atmosphere 

and fix it in forms easily used by the soybean plant. Since the bacteria are not native to 

U.S. soils, inoculation of the soybean seed is recommended when soybean has not been 

grown in a field for three to five years. Nitrogen fertilizer applications at planting 

generally do not improve yield and decreases nodulation while increasing the plant’s 

dependency on the soil for nitrogen (Pedersen, 2008a). Therefore, nitrogen fertilizer 

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 requirements 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 Midwest, 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 differences in crop rotations and soil types. 

Although not common, deficiencies can occur in secondary nutrients (calcium, 

magnesium, and sulfur) or micronutrients (boron, chloride, copper, iron, manganese, 

molybdenum, and zinc). The availability of soil nutrients is dependent on soil acidity or 

pH level. Soybean is adversely affected when 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 adapted to certain geographical zones and are 

separated into ten maturity groups – Group 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 latitude 46o north. Succeeding groups are adapted further south 

with Groups I and II within latitudes 41o and 46o north, and Group III within latitudes 38o 

and 41o North. Group 00 through Group IV soybean varieties are planted in the Midwest 

and Eastern Coastal regions. Groups II, III and IV account for approximately 75%, 24%, 

36%, and 16%, respectively of the soybean planted in the U.S., with Group III having the 

largest acreage (T. Schlueter, personal communication, 2008). Groups IV through VIII 

are planted in the southern states with Groups V, VI and VII representing 7%, 2%, and 

2% of the planted soybean, respectively (T. Schlueter, personal communication, 2008). 

Soybean variety selection is crucial for high yield and quality, and is the foundation of an 

effective management plan (Pedersen, 2008a). Soybean characteristics to consider in 

selecting a variety include maturity, yield potential, disease and pest resistance, iron 

deficiency tolerance (chlorosis), lodging score, height, and specific soybean quality traits, 

such as protein and oil content. If a field has a history of a particular disease or pest, 

planting soybean varieties that have resistance or tolerance to these pests and diseases can 

be an effective and economical method of control. 

Row spacing is important to maximize soybean yield. Research in the Midwest over the 

past 20 years consistently shows that row spacing of less than 20 inches is preferred for 

soybean regardless of tillage system, rotation sequence or planting date (Pedersen, 

2008a). In the Southern states, the advantage from narrow rows is less consistent and 

less beneficial. In 2000, approximately 40% of soybean was planted in row spacing of 10 

inches or less, 27% in 10.1 to 28.5 inches, and 33% in rows wider than 28.5 (Hoeft et al., 

2000). 

Planting and Early Season 

An understanding of the growth stages of soybean is also important for the proper timing 

of certain management practices, such as herbicide and insecticide applications. In 

addition, the impact of certain weather conditions, insect pests, and diseases on soybean 

yield is dependent on growth stage. The system of soybean growth stages divides plant 

development into vegetative (V) and reproductive (R) stages (Pedersen, 2008a). The 

vegetative stages begin with VE, which designates emergence. V stages continue and are 

numbered according to how many fully developed trifoliate leaves are present (i.e., V1, 

V2, etc.). The reproductive (R) stages begin at flowering (R1) and include pod 

development and plant maturation. Full maturity is designated as R8. 

Adequate soil moisture and warm temperatures facilitate rapid seed germination and 

emergence. The ideal soil temperature for soybean germination and emergence is 77 oF 

(Pedersen, 2008a). However, waiting for soils to reach this soil temperature will delay 

planting beyond the optimum planting date that will maximize yield. Soybean can 

germinate at a soil temperature of 50 oF when planted at a depth of two inches. However, 

emergence is slow and can take up to three weeks in Northern climates. Because of 

fluctuations in soil temperature in early spring, soil temperature should not be the only 

criteria for optimum planting time. Planting into a good seedbed is the most important 
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consideration. Planting into soil that is too wet will reduce emergence and plant 

population, and can lead to reduced yield. 

Planting date has the greatest impact on yield according to research conducted in the 

Northern states (Hoeft et al., 2000). Highest yields generally are obtained when planting 

is in early to mid May. Yields begin to drop off quite rapidly when planting is delayed 

until late May. For example, the optimum planting dates for soybean in Iowa are the last 

week of April in the southern two-thirds of the state and the first week of May in the 

northern one-third of the state (Pedersen, 2008a). In the Southern U.S., planting adapted 

varieties before late April results in shorter plants and, in many cases, lower yields than 

when the same varieties are planted in May or early June. Planting after early June 

generally decreases plant height and yield due to water shortages in July and August. 

Variations in plant spacing through row spacing and plant population have a significant 

effect on canopy development and soybean yield. Soybean has the ability to produce 

good yield over a wide range of plant populations. Most soybean varieties have the 

ability to branch and adjust the number of pods on branches to compensate for large 

differences in seeding rate. Maximum yields generally require planting rates that result 

in about 2.5 to five plants per square foot (Hoeft et al., 2000). Therefore, a full stand of 

soybean is approximately eight to 10 plants per foot of row at harvest for 40-inch rows, 

six to eight plants per foot of row in 30-inch rows, four to six plants in 20-inch rows, and 

two to three plants in 10-inch rows. This translates to 109,000 to 218,000 plants per acre 

at harvest. Higher populations are recommended in narrow rows for maximum yields 

because plants are more uniformly spaced in narrow rows. Seeding rates are generally 10 

to 25% higher than the desired harvest population, especially in no-till, to account for the 

losses in germination, emergence, and seedling diseases. The accuracy of the planting 

equipment also can impact the decision on seeding rate. Soybean seed traditionally has 

been sold by weight. Therefore, the farmer must know the number of seeds per pound for 

the particular soybean varieties being planted for accurate seeding rates. 

Treating soybean seed with a fungicide (e.g., pyraclostrobin, metalaxyl, mefenoxam) to 

prevent damping-off diseases may be beneficial when planting in cold, wet soils, using 

reduced till and no-till planting systems, and when planting seed with a low germination 

rate (<80%) or low seed vigor. 

Annual and perennial weeds are considered to be the greatest pest problem in soybean 

production (Aref and Pike, 1998). In order to maximize yields, weeds must be controlled 

during the early growth stages of soybean because weeds compete with soybean for 

water, nutrients, and light. A combination of tillage and herbicides are 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 follows 
Monsanto Company 09-SY-200U Page 155 of 395 

night temperatures around 70 oF. Soybean varieties differ in their response and tolerance 

to temperatures. 

Soybean is photoperiod sensitive, which means that it transitions from vegetative to 

flowering stage in direct response to length of daylight (Scott and Aldrich, 1970). Most 

soybean varieties begin flowering soon after the day length begins to shorten. Flowering 

of southern varieties is initiated by a shorter day than varieties adapted to the north. The 

extent of vegetative growth occurring after the initiation of flowering depends not only 

on environmental factors but also on the growth habit. Soybean varieties are described as 

either indeterminate or determinate in their growth habit (Scott and Aldrich, 1970). 

Indeterminate varieties increase their height by two to four times after flowering begins. 

These are grown in the Northern and Central U.S. Determinate varieties increase their 

height very little after flowering and generally are grown in the Southern U.S. 

Indeterminate and determinate varieties also differ in flowering characteristics. 

Indeterminate plants generally bloom first at the fourth or fifth node and progress 

upward. Flowering on determinate plants begins at the eight or tenth node and progresses 

both downward and upward. 

The first appearance of flowers signals the beginning of the reproductive stage, namely 

the R1 stage (Hoeft et al., 2000). The reproductive period consists of flowering, pod set, 

and seed formation. Climatic conditions such as temperature and moisture supply during 

the flowering period will affect the number of flowers. The soybean plant does not form 

a pod for each flower. It is common for the soybean plant to have 75% of the flowers fail 

to develop a pod (Scott and Aldrich, 1970). This characteristic makes soybean less 

susceptible than corn to short periods of adverse weather during flowering. Under 

normal conditions, pod set occurs over about a three-week period. Good soil moisture is 

most critical during the pod-filling stages to prevent pod abortion and to ensure high 

yields (Hoeft et al., 2000). Another critical period is during the seed-filling stages to 

assure high rates of photosynthesis. High humidity and temperatures during seed 

development and maturity can result in poor seed quality 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 weather conditions, seed moisture content will drop to 13 to 

14% in 10 to 14 days from physiological maturity (Hoeft et al., 2000). Soybean can be 

harvested when the moisture content drops below 15%. However, soybean should be at 

13% moisture to be stored without artificial drying (Scott and Aldrich, 1970). Moisture 

content below 12% may increase seed cracking and seed coat damage. 

Pre-harvest losses are influenced by variety, weather, and timeliness of harvest (Scott and 

Aldrich, 1970). Timely harvest when the moisture content is 13 to 14% also will 

minimize losses. Proper operation and adjustment of the combine is essential to 

minimizing harvest losses in the field. 
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IX.D. Weed Management 

Annual weeds are 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 harbor insects and diseases, and can also 

interfere with harvest, causing extra wear on harvest equipment (Pedersen, 2008a). The 

primary factors affecting soybean yield loss from weed competition are the weed species, 

weed density, and the duration of the competition. When weeds are left to compete with 

soybean for the entire growing season, yield losses can exceed 75% (Dalley et al., 2001). 

Generally, the competition increases with increasing weed density. The time period that 

weeds compete with the soybean crop influences the level of yield loss. In general, the 

later the weeds emerge, the less impact the weeds will have on yield. Soybean plants 

withstand early-season weed competition longer than corn, and the canopy closes earlier 

in soybean than corn. In addition, canopy closure is much sooner when soybean is drilled 

or planted in narrow rows. 

Crop rotations and environment have a significant impact on the adaptation and 

occurrence of weeds in soybean. Foxtail spp. (foxtail species group), pigweed, 

velvetleaf, lambs quarters, and cocklebur are common weeds in Midwest corn and 

soybean fields. However, growers consider giant ragweed (Ambrosia artemisiifolia), 

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 over weeds. Although the primary purpose of 

tillage is for seedbed preparation, tillage still is used to supplement weed control with 

selective herbicides in soybean production. Approximately 98 percent of the soybean 

acreage received an herbicide application in 2006, indicating the importance of excellent 

weed control in maximizing soybean yield (USDA-NASS, 2007b). Herbicide-tolerant 

soybean was introduced to provide growers with additional options to improve crop 

safety and/or improve weed control. The Roundup Ready soybean system, i.e., planting 

Roundup Ready soybean and applying glyphosate in crop has become the standard weed 

control program in U.S. soybean production. Currently, Roundup Ready soybean is 

planted on 91 percent of the soybean acreage (USDA-NASS, 2007a). Consequently, 

glyphosate is the most widely used herbicide in soybean, being applied on 96 percent of 

the soybean acreage in 2006 (USDA-NASS, 2007b). 
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IX.E. Management of Insects 

Although insects are typically as less problematic than weeds in U.S. soybean production, 

management of insect pests during the growth and development of soybean is important 

for protecting the yield of soybean (Aref and Pike, 1998). Understanding the impact of 

insects on soybean growth is essential for proper management (Way, 1994). It is 

important to understand the way that insects injure soybean as well as how the soybean 

plant responds to insect injury. Insect injury can impact yield, plant maturity, and seed 

quality. Injury is defined as a stimulus producing an abnormal change in plant 

physiological processes. Injury may produce stress which is a departure from optimal 

physiological conditions (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 injury is essential in establishing economic 

injury levels (Way, 1994). Most often, soybean insects are categorized or defined by the 

plant parts they injure, namely root-feeding, stem-feeding, leaf-feeding, or pod-feeding 

insects. The root- and stem-feeding insect groups are often the hardest to scout and 

typically are not detected until after they have caused their damage. The leaf-feeding 

insects comprise the biggest group of insects, but not necessarily the most damaging 

insects. Recent research on defoliation has determined that a major effect of injury is to 

reduce light interception by the soybean canopy which in turn can have a significant 

effect on yield (Way, 1994). Soybean has an extraordinary capacity to withstand 

considerable defoliation early in the season without significant 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 soybean plant can sustain a 35% leaf loss prior 

to the pre-bloom period without lowering yield (NDSU, 2002). However, from pod-set 

to maturity, the plant can tolerate only a 20% defoliation level before yield is impacted. 

IX.F. Management of Diseases and Other Pests 

More than 100 pathogens are known to affect soybean, of which 35 are considered to be 

of economic importance (Bowers and Russin, 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 yield. The extent of 

losses depends upon the pathogen, the state of plant development and health when 

infection occurs, the severity of the disease on individual plants, and the number of plants 

affected (Bowers and Russin, 1999; and Funderburk et al., 1999). 

One or more diseases generally can be found in fields wherever soybean is grown 

(Bowers and Russin, 1999; and Funderburk et al., 1999). However, a pathogen may be 

very destructive one season and difficult or impossible to find the next season. The 

extent and severity of soybean diseases depend on the degree of compatibility between 

the host and the pathogen and the influence of the environment. 
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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). Phytophthora root and stem rot (Phytophthora sojae), 

brown stem rot (Phialophora gregata), sclerotinia stem rot (Sclerotinia sclerotiorum), 

and seedling diseases followed in economical importance. As expected, yield losses 

varied by region. Sclerotinia stem rot caused yield losses in several northern states, but 

not in other states. Rhizoctonia foliar blight losses were greatest in Arkansas, Louisiana, 

and Texas where humidity and temperature conditions are suitable for disease 

development. 

Selecting resistant varieties is the primary tool growers have for disease control (Bowers 

and Russin, 1999; and Funderburk et al., 1999). Resistant varieties may have 

morphological or physiological characteristics that provide immunity, resistance, 

tolerance or avoidance to certain pathogens. Cultural practices play an important role in 

disease management by reducing initial inoculums or reducing the rate of disease 

development (Bowers and Russin, 1999; and Funderburk et al., 1999). Preplant tillage 

can bury crop residue, which encourages the decomposition of fungal-resting structures. 

Crop rotation is routinely recommended as a disease management strategy. Rotating 

crops interrupts the disease cycle and allows time for the decomposition of inoculums. 

One exception is Rhizontonia, a soil-inhabitant pathogen that grows on a wide variety of 

crops and can survive sufficiently in the soil to make crop rotation as a means of 

controlling this pest impractical. Row spacing, plant population, and planting date 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 about $1.5 billion in the U.S. (Pedersen, 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 nematodes feed on the roots causing severely 

stunted and yellow plants. The simplest, least expensive method to reduce populations of 

this pest is to rotate soybean with a non-host crop, such as corn, small grains, or sorghum. 

Planting resistant varieties is regarded as the best and most effective management 

practice to prevent losses from this pest. Several public and private soybean varieties 

offer sources of resistance to certain races of nematode. Alternating varieties with 

different sources of resistance may be beneficial. 

High-quality seed is essential for controlling seedling diseases. The most important 

seedling diseases in soybean are Phytophthora, Pythium, Rhizoctonia, and Fusarium 

(Pedersen, 2008a). Many soybean varieties have race-specific resistance to 

Phytophthora. Treating soybean seed with a fungicide (e.g., pyraclostrobin, metalaxyl, 

mefenoxam) is effective against damping-off disease (seedling blight) caused by common 

soil fungi, such as Phytophthora and Pythium. Fungicide seed treatments are 

recommended where there is a history of these seedling diseases. 

Asian soybean rust is a foliar fungal disease that typically infests soybean during 

reproductive stages of development and can cause defoliation and reduce yields 

significantly in areas such as Brazil (Dorrance et al., 2007). Soybean rust is caused by 
Monsanto 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 application of fungicides 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 producing soybean seed. According to USDANASS statistics, fungicides were applied on approximately 4% of the soybean acreage in 

2006 (2007b). 

IX.G. Crop Rotation Practices in Soybean 

The well-established farming practice of crop rotation is still a key management tool for 

growers. The purpose of growing soybean in rotation 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, increase residue cover, mitigate or break disease, 

insect, and weed cycles, reduce soil erosion, 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 Research Service crop 

residue management studies, 95% of the soybean-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 growers the opportunity to diversify farm production 

in order to minimize market risks. 

Continuous soybean production is not a common practice in the Midwest and is 

discouraged by most extension soybean specialists to reduce the risk of diseases and 

nematodes (Al-Kaisi et al., 2003; Hoeft et al., 2000). Corn and soybean occupy more 

than 80% of the farmland in many of the Midwestern states, and the two-year cropping 

sequence of a soybean-corn rotation is used most extensively 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 after a number of years without soybean. The 

yields of both corn and soybean are approximately 10% higher when grown in rotation 

than when either crop is grown continuously (Hoeft et al., 2000). 

A combination of conservation tillage practices and crop rotation has been shown to be 

very effective in improving soil physical properties. Long-term studies in the Midwest 

indicate that a corn-soybean rotation improves yield potential of no-till systems compared 

to continuous corn production (Al-Kaisi, 2001). The reduction in yield of continuous 

corn production in no-till systems is attributed to low soil temperature during seed 

germination, which is especially evident on poorly drained soils under no-till practices. 

Unique to the southern portion of the Midwest region and the mid-south states, soybean 

is grown in a double-cropping system. Double-cropping refers to the practice of growing 
Monsanto 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 plant canopy most of the year (Hoeft et al., 

2000). In the Midwest, winter wheat is harvested in late June or July, and then soybean is 

planted into the wheat stubble in a no-till system to conserve moisture. Due to the 

uncertainty of double-cropping yields, farmers sometimes 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-cropping 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 remain in a field after soybean harvest as a 

result of pods splitting before or during harvest. Soybean seeds also can remain in a field 

when pod placement on the plants is too close to the ground for the combine head to 

collect all the pods or the combine is improperly adjusted for efficient harvesting. 

Volunteer soybean in rotational crops is typically not a concern in the Midwest region 

because the soybean seed is typically not viable after the winter period (Carpenter et al., 

2002; OECD, 2000). In southern soybean growing areas of the U.S. where the winter 

temperatures are milder, it is possible for soybean seed to remain viable over the winter 

and germinate the following spring. 

Volunteer soybean is normally not a concern in rotational crops such as corn, cotton, rice, 

and wheat that are the significant rotational crops following soybean due to control 

measures that are available for volunteer soybean when they arise (Carpenter et al., 2002; 

OECD, 2000). Preplant tillage is the first management tool for control of emerging 

volunteer soybean in the spring. If volunteer soybean should emerge after planting, 

shallow cultivation will control most of the plants and effectively reduce competition 

with the crop. Several postemergence herbicides also are available to control volunteer 

soybean (conventional or glyphosate-tolerant soybean) in each of the major rotational 

crops. Table IX-4 provides control ratings on volunteer glyphosate tolerant soybean for 

several herbicides used in the major rotational crops. 

To provide control of volunteer soybean in corn, postemergence applications of AAtrex

(atrazine), Clarity (dicamba), Distinct (diflufenzopyr + dicamba), Hornet (flumetsulam + 

clopyralid) and Widematch (clopyralid + fluroxypyr) provide excellent control 

(Zollinger, 2005). In wheat, Bronate Advanced (bromoxynil), Clarity (dicamba) and 

Widematch postemergence provide excellent control of volunteer soybean (Zollinger, 

2005). 

Volunteer soybean in cotton is normally not a concern. However, hurricanes or other 

extreme weather conditions can damage a soybean crop preceding cotton production in 

the Mid-south states, where the unharvested soybean seed can produce volunteer plants. 

Preplant applications of paraquat or herbicide mixtures containing paraquat will 

effectively control volunteer glyphosate-tolerant soybean (Montgomery et al., 2002; 

Murdock et al., 2002). Recent research in North Carolina indicates Envoke 

(trifloxysulfuron) will provide excellent postemergence control of soybean with traits for 
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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 applications of Grasp (penoxsulam), Permit 

(halosulfuron) and Regiment (bispyribac) typically used for weed control in rice will 

effectively alleviate competition from volunteer soybean (Dillon et al., 2006). 
Monsanto Company 09-SY-200U Page 162 of 395 

Table IX-4. Ratings for Control of Volunteer Glyphosate-Tolerant Soybean in 

Labeled Rotational Crops1

Product Rate 

(Product/Acre)

Soybean 

V2 – V3 

Soybean 

V4- V6 

Corn2 

AAtrex 0.38 qts E P 

0.50 qts E F 

Clarity 4 fl oz E E 

5 fl oz E E 

Distinct 1 oz E G 

 2 oz E E 

Hornet 1 oz E F 

 2 oz E F-G 

Widematch 0.25 pt E G 

Wheat2 

Bronate Advanced 0.8 pt E E 

Clarity 4 fl oz E E 

 5 fl oz E E 

Widematch 0.25 pt E G 

Cotton3 

Envoke 0.1 oz E E 

Rice4 

Grasp 2 oz E NA 

Permit 1 oz E NA 

Regiment 0.4 oz E E 

NA denotes “not applicable.” 

1 Weed control ratings: E = Excellent (90 to 99% control), G = Good (80 to 90% control), F = 

Fair (65 to 80 control), and P = Poor (40 to 65% control). 

2

 Zollinger, 2005. 

3 York et al., 2005. 

4 Dillon et al., 2006. 

IX.I. Stewardship of MON 87769 

Monsanto Company is firmly committed to its legal, ethical, and moral obligation to 

ensure that its products and technologies are safe and environmentally responsible. 

Monsanto demonstrates this commitment by implementing product stewardship processes 

throughout the lifecycle of a product and by participation in the Excellence Through 

StewardshipSM Program (http://www.excellencethroughstewardship.org/). These policies 

and practices include rigorous field compliance and quality management systems and 

verification through auditing.
Monsanto Company 09-SY-200U Page 163 of 395 

As with all of our products, Monsanto is committed to the rigorous product stewardship 

of MON 87769. In keeping with past practice, Monsanto will seek regulatory approval 

for MON 87769 in all key soybean import countries with a functioning regulatory system 

to assure global compliance and support the flow of international trade. Monsanto 

continues to monitor other countries that are key importers of soybean from the U.S., for 

the development of formal biotechnology approval processes. If new functioning 

regulatory processes are developed, Monsanto will make appropriate and timely 

regulatory submissions. 

Once appropriate approvals are received, as with other value-added specialty soybean 

products, MON 87769 will be grown using an appropriate IDP system based on 

established practices as described in Section IX.B.3. IDP practices are implemented for 

value-added specialty soybean to capture the enhanced value of the product and ensure 

that the enduser or processor receives the soybean with the identity, fatty acid 

composition of the oil, and desired quality. 

Prior to obtaining all key global regulatory approvals, Monsanto will conduct seed testing, 

variety development and production, and oil manufacturing, testing and commercial 

activities leading up to the commercial introduction 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, harvest, processing and use of the product. 

Grain production processes will include mass balance and accounting of all planting, 

harvested seed, secured storage facilities, labeling of all soybean, training of personnel, 

identification and audit of field production sites and acreage, spatial isolation of fields, 

equipment clean out procedures and documentation. Soybean processing will include 

segregation, control and traceability of handling, processing, packaging, and shipping of 

products and co-products. 

Before implementing a closed loop system, Monsanto will dialogue with the appropriate 

value chain stakeholders in the countries of production and 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 system without adequate assurance that the 

system will be effective in containing, preventing the escape of, and controlling the 

disposition of MON 87769, so it is not comingled 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 trade, processing industry and food industry 

throughout the regulatory and product development process. 

IX.J. Impact of the Introduction of MON 87769 on Agricultural Practices 

Introduction of MON 87769 is expected to have 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 fatty 
Monsanto Company 09-SY-200U Page 164 of 395 

acid composition (presence of SDA and GLA in MON 87769 seed). MON 87769 is a 

nutritionally improved specialty soybean product that is expected to bring added value to 

consumers. The added value of MON 87769 is expected to bring higher returns to 

growers compared to commodity soybean. 
Monsanto Company 09-SY-200U Page 165 of 395 

X. ENVIRONMENTAL CONSEQUENCES 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 practices. 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. 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. 

The definition of “plant pest” in the Plant Protection Act (PPA) includes living organisms 

that could directly or indirectly injure, damage, or cause disease in any plant or plant 

product [7 U.S.C. § 7702(14)]. Information in this petition related to plant pest risk 

characteristics include disease and pest susceptibilities, expression and characteristics of 

the gene products (PjΔ6D and NcΔ15D proteins), impacts to NTOs, changes to plant 

metabolism, weediness of the regulated article, 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 biotechnology-derived crop products is not 

zero risk, but rather a determination that deregulation of the regulated article is not likely 

to pose a plant pest risk. The plant pest risk assessment of MON 87769 is based 

primarily on eight lines of evidence: (1) modern soybean has inherently low plant pest 

potential, (2) insertion of a single functional copy of the inserted expression cassette, (3) 

characterization of the PjΔ6D and NcΔ15D proteins expressed in MON 87769 and the 

altered soybean oil profile, (4) mode of action and safety of the PjΔ6D and NcΔ15D 

proteins and the altered soybean oil profile, (5) compositional equivalence of 

MON 87769 seed as compared to conventional soybean, with the exception of intended 

fatty acid change, (6) phenotypic and agronomic characteristics demonstrating no 

increased plant pest potential, (7) negligible risk to NTOs and threatened or endangered 

species, and (8) no greater likelihood to impact agronomic practices, including land use, 

cultivation practices, or the management of weeds, diseases, and insects than 

conventional soybean. 

As part of the plant pest risk assessment, the genetic construct inserted into MON 87769 

was evaluated to determine if those sequences 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 currently on the market. Agronomic practices 

associated with MON 87769 were considered relative to potential changes that could lead 

to increased plant pest potential. The potential for gene flow and introgression of the 

genetic construct into other plant varieties or wild relatives also was evaluated to 

determine the potential of increased weedy or invasive characteristics in sexually 
Monsanto 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) compared to conventional soybean and the potential 

for horizontal gene transfer were evaluated. Using this risk assessment process, the data 

and analysis presented in this petition lead to a conclusion 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 endangered 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 PjΔ6D and NcΔ15D proteins to other desaturases present in plants, the 

functional activity of the PjΔ6D and NcΔ15D proteins, and the expression level of the 

PjΔ6D and NcΔ15D proteins in MON 87769. The evaluation 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 and impact from gene flow. Due to the lack of sexually 

compatible relatives in the U.S., pollen-mediated gene flow is expected to occur only 

within cultivated soybean. Given the reproductive biology of soybean, pollen-mediated 

gene flow is expected to be negligible within cultivated soybean. The probability for 

horizontal gene flow is exceedingly small. Even if it were to occur, the consequences 

would be negligible because the genes introduced into MON 87769 (Pj.D6D and

Nc.Fad3) are of plant and fungal origin and the proteins produced from the introduced 

genes (PjΔ6D and NcΔ15D) 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 current 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 CFR § 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 agriculture, MON 87769 also would not be considered a “noxious 

weed” as defined by the Plant 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 binary transformation plasmid PV-GMPQ1972 

(Section IV: Figure IV-1and Table IV-1). Molecular analyses indicate that MON 87769 

contains one copy of the insert at a single integration locus. No additional elements from 

the transformation vector were detected in the genome of MON 87769, including 

T-DNA II or backbone sequence from plasmid PV-GMPQ1972. Additionally, the data 
Monsanto Company 09-SY-200U Page 167 of 395 

confirm the organization and sequence of the insert, and demonstrate the stability of the 

insert over several generations. On the basis of these data, it is concluded that only the 

expected PjΔ6D and NcΔ15D proteins are produced from the inserted DNA. Molecular 

analysis confirmed that MON 87769 does not contain elements that promote plant disease 

(refer to Table IV-1). 

X.A.2. Mode of Action and Safety of PjΔ6D and NcΔ15D Proteins 

The PjΔ6D and NcΔ15D proteins expressed in MON 87769 are members of a family of 

integral membrane fatty acid desaturases found in all eukaryotic organisms (plants, 

animals, and fungi) and some prokaryotes, e.g. cyanobacteria (Hashimoto et al., 2008; 

Los and Murata, 1998). The PjΔ6D and NcΔ15D 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 PjΔ6D and NcΔ15D proteins are well understood (see 

Section IV.A). PjΔ6D catalyzes the desaturation of ALA (18:3) to form SDA (18:4) in 

the seed of MON 87769 and also converts LA (18:2) to GLA (18:3). NcΔ15D catalyzes 

the desaturation of LA to ALA and, therefore, reduces the substrate pool for GLA 

production and increases the substrate pool for SDA production (Figure VII-1). 

The safety assessment of the PjΔ6D and NcΔ15D 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 limit of detection of 0.1 – 0.2 μg/g of tissue 

on a fresh weight (FW) basis for PjΔ6D and 0.5 – 1.0 μg/g on a FW basis for NcΔ15D. 

The mean PjΔ6D protein levels in immature seed, mature seed, and forage were 100, 1.8 

and 16 μg/g DW, respectively. The mean NcΔ15D protein levels in immature seed, 

mature seed, and forage were 200, 10 and 14 μg/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 soybean seed. Thus, it was expected that the 

PjΔ6D and NcΔ15D 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 PjΔ6D and NcΔ15D proteins are readily digestible in simulated gastric and 

intestinal fluids and lack acute oral toxicity in mice (see Sections VI.D and VI.E.). 

Collectively, these data establish the safety of the PjΔ6D and NcΔ15D proteins. From 

these data, it can be concluded with reasonable certainty that the PjΔ6D and NcΔ15D 

proteins have no meaningful toxic potential to exposed organisms in the environment. 

Furthermore, these integral membrane proteins are structurally and functionally related to 

other desaturases that are widely distributed in plants and 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 soil-dwelling organisms when postharvest soybean biomass or detritus 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 ten commercial soybean varieties. 

Compositional analyses compared a total of 75 different analytical components, seven in 

forage and 68 in seed, between MON 87769 and a conventional soybean control with 

genetics comparable to MON 87769, but lacking the introduced trait. Data presented in 

Section VII indicate that there are no meaningful 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 were some statistical differences between 

MON 87769 and the conventional control, it is concluded that the statistical differences 

represent the natural variability for these analytes in soybean such that they were not 

regarded as biologically meaningful. Seed and forage analytical component values also 

were comparable to published scientific literature 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 for 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 that play a role in 

most organisms in metabolic energy storage, 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 Sprecher, 1991; Watkins, 1991and 1995). In 

insects, PUFAs also serve as precursors in the biosynthesis of waxes, sex pheromones, 

and as components of defensive secretions (Stanley-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) because 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 relates to fatty acid biosynthesis, is their ability or inability to 

biosynthesize LA. Most insects have a dietary 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 supplied 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. Deficiencies of LA in poultry have been reported 

to include retarded growth, increased water consumption, reduced disease resistance, 

enlarged livers, and reduced testes and delayed development of secondary sexual 

characteristics in males. Decreased egg size and weight and changes in yolk fatty acids 

result from LA-deficient laying hens (Watkins, 1991). 
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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 (Canavoso 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 derived from a dietary source. Primary 

routes of metabolism include β-oxidization in the mitochondria 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 (Pan et al., 1994; Watkins, 1991; StanleySamuelson, 1988). Generally, the desaturation/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 intermediate 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 intermediate in the conversion of LA to dihomo-γlinolenic acid (DGLA) and arachidonic acid following the same pathway. C20 PUFAs, 

particularly EPA, DGLA, and AA, play important metabolic roles in most animals as 

precursors in the biosynthesis of physiologically active eicosanoids, which include the 

prostaglandins (PG), thromboxanes, leukotrienes, and lipoxins (Beare-Rogers et al., 

2001). 

Thus, PUFAs are important compounds in most animals. They are oxidized in energy 

production, incorporated in phospholipids and are essential for cellular membrane 

formation and function, desaturated/elongated, and oxygenated into physiologically 

active eicosanoids. SDA and GLA are PUFAs that are present in MON 87769 seed and 

are also consumed from other sources in the environment including plants, marine algae, 

fish/fish oil (see Section X.A.3.2). Therefore, these PUFAs in MON 87769 seed are 

expected to be absorbed, transported, and metabolized 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 field 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 fatty acids present in MON 87769 are the same 

as those present in conventional soybean. Thus, the analysis here focuses 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 through trophic interactions. However, 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, exist in many sources in the 

environment without known adverse ecological effects. SDA and GLA are produced in 

many plant seed oils. More than 230 species from among 17 plant families are 
Monsanto Company 09-SY-200U Page 170 of 395 

documented to produce SDA in seed oil at concentrations 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 produce 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 concentrations of other related PUFAs, 

such as ALA and LA (Table X-1). The GLA content in many of these species exceeds 

that found in MON 87769. GLA is also present 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 linseed oil. The oil from flaxseed 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 ingredient 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 fertilizer for crop production, 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 cod 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 Database for Standard Reference).9

 Fish oil is used for 

human consumption and in animal feed and industrial applications, including soaps, 

lubricating greases, and waterproofing agents (Bimbo and Crowther, 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 supplements, along with plant-based sources of 

SDA, such as black currant. GLA may be found 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, respectively (Ishihara et al., 2000). 

 

7

 Federal Research Centre for Nutrition and Food – Institute 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 – Institute 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 conventional soybean seed is not a source of SDA and GLA 

exposure to the environment, conventional soybean 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 history of exposure in 

soybean without known adverse 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 characteristics 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 animal 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 soybean, beans, peas and other plants, is a 

major transformation agent for PUFAs (Gardner, 1995; Feussner and Wasternack, 2002). 

PUFAs can also undergo non-enzymatic oxidative changes such as peroxidation, photooxidation, thermal oxidation, oxidative fission, hydrolysis and other reactions that 

convert the fatty acids into breakdown products (DeMan, 2000). The decomposition of 

PUFAs is primarily a result of their reactivity towards oxygen. The more unsaturated 

fatty acids are more reactive, 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; linoleic (18:2) = 

1200; and linolenic (18:3) = 2500 (DeMan, 2000). It is expected that SDA (18:4) and 

GLA (18:3) are at least as reactive, and likely more reactive 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 Stearidonic Acid (SDA), Gamma-Linolenic Acid 

(GLA), and Other Polyunsaturated Fatty Acids (PUFAs) 

Fatty acid composition of seed oil (%) 

Taxa1

C18:2n-6 

(Linoleic) 

C18:3n-6 

(γ-Linolenic) 

C18:3n-3 

(α-Linolenic)

C18:4n-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 

Table X-2. Fish that Contain Stearidonic Acid (SDA) 

Fish % SDA1 Fish % SDA Fish Anchovy 0.055 Mullet 0.082 Shark Bluefish 0.167 Ocean perch, Atlantic 0.024 Sheepshead Bass, freshwater 0.039 Perch, mixed species 0.008 Smelt Carp 0.058 Pollock, Atlantic 0.005 Snapper Catfish, farmed 0.037 Rockfish, Pacific 0.016 Spot Catfish, wild 0.013 Roe 0.012 Sturgeon Cisco 0.029 Roughy, orange 0.001 Sucker, white Cod, Atlantic 0.001 Sablefish 0.114 Tilapia Croaker 0.011 Salmon, Atlantic, farmed 0.121 Trout, mixed species Dolphinfish 0.005 Salmon, Atlantic, wild 0.083 Trout, rainbow, farmed Flatfish 0.016 Salmon, chinook 0.188 Trout, rainbow, wild Haddock 0.003 Salmon, chum 0.068 Tuna, fresh, bluefin Halibut, Atlantic/Pacific 0.039 Salmon, coho, farmed 0.112 Tuna, fresh, skipjack Halibut, greenland 0.132 Salmon, coho, wild 0.119 Tuna, fresh, yellowfin Herring, Atlantic 0.214 Salmon, pink 0.09 Whitefish Herring, Pacific 0.248 Salmon, sockeye 0.102 Whiting Mackerel, king 0.278 Seabass 0.046 Wolffish Mackerel, Atlantic 0.09 Seatrout 0.008 Mackerel, Pacific 0.125 Shad, American 0.328 

Source: USDA-ARS National Nutrient Database for Standard Reference (http://www.ars.usda.gov/nutrientdata). [Accessed on 8 De1 Concentration 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 biotechnology-derived crop. An assessment of 

MON 87769 for potential risks to NTOs was conducted based on the finding that the 

presence of the inserted genes, the expression of the PjΔ6D and NcΔ15D proteins, and 

the production of the expected fatty acids (SDA and GLA) in MON 87769 seed were 

meaningful differences compared to conventional 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 assessment considered pertinent product 

characterization information, information from the protein safety assessments, history of 

environmental exposure to SDA and GLA (the additional fatty acids present in 

MON 87769 seed), and results from the ecological interaction assessment. Based on the 

weight of evidence of all the information reported 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 that MON 87769 contains one copy of the insert at a single 

integration locus. No additional elements from the transformation vector were detected 

in the genome of MON 87769. Additionally, the data confirm the organization and 

sequence of the insert and demonstrate the stability of the insert over several generations. 

On the basis of these data, it is concluded that only the expected PjΔ6D and NcΔ15D 

proteins are produced from the inserted DNA (Section X.A.1). 

From the protein safety assessment data (Sections VI and X.A.2), it can be concluded 

with reasonable certainty that the PjΔ6D and NcΔ15D proteins have no meaningful toxic 

potential to exposed organisms in the environment. Furthermore, these integral 

membrane proteins are structurally and functionally related to other desaturases that are 

widely distributed in plants and 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 and GLA are not typically produced in soybean, they are 

already present in the environment (Section X.A.3). Importantly, the history of exposure 

across a wide taxonomic range provides evidence that these fatty acids 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 other animals. Their lack of toxicity is further supported by 

the known biological role and metabolism of polyunsaturated fatty acids, the ecological 

interaction assessments of MON 87769 conducted 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 and diseases under field 

conditions over two consecutive years, including over 500 observations of plant damage 

caused by various arthropods and diseases, and 260 observations of the abundance of 

various pest and beneficial arthropods were conducted (see Section VIII.D.2.2.). Data 

support the conclusion that MON 87769 does not have increased susceptibility or 

tolerance to the evaluated arthropods or diseases or altered interactions with the evaluated 

pest and beneficial arthropods, as indicated by the abundance data, when compared to 

conventional soybean. In particular, there 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 PjΔ6D and 

NcΔ15D proteins and the additional fatty acids present in MON 87769 immature and 

mature seed. Thus, the results of the environmental interactions assessment further 

corroborate the hypothesis that the introduced genes (Pj.D6D and Nc.Fad3), the proteins 

produced from the introduced genes (PjΔ6D and NcΔ15D), 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 invading established ecosystems. Soybean is not 

listed as a weed in the major weed references (Crockett, 1977; Holm et al., 1979; 

Muenscher, 1980), nor is it identified as a noxious weed species on the list distributed by 

the federal government (7 CFR § 360). Soybean does not possess any of the attributes 

commonly associated with weeds (Baker, 1965), such as long persistence of seed in the 

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

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

absence of dormancy, a trait that has been removed through commercial breeding, 

soybean seed can germinate quickly under adequate temperature and moisture and can 

potentially grow as volunteer plants. However, volunteer plants likely would be killed by 

frost in the soybean growing regions during autumn or winter of the year they were 

produced. If they did become established, volunteer plants would not compete well with 

the succeeding crop, and could be controlled readily by either mechanical or chemical 

means (OECD, 2000). In addition, since 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 alter their weediness potential. 

In comparative studies between MON 87769 and a conventional soybean control, 

phenotypic, agronomic and environmental interaction data were evaluated (Section VIII) 

for changes that would impact the plant pest potential and, in particular, plant weediness 

potential. Results of these evaluations indicate that there is no fundamental difference 

between MON 87769 and the conventional control for traits associated with weediness. 

Collectively, these findings support the conclusion that MON 87769 has no increased 

weediness potential compared to conventional soybean. Data on environmental 
Monsanto Company 09-SY-200U Page 176 of 395 

interactions also indicate that MON 87769 does not confer any biologically meaningful 

increased susceptibility or tolerance to specific disease, arthropod, or abiotic stressors. 

X.C. Potential for Pollen-mediated Gene Flow 

Gene flow and gene introgression are processes whereby one or more genes successfully 

integrate into the genome of a recipient plant. Introgression is affected by many factors, 

including the frequency of the initial pollination event, environmental factors, sexual 

compatibility of pollen donor and recipient plants, pollination biology, flowering 

phenology, hybrid stability and fertility, selection and the ability to backcross repeatedly. 

Since gene introgression is a natural biological process, it does not constitute an 

environmental risk in and of itself (Sunderland 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 transgenes and their subsequent 

transfer to recipient plants will result 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-pollinated 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-pollination has been found to be generally very low. Most 

outcrossing occurred with surrounding plants and cross-pollination frequencies vary, 

depending on growing season and genotype. Insect activity does increase the outcrossing 

rate, but soybean generally is not a preferred plant for pollinators (Erickson, 1975 and 

1984). 

Numerous studies on soybean cross-pollination have been conducted, and the published 

results (with and without supplemental pollinators) are summarized in Table X-3. Under 

natural conditions, cross pollination among adjacent 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 ranged from 0.5 to 7.74% in adjacent plants or adjacent rows. 

However, cross-pollination does not occur at these levels over long distances. 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 reported: 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 limited. This is recognized in certified seed 

regulations for Foundation seed in the U.S., which permit any distance between different 

soybean cultivars in the field so long as the 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 evaluations have shown that 
Monsanto Company 09-SY-200U Page 177 of 395 

the introduced genes (Pj.D6D and Nc.Fad), the proteins produced from the introduced 

genes (PjΔ6D and NcΔ15D) and the intended fatty acid change are not likely to enhance 

weediness or plant pest potential. Therefore, 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 Literature on Soybean Cross-Pollination 

Distance 

from Pollen 

Source 

% CrossPollination Comments Reference 

0.3 m 0.04% 

(estimated per 

pod)

Interspaced plants within a row. Experiment 

conducted in a single year. Single male and 

female parental varieties. Percent 

outcrossing calculated per pod rather than 

per seed. 

Woodworth, 

1922

0.8 m 0.07 to 0.18% Adjacent rows. Experiment conducted over 

two years. Several male and female parental 

varieties. 

Garber and 

Odland, 1926

0.1 m 0.38 to 2.43% Adjacent plants within a row. Experiment 

conducted in a single year. Several male and 

female parental varieties.

Cutler, 1934

0.1 m 0.2 to 1% Adjacent plants within a row. Experiment 

conducted in single year at two locations. 

Several male and female parental varieties.

Weber and 

Hanson, 1961

0.9 m

2.7 – 4.6 m

6.4 – 8.2 m

10 – 15.5 m

0.03 to 0.44 %

0.007 to 0.04% 

0 to 0.02%

0 to 0.01%

Frequency by distance was investigated. 

Experiment conducted over three years. 

Single male and female parental varieties.

Caviness, 1966

0.8 m 0.3 to 3.62% Various arrangements within and among 

adjacent rows. Experiment conducted over 

three years. Several male and female 

parental varieties.

Beard and 

Knowles, 1971

One row

(undefined)

1.15 to 7.74% Bee pollination of single-row, small-plots of 

pollen receptor surrounded by large fields 

(several acres) of pollen donor soybean. 

Soybean is not a preferred flower for 

honeybee. 

Abrams et al., 

1978

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., 2003

 
Monsanto Company 09-SY-200U Page 179 of 395 

X.C.1.2. Hybridization with the Wild 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 “natural hybrids between G. max and G. 

soja are rare ... and hybrid swarms involving both species have never been reported.” 

Many barriers exist to natural hybridization between soybean and wild relatives including 

the highly selfing nature of both plants, required proximity of wild soybean to cultivated 

soybean, synchrony of flowering, and presence of pollinators. As such, it is unlikely that 

naturally occurring, pollen-mediated gene flow and transgene introgression into wild 

soybean relatives from incidentally released biotechnology-derived soybean will occur at 

any meaningful frequency. 

As described earlier, the subgenus Soja also contains an unofficial species, G. gracilis 

(Hymowitz, 2004). G. gracilis is known only from Northeast China, and is considered a 

weedy or semi-wild form of G. max, with some phenotypic characteristics intermediate to 

those of G. max and G. soja. G. gracilis may be a hybrid between G. soja and G. max

(Hymowitz, 1970). Interspecific fertile hybrids formed by intentional crosses between G. 

max and G. soja and between G. max and G. gracilis have been easily obtained 

(Dorokhov et al., 2004). Although hybridization between G. max and members of the 

subgenus G. soja can take place, G. soja is not found in North or South America, and it is 

highly unlikely that gene transfer will occur. 

X.C.1.3. Hybridization with Wild Perennial Species of Subgenus Glycine

The wild perennial species of the Glycine subgenus occur in Australia, West Central and 

South Pacific Islands, China, Papua New Guinea, Philippines, and Taiwan. Therefore, 

the only opportunities for inter-subgeneric hybridization would occur in areas where 

those species are endemic (Hymowitz et al., 1992; Hymowitz and Singh, 1992). 

Nonetheless, the likelihood of interspecific hybridization between G. max and the wild 

perennial Glycine species is extremely low because they are genomically dissimilar (see 

Table II-2) and pod abortion is common. From time to time, immature seeds of the 

crosses could be germinated aseptically in vitro, but the resulting F1 hybrids are slowgrowing, morphologically weak, and completely sterile. Their sterility is due to poor 

chromosome pairing. Furthermore, species distantly related usually produce nonviable 

F1 seeds that either have premature death of the germinating seedlings or suffer from 

seedling and vegetative lethality (Kollipara et al., 1993; Singh and Hymowitz, 1989). In 

North and South America, it is not possible for gene transfer 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 other 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 PjΔ6D and NcΔ15D proteins as well as the two fatty acids 

not previously produced in soybean (SDA and 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 disease to any plant or plant 

product [7 U.S.C. § 7702(14)]. Characterization 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 (PjΔ6D and NcΔ15D), and the intended 

fatty acid change, MON 87769 is not different from conventional soybean in its 

phenotype, environmental interactions, or susceptibility to pest or disease. A plant pest 

assessment was conducted to assess the potential impact of the introduced genes (Pj.D6D 

and Nc.Fad3), the proteins produced from the introduced genes (PjΔ6D and NcΔ15D), 

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 concluded that the potential 

risk of MON 87769 to cause adverse effects 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 pollen-mediated gene flow from MON 87769 to 

other sexually compatible plants is considered negligible because the potential for gene 

flow is low. Evaluations have shown that the introduction of the genes (Pj.D6D and 

Nc.Fad3), the proteins produced from the introduced genes (PjΔ6D and NcΔ15D), and 

intended fatty acid change is not likely to enhance weediness or plant pest potential. 

There are no changes expected in agronomic practices for MON 87769 and the 

production systems currently used with specialty soybean. Thus, compared to 

conventional soybean, there are no increased plant pest characteristics 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, Monsanto 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 observations associated with MON 87769 that 

indicate there would be an adverse environmental consequence from the introduction of 

MON 87769. MON 87769 is an alternate source of omega-3 fatty acid. As demonstrated 

by field results and laboratory tests, the only phenotypic difference between MON 87769 

and conventional soybean is the intended change in seed fatty acid composition and the 

presence of the two desaturase proteins (PjΔ6D and NcΔ15D). 

The data and information presented in this petition demonstrate that MON 87769 is 

unlikely to pose an increased plant pest potential or to have an adverse environmental 

consequence compared to conventional soybean. This conclusion is reached based on 

multiple lines of evidence developed from a detailed characterization of the product 

compared to conventional soybean, followed by risk assessment on detected differences. 

The characterization evaluations included molecular and protein analyses, which 

confirmed the insertion of a single functional copy of the Pj.D6D and Nc.Fad3

expression cassette at a single locus within the soybean genome and that PjΔ6D and 

NcΔ15D 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 PjΔ6D and NcΔ15D proteins concluded that the PjΔ6D and NcΔ15D 

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, animals, and other NTOs from MON 87769 are 

negligible under the conditions of use. 

The introduction of MON 87769 will not impact cultivation practices and the 

management of weeds, diseases, and insects in soybean production systems. Successful 

adaptation of MON 87769 will provide growers 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. Harris. 1978. Yields and cross-pollination of 

soybeans as affected by honey bees and alfalfa leafcutting bees. American Bee Journal 

118:555-560. 

Abud, S., P.I. Mello de Souza, C.T. Moreira, S.R.M. Andrade, A.V. Ulbrich, G.R. 

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APPENDICES 

Appendix A. USDA Notifications 

Field trials of MON 87769 were 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 MON 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 preparation. A list of trials conducted under 

USDA notification and the status of the final reports for these trials are provided in 

Table A-1. 

 
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Table A-1. USDA Notification Approved 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(11), 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 USDA 
Monsanto Company 09-SY-200U Page 201 of 395 

Table A-1 (continued). USDA Notification 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 USDA 
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Table A-1 (continued). USDA Notification 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 USDA 
Monsanto Company 09-SY-200U Page 203 of 395 

Appendix B. Materials and Methods Used for Molecular Analyses of MON 87769 

B.1. Materials 

The DNA used in the molecular analyses was 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 generations of MON 87769 seed and used in 

generational stability analyses. The control DNA was isolated from leaf tissue from 

conventional soybean with the same genetic background (seed lot number GLP-0509-

16625-S). The reference substances included the PV-GMPQ1972 plasmid, probe 

templates generated from this plasmid, and the size estimation molecular weight 

standards. As a positive control on Southern 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 substance digested with appropriate enzyme(s). 

Probe templates generated from this plasmid also served as positive hybridization 

controls. Digested plasmid DNA or probe templates were added to predigested 

conventional soybean genomic DNA. The molecular weight standards include the 1 kb 

DNA Extension Ladder (Invitrogen, Carlsbad, CA) and λ DNA/Hind 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 identity of the materials was confirmed by the 

generation stability Southern blots themselves. The stability of the genomic DNA was 

confirmed in each Southern analysis by observation of the digested DNA sample on an 

ethidium bromide-stained agarose gel. 

B.3. DNA Isolation for Southern Blot and PCR Analyses 

Genomic DNA samples from MON 87769 and conventional soybean control were 

isolated from leaf tissue that was processed into a fine powder using a mortar and pestle 

under liquid nitrogen. DNA was extracted from the processed seed using a 

hexadecyltrimethylammonium bromide (CTAB) 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 extraction buffer (1.5% CTAB, 75 mM Tris pH 8.0, 

100 mM EDTA, 1.05 M NaCl, 0.75% PVP [40K]) and 5-10 μL of 10 mg/mL RNase was 

added. The samples were incubated at 65o

C for 40-50 minutes and mixed halfway 

through the incubation. The samples were cooled to room temperature, and divided 

between two tubes. 20 mL of 24:1 chloroform: isoamyl alcohol was added and the 

samples were mixed for 2-3 minutes and 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 samples 
Monsanto Company 09-SY-200U Page 204 of 395 

were then centrifuged at 20-25o

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 HCl, pH 8.0, 1 mM EDTA, pH 8.0). To 

facilitate resuspension of the DNA, additional 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 performed using a Hoefer DyNA Quant 200 

Fluorometer with Roche molecular size marker IX as a DNA calibration standard. 

B.5. Restriction Enzyme Digestion of Genomic DNA 

Approximately 10 or 20 μg of genomic DNA extracted from the test and control 

substances were used for restriction enzyme digestions. When digesting genomic DNA 

with Lgu I (Fermentas, Hanover, MD) or the enzyme combination BsrG I and PshA I 

(New England BioLabs, Ipswich, MA), Tango Buffer (Fermentas) was used. When 

digesting genomic DNA with the enzyme combination Lgu I and BstX 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 μl 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 32P-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 electrophoresis. Except for 

generational stability analyses, DNA samples were loaded on the gels for a long run and a 

short run in an effort to provide better resolution 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 Southern 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 insert. The PCR analyses were conducted 
Monsanto 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 μl reaction volume containing a final 

concentration of 1.8 mM MgCl2, 0.2 μM 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, Product 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 following 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 performed 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 were 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 PjΔ6D and NcΔ15D Proteins Produced in 

MON 87769 – Materials, Methods and the Results 

C.1. Materials and Methods Used for the Characterization of PjΔ6D Protein 

C.1.1. Protein Purification 

The PjΔ6D protein was purified from immature MON 87769 seed prior to initiation of 

this characterization plan. The purification of PjΔ6D 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 were followed. All operations were carried 

out at 4 °C unless otherwise indicated. 

A total of 11 kg of immature MON 87769 seed (Orion Lot No. 10002214) was used for 

the isolation of PjΔ6D. The seed was homogenized for membrane 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 and separated on a 3,000-mL cation exchange 

chromatography column (a total of three runs) followed by immuno-affinity purification 

(a total of 30 runs). The MON 87769-produced PjΔ6D protein isolated from the different 

batches were combined as a final preparation. After preliminary purity and concentration 

determination, the protein preparation was submitted 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 (Waring Commercial, 

Torrington, CT) for 3 min in 4 L 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 μM leupeptin, and 1.0 

mM sodium benzamidine hydrochloride. After 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-suspended in ice cold de-ionized water (DI 

H2O) at a total protein concentration 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 MgCl2, 0.5 mM PMSF, 1 μM leupeptin, and 1.0 mM 

benzamidine hydrochloride). Immediately after 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-suspended 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]. Solid Fos-choline 12 was added to a final 

concentration of 2% and the membrane/detergent solution was incubated at 4 °C for 2 hr 

with stirring. The mixture 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-Sepharose column (bed 
Monsanto 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% glycerol (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. Fractions containing the protein of interest 

were pooled, and stored at -80 °C. 

Immuno-affinity chromatography - Immuno-affinity resin used in the purification was 

prepared by immobilizing anti-PjΔ6D peptide IgG to agarose resin using an IgGorientation kit from Pierce (Cat. No. 44990, Rockford, IL), according to the 

manufacturer’s instructions. The anti-PjΔ6D antibody (Lot Numbers: 7580967 and 

7580957) was used for immunoaffinity resin preparation (total vol. of resin ~28 mL). 

The immuno-affinity column was equilibrated with Immunoaffinity Binding and 

Washing (IABW) Buffer consisting of 1X PBS, pH7.2, 1mM MgCl2, 10% glycerol (v/v) 

and 0.1% Fos-choline 12 (w/v). Before applying 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 through was repeated three times. The column 

was washed extensively with the IA washing buffer and PjΔ6D was eluted with 0.1 M 

Glycine, pH 2.8 containing 1 mM MgCl2, 10% glycerol (v/v) and 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-purified 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 previously 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 PjΔ6D 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 PjΔ6D 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 column, the flow through fraction was collected 

and saved as the final PjΔ6D protein preparation. After preliminary purity and 

concentration determination, the final preparation 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 PjΔ6D protein sample was determined 

by amino acid analysis (AAA) using AccQ-Tag derivatization (Waters Corporation, 

Milford, MA), a pre-column derivatization method which allows for high sensitivity 

fluorescent detection of amino acids. In order to avoid the interference from buffer 

components during protein hydrolysis, protein samples were precipitated using ethanol. 

The sample preparation was as follows: in a hydrolysis tube (~300 μl), approximately 

1 μg of a protein sample was mixed with 200 μL of chilled 95% ethanol. After 

incubation overnight at -20 oC, each sample was centrifuged for 45 min at 2-4 °C at 
Monsanto 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 μL of both 

chilled acetone and water. Replicates of MON 87769-produced PjΔ6D protein, a 

hydrolysis blank, four dilutions of a calibration 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 tubes 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. Samples 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 using a Speed-Vac concentrator. A 20 μL volume 

of reconstitution solution (20 mM HCl) was added 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 data were collected using Atlas software 

(Thermo Electron Corp.). 

C.1.3. Protein Identity 

Immunoblot Analysis

Immunoblot analysis was performed to confirm the identity of PjΔ6D protein purified 

from MON 87769. Based on the concentration and purity, aliquots of the purified PjΔ6D 

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 Buffer (LB) (312.5 mM Tris-HCl, 25% (v/v) 

2-mercaptoethanol, 10% (w/v) sodium dodecyl sulfate, 0.025% (w/v) bromophenol blue, 

50% (v/v) glycerol, pH 6.8 and loaded directly onto gels without boiling. Membrane 

proteins in (a portion) the presence of detergents 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 the 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-stained molecular weight markers (Precision 

Plus protein standard, BioRad, Hercules, CA) were used to confirm 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 probed with a 1:5000 dilution of goat anti-PjΔ6D 

antibody (APS Lot No. 7580957 ̧ Orion number 10000821) in PBST containing 2% (w/v) 

NFDM for 1 hr. The IgG was purified from crude antiserum using immobilized PjΔ6D 

protein and was used at dilution of 1:5000. Rabbit anti-goat IgG conjugated with horse 
Monsanto Company 09-SY-200U Page 209 of 395 

radish peroxidase (Pierce, Rockford, IL) was used as secondary antibody at a dilution of 

1:10,000. Excess antibody was removed using five 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, Piscataway, NJ) and exposed 

(30 and 60 sec) to Hyperfilm ECL (G. E. Healthcare, Piscataway, NJ). Films were 

developed using a Konica SRX-101A automated film processor (Tokyo, Japan). The 

MW of the immunoreactive bands was estimated 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 PjΔ6D. 

Protein Blot for N-Terminal Analysis

An aliquot of MON 87769-produced PjΔ6D 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 directly onto gels without boiling. SDS-PAGE was 

conducted using 4-20% precast polyacrylamide gels (Invitrogen, 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 electrotransferred to a PVDF (Invitrogen, Carlsbad, 

CA) membrane in the electrotransfer buffer containing 10 mM CAPS, pH 11, and 10% 

methanol at a constant voltage of 26 V for 120 min. Pre-stained molecular weight 

markers (Precision Plus protein 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 

PjΔ6D protein was excised from the blot and N-terminal sequence analysis was 

performed for 15 cycles using automated Edman degradation chemistry (Hunkapiller and 

Hood, 1983). An Applied Biosystems 494 Procise Sequencing System with 140C 

Microgradient system and 785 Programmable Absorbance Detector and Procise Control 

Software (version 1.1a) were used. Chromatographic data were collected using Atlas 

software (version 3.59a, LabSystems, 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 PjΔ6D 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 PjΔ6D protein. 

Protein Gel for Tryptic Mass Map Analysis

A 100 μL aliquot of MON 87769-produced PjΔ6D 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 directly onto gels without boiling. SDS-PAGE was 

conducted using 4-20% precast polyacrylamide gels (Invitrogen, 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 Coomassie blue dye R-250 (BioRad, Hercules, CA) 

to visualize the protein of interest. The band corresponding to the MON 87769-produced 

PjΔ6D protein was excised from five lanes of the gel, destained, reduced, and alkylated. 

Briefly, each gel band was destained for 30 min by incubation in 100 μL of destaining 

solution (40% methanol, 10% acetic acid in water) in a microfuge tube. This step was 

repeated four times with the final destaining step proceeding for 60 minutes. Following 

destaining, the gel band was incubated in 100 μL of 100 mM ammonium bicarbonate 

buffer for 30 min at room temperature. The protein was reduced in 100 μL of 10 mM 

DTT solution for 2 h at 37 °C. The protein was alkylated by the addition of 100 μL of 20 

mM iodoacetic acid. The alkylation reaction was allowed to proceed at room temperature 

for 20 min in the dark. Gel band was incubated in 200 μL of 25 mM ammonium 

bicarbonate buffer for 15-25 min at room temperature. This step was repeated two 

additional times, following which the gel band was dried using a Speed Vac concentrator 

(Ramsey, MN). A single gel band was rehydrated with 20 μL of 0.02 μg/μL trypsin in 

25 mM ammonium bicarbonate, 10% acetonitrile, and was incubated for about 1 h at 

room temperature, after which excess liquid was removed and the sample was incubated 

overnight at 37 °C in 40 μL 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 concentrator (Extract 1). The gel band was resuspended in 30 μL 60% acetonitrile, 0.1% trifluoroacetic 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 μL 0.1% trifluoroacetic acid and then dried using a Speed Vac concentrator. Extract 1 

was resuspended in 5 μL of 50% acetonitrile, 0.1% trifluoroacetic acid, while Extract 2 

was re-suspended in 10 μL 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 mixture (CalMix 2; Applied Biosystems). 

Extract 1 and Extract 2 samples (0.3 μL) were co-crystallized with 0.70 μL 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 acid) at separate 
Monsanto 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. The laser intensity 

setting is a unit-less MALDI-TOF instrument-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 acid matrix were analyzed in the 850 to 7500 

Da range using 200 shots at a laser intensity setting of 3200. Protonated (MH+) peptide 

masses were observed monoisotopically in reflector 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 PjΔ6D protein sequence based upon the nucleotide sequence. 

Masses were calculated for each theoretical peptide and compared to the raw mass data. 

Experimental masses (MH+) were assigned to peaks in the 500 to 1000 Da range if there 

were two or more isotopically resolved peaks, 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 than approximately twice the baseline noise, or when a mass 

could not be assigned due to overlap with a stronger signal ±2 Da from the mass analyzed. 

C.1.7. Molecular Weight and Purity Estimation – SDS-PAGE 

Aliquots of the MON 87769-produced PjΔ6D protein were diluted with Milli-Q water 

and mixed with 5X LB to a final protein concentration of 0.2 μg/μL. The MON 87769-

produced PjΔ6D protein was analyzed in duplicate at 2, 3, and 4 μg of total protein per 

lane. Molecular weight 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). Pre-stained molecular 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 minutes. Proteins were 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 apparent molecular weight of the MON 87769-

produced PjΔ6D protein. For the purity evaluation, all visible bands within each lane 

were quantified. The purity and estimated molecular weight of the MON 87769-

produced PjΔ6D protein were reported as the average of the six values obtained by 

densitometric analysis. 

C.1.8. Glycosylation Analysis 

The GE Healthcare Glycosylation Detection Module (Piscataway, NJ) was used to detect 

carbohydrate covalently bound to the MON 87769-produced PjΔ6D proteins after it was 

resolved by SDS-PAGE and electrotransferred onto PVDF membrane. The kit utilizes 

the specific labeling of biotin to the bound carbohydrate moiety followed by probing with 

streptavidin-conjugated horse radish 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 moieties in the glycoproteins 
Monsanto Company 09-SY-200U Page 212 of 395 

to aldehydes which then react with biotin, resulting 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 PjΔ6D protein in SP-buffer A were 

mixed with 5X LB and loaded onto a 4-20% precast polyacrylamide gel (Invitrogen, 

Carlsbad, CA). Along with the PjΔ6D samples, 25, 50 100 and 200 ng of the 

glycosylated control protein, transferrin, were loaded in parallel. The PjΔ6D samples 

were not heated, 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. Proteins in the gel were electrotransferred to a PVDF membrane (Invitrogen, 

Carlsbad, CA) at a constant voltage of 25 V for 90 min. Pre-stained molecular weight 

markers (Precision Plus Dual Color protein 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. Transferrin (~76 – 81 kDa, Sigma-Aldrich, St. Louis, MO) 

was loaded in parallel as a positive control of glycosylated protein in series of dilution. 

The labeling and detection of carbohydrate was carried out according to the 

manufacturer’s instructions and all the reagents except PBS were provided by the kit. 

Following electrotransfer to 0.45 μm PVDF membrane, the blot was incubated first in 30 

mL of PBS for 10 min, then in 10 mM NaIO4 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 two 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 min 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 incubation, the excess detection solution was removed by 

blotting with paper towel and the blot was exposed to Hyperfilm ECL (G. E. Healthcare, 

Piscataway, NJ). The film was developed using a Konica SRX-101A automated film 

processor (Tokyo, Japan). The blot images were captured using a Bio-Rad GS-800 

densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). 

C.1.9. Storage Stability 

The short-term stability of the MON 87769-produced PjΔ6D protein was evaluated by 

comparing the purity and molecular weight values before and after storage for 29 days in 

a 4 oC refrigerator, and in -20 oC, and -80 oC freezers. At the end of storage, aliquots of 

the PjΔ6D protein samples were removed and subjected to SDS-PAGE followed by 

staining, and subsequent purity and MW estimation. The MON 87769-produced PjΔ6D 

protein was analyzed in duplicate at 2, 3, and 4 μg 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, Hercules, 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 stained using colloidal Coomassie Brilliant Blue 

stain (Sigma-Aldrich, St. Louis, MO). 

Analysis of each 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 apparent molecular weight of the MON 87769-

produced PjΔ6D protein. 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.2. Materials and Methods Used for the Characterization of NcΔ15D Protein 

C.2.1. Protein Purification 

The NcΔ15 protein was purified from immature MON 87769 seed prior to initiation of 

this characterization plan. The purification was not performed under a GLP study or 

plan; however, all procedures were documented 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 seed was used for the isolation of NcΔ15D 

protein in two separate batches. Approximately 2.5 kg of seed (Seed Lot No. GLP-0509-

16624-S) were homogenized in four batches (500-800 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 isolation at a larger scale with 1,000-2,500 g 

of seed per extraction (a total of six runs). The solubilized membranes were then 

separated on a 3,000-mL cation exchange chromatography column (a total of three runs) 

followed by immuno-affinity purification (a total of 30 runs). The NcΔ15D protein 

preparations by both small-scale and large-scale operations were combined. Although at 

different scales, conditions (e.g., buffer to sample ratio) used to purify NcΔ15D protein at 

both small and large scales were proportionally comparable and therefore practically 

identical. After preliminary purity and concentration determination of the purified 

NcΔ15D 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), approximately 400 g of 

immature MON 87769 seed was homogenized 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 μM leupeptin, and 1.0 mM sodium benzamidine hydrochloride. 

The process was repeated to homogenize the required amount seed (typically 1,000 g to 

2,000 g seed per extraction). After filtration of the homogenate through four layers of 
Monsanto 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 pellet re-suspended in ice-cold DI H2O at a membrane 

concentration of approximately 40 mg/mL (fw/v). The membrane suspension was then 

mixed with an equal volume of 2X Carbonate Wash Buffer (200 mM sodium carbonate, 

pH 11.5, 2 mM MgCl2, 0.5 mM PMSF, 1 μM leupeptin, and 1.0 mM benzamidine 

hydrochloride). Immediately after mixing, the mixture was centrifuged at 37,000 x g for 

30 min. The supernatant was discarded and the membrane pellets 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 μM 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 solubilized 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 consisting 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 pooled, and stored at -80 °C. 

Immuno-affinity chromatography 

Immuno-affinity resin used in the purification was prepared by immobilizing antiNcΔ15D IgG to agarose resin using an IgG-orientation kit (Pierce Cat. No. 44990, 

Rockford, IL) according to the manufacture’s instructions. The anti-NcΔ15D 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 phosphate, pH 7.2, 150 mM NaCl, 1 mM MgCl2, 

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 extensively with the IAWB and the NcΔ15D protein was 

eluted with 0.1 M glycine, pH 2.8 containing 1mM MgCl2, 10% glycerol (v/v) and 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. 

Concentration of Protein solution 

To concentrate the immuno-affinity-purified NcΔ15D 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 equilibrated with the SP-Buffer A. The flow 

rate was typically at 4.0 mL/min. The protein was eluted with 0.5 M NaCl in the SPBuffer A. Fractions containing the NcΔ15D 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 concentration of the MON 87769-produced NcΔ15D protein preparation 

was determined by amino acid analysis (AAA) using AccQ-Tag derivatization (Waters 

Corporation, Milford, MA), which allows for high sensitivity fluorescent detection of 

amino acids. In order to avoid the interference from buffer components during protein 

hydrolysis, protein was precipitated using ethanol. The sample preparation was as 

follows: in a hydrolysis tube (~300 μl), approximately 1 μg of protein samples (~10 μL) 

were mixed with 200 μL of chilled 95% ethanol. After incubation overnight at -20 oC, 

samples were centrifuged at 12,000 rpm in a microcentrifuge for 30 min at 2-8 °C. The 

supernatant was removed and discarded. Precipitates were then washed sequentially with 

100 μL of both chilled acetone and water. Along with replicates of the test sample, a 

hydrolysis blank, four dilutions of a calibration 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 tubes using a Speed-Vac 

concentrator. 500 μL of hydrolysis solution (6N HCl/1% phenol) was added and the 

tubes were transferred to a vacuum chamber. 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 reconstituted 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 autosampler vials, capped, and heated to 55 °C 

for 10 min. Samples were analyzed using 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 NcΔ15D protein purified 

from MON 87769. Based on the concentration and purity, aliquots of the purified 

NcΔ15D 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 μL, respectively. The samples (10 μL) were then mixed with 2.5 μL 

of 5X Loading Buffer (312 mM Tris-HCl, pH 6.8, 25% 2-mercaptoethanol, 10% sodium 

dodecyl sulfate, 0.025% bromophenol blue, 50% glycerol) and separated on the SDSPAGE at 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 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 transfer of proteins and estimate 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 

NcΔ15D antibody (Lot No. 7580958) in PBST containing 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 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 15 min washes with PBST, immunoreactive bands were 

visualized using the ECL Detection System (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 automated film processor (Tokyo, Japan). 

The MW of the immunoreactive bands were estimated using a Bio-Rad GS-800 

densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). 

C.2.4. N-Terminal Sequencing 

Edman degradation was used to confirm the N-terminal identity of the MON 87769-

produced NcΔ15D 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 NcΔ15D protein preparation were removed 

from storage, mixed with 5 μL of 5X LB and 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 the gel were electrotransferred to PVDF 

membrane (Invitrogen, Carlsbad, CA) at a constant 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, Hercules, CA) to visualize the protein of 

interest. 

N-Terminal Sequencing

Following electroblotting and staining, the band corresponding to the NcΔ15D protein 

was excised from the blot and N-terminal sequence analysis was performed for 15 cycles 

using automated Edman degradation chemistry (Hunkapiller and Hood, 1983). An 

Applied Biosystems 494 Procise Sequencing System with 140C Microgradient system 

and 785 Programmable Absorbance Detector and Procise Control Software (version 1.1a) 

were used. Chromatographic data were collected using Atlas99 software (version 3.59a, 

LabSystems, Altrincham, Cheshire, England). A 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, 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 NcΔ15D protein preparation were removed 

from storage, mixed with 5 μL of 5X LB and loaded in five lanes (10.25 μg/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 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. Pre-stained MW markers were loaded to 

estimate the size of the protein bands. Following electrophoresis, the gel was stained 

with Coomassie blue dye R-250 (BioRad, Hercules, CA). The bands corresponding to 

the MON 87769-produced NcΔ15D 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 μL of destaining solution in a microfuge tube. This step was repeated 

three times with the final destaining step proceeding for 60 minutes. Following 

destaining, the gel bands were incubated in 100 μL per band of 100 mM ammonium 

bicarbonate buffer for 30 min at room temperature. The protein was reduced in 100 μL 

of 10 mM dithiothreitol solution for 2 h at 37 °C. After removing the reducing solution, 

the protein in the gel was alkylated by incubating in 100 μL 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 protein band was incubated in 200 μL of 25 mM ammonium 

bicarbonate buffer for 15-45 min at room temperature. 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 μL of 0.02 μg/μL 

trypsin in 25 mM ammonium bicarbonate and 10% acetonitrile, and was incubated for 

about 1 h at room temperature. Following the incubation, the excess solution was 

removed and the gel/trypsin reaction mixture was incubated overnight at 37 °C in 40 μL 

of 25 mM ammonium bicarbonate and 10% acetonitrile. 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 μL of a solution 

consisting of 60% acetonitrile, 0.1% trifluoroacetic acid and 0.1% octyl-β-Dglucopyranoside, and sonicated for 5-10 min. After transfer of the supernatant to a new 

tube, this step was repeated one more time, and the combined supernatants were dried 

using Speed Vac concentrator (Extract 2). Extracts 1 and 2 were separately dissolved in 

20 μL 0.1% trifluoroacetic acid and then dried using a Speed Vac concentrator. Finally, 

Extract 1 was dissolved in 5 μL of 50% acetonitrile/0.1% trifluoroacetic acid, while 

Extract 2 was dissolved in 10 μL 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 mixture (CalMix 2; Applied Biosystems, Foster 

City, CA). The samples Extract 1 and Extract 2 (0.1-0.25 μL) were co-crystallized with 

0.75 μL each of the following matrix solutions: 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 using 200 shots at a laser intensity setting of 

3,001. The laser intensity setting is a unit-less 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 setting of 2,601. The samples in sinapinic acid matrix were 

analyzed in the 900 to 8,000 Da range using 200 shots at a laser intensity setting of 3,100. 

Protonated (MH+) peptide masses were observed monoisotopically in reflector mode 

(Aebersold, 1993; Billeci and Stults, 1993), except above 3,000 Da, where mass-averaged 

values were used. GPMAW32 software (Applied Biosystems) was used to generate a 

theoretical trypsin digest of the deduced NcΔ15D amino acid 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 1,000 Da range if there were two or 

more isotopically resolved peaks, and in the 1,000 to 8,000 Da range if there were three 

or more isotopically resolved peaks in the spectra. Peaks were not assessed if the peak 

heights were less than approximately twice the 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 Purity Analysis by SDS-PAGE 

Aliquots of the MON 87769-produced NcΔ15D 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 μg/μL. The 

NcΔ15D protein was analyzed in duplicate at 1, 2, and 3 μg 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). 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 NcΔ15D protein. For the purity evaluation, all 

visible bands within each lane were quantified. The purity and estimated MW of the 

MON 87769-produced NcΔ15D protein were reported as the average of the six values 

obtained by densitometric analysis. 

C.2.8. Glycosylation Analysis 

Glycosylation Detection Module (Cat. No. RPN 2190, GE Healthcare, Piscataway, NJ) 

was used to detect carbohydrate covalently bound to MON 87769-produced NcΔ15D 

after the proteins were resolved by SDS-PAGE and electrotransferred onto PVDF 

membrane. The kit utilizes the specific labeling of biotin to the bound carbohydrate 

moiety followed by probing with streptavidin-conjugated horse radish peroxidase (StrepHRP) 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 
Monsanto Company 09-SY-200U Page 219 of 395 

moiety of the glycoprotein. This biotin-labeling procedure is specific 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 detecting glycoprotein on 

membrane blots is widely used for the determination of glycoproteins. 

Aliquots of 0.5 μg and 1.0 μg of NcΔ15D protein were mixed with 5X LB and loaded 

onto a 4-20% precast polyacrylamide gel (Invitrogen, Carlsbad, CA). Along with the 

NcΔ15D samples, 25, 50, 100, and 200 ng of the glycosylated control protein, transferrin, 

were loaded in parallel. The NcΔ15D samples were not heated, 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 μM 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 to verify electrotransfer of proteins and estimate the size of the 

bands on the blot. 

The labeling and detection of carbohydrate was carried out according to the 

manufacturer’s instruction and all the reagents 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 NaIO4 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% blocking 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 min washes with 15 mL PBS, the membrane 

was then developed with ECL detection reagents by mixing 2 mL of the Reagent 1 and 2 

mL of Reagent 2. After 1 min incubation, the excess detection solution was removed by 

blotting with paper towel and the blot was exposed to Hyperfilm ECL (G. E. Healthcare, 

Piscataway, NJ). The film was developed using a Konica SRX-101A automated film 

processor (Tokyo, Japan). The blot images were captured using a Bio-Rad GS-800 

densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). 

C.2.9 Storage Stability 

The short-term stability of the MON 87769-produced NcΔ15D 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 

NcΔ15D protein samples equivalent to 1, 2, and 3 μg of the purity-corrected NcΔ15D at 

Day 0 were removed and analysed by SDS-PAGE followed by staining, and subsequent 

purity and MW estimation. The MON 87769-produced NcΔ15D protein was analyzed in 

duplicate at 1, 2, and 3 μg 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). Electrophoresis was 

performed at a constant voltage 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 Brilliant Blue G stain (Sigma-Aldrich, St. Louis, 

MO). 

Analysis of each 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 apparent molecular weight of the MON 87769-

produced NcΔ15D 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 PjΔ6D Protein 

To characterize the purified PjΔ6D protein produced in MON 87769, a number of 

analyses were performed as described in Section C.1. The analyses employed for the 

characterization of MON 87769-produced PjΔ6D protein included: 

1. N-terminal sequence analysis. 

2. MALDI-TOF to generate a tryptic peptide map. 

3. Immunoblot analysis to establish protein identity through immunoreactivity with 

PjΔ6D–specific antibody. 

4. SDS-PAGE to assess the apparent molecular weight of the protein. 

5. Glycosylation analysis to evaluate potential 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 PjΔ6D antibody (Figure C-2) resulted in the expected 

sequence for the PjΔ6D protein (Table C-1). 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 aminopeptidase, is a common modification that occurs co-translationally 

before completion of the nascent protein chain in many organisms and its removal has no 

effect on protein structure or activity (Arfin 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 which 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 PjΔ6D protein. The protein preparation was not highly 

purified; therefore the presence of additional peptide fragments is not unexpected. The 

N-terminal sequence information, therefore, confirms the identity of the PjΔ6D protein 

isolated from MON 87769 and that its N-terminus is intact. 
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C.3.2. MALDI-TOF Mass Spectrometry Analysis 

The identity of the MON 87769-produced PjΔ6D protein was further confirmed by 

tryptic peptide mass mapping analysis using MALDI-TOF MS. In general, protein 

identification made by peptide mapping is considered to be reliable if the measured 

coverage of the sequence is 15% or higher with a minimum of five matched peptides 

(Jensen et al., 1997). Observed tryptic peptides were considered a match 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 

MON 87769-produced PjΔ6D protein sample was heat-denatured, chemically reduced, 

alkylated, digested with trypsin, guanidinated, and the mass of the tryptic peptides were 

measured. 

There were 30 unique protein fragments identified that matched the expected masses of 

the PjΔ6D trypsin-digested peptides. The identified protein fragments were used to 

assemble a coverage map of the entire PjΔ6D protein (Figure C-3), resulting in ~42% 

(188 out of 446 amino acids) coverage of the total protein. This analysis confirmed the 

identity of the MON 87769-produced PjΔ6D protein. 

 
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Figure C-1. SDS-PAGE of the MON 87769-Produced PjΔ6D Protein 

Aliquots of the MON 87769-produced PjΔ6D protein were separated by SDS-PAGE, 

followed by Coomassie blue staining. Approximate molecular weights (kDa) are shown 

on the left and correspond to the marker loaded in lanes 2 and 9. Arrow indicates PjΔ6D 

protein. 

Lane Sample Amount loaded (μg) 

1 Blank 0 

2 BioRad Broad Range Marker 4.5 

3 MON 87769-produced PjΔ6D protein 2.0 

4 MON 87769-produced PjΔ6D protein 2.0 

5 MON 87769-produced PjΔ6D protein 3.0 

6 MON 87769-produced PjΔ6D protein 3.0 

7 MON 87769-produced PjΔ6D protein 4.0 

8 MON 87769-produced PjΔ6D protein 4.0 

9 BioRad Broad Range Marker 4.5 

10 Blank 0 

 
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Figure C-2. Western Blot Analysis of MON 87769-Produced PjΔ6D Protein 

Aliquots of the purified, MON 87769-produced PjΔ6D protein were separated by SDSPAGE, and electro-transferred to a PVDF membrane. The membrane was probed with 

goat anti-PjΔ6D antibody and developed using an ECL system (GE Healthcare, 

Piscataway, NJ). Approximate molecular weights (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 PjΔ6D protein 4 

4 MON 87769-produced PjΔ6D protein 6 

5 MON 87769-produced PjΔ6D protein 8 

6 MON 87769-produced PjΔ6D protein 10 

7 MON 87769-produced PjΔ6D protein 12 

8 Empty - 

9 Empty - 

10 Empty - 

 
Monsanto Company 09-SY-200U Page 224 of 395 

Table C-1. N-terminal Amino Acid Sequence Analysis of the MON 87769-Produced 

PjΔ6D Protein 

Amino acid 

residue # from 

the Nterminus 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Predicted PjΔ6D 

Sequence1,2 M T K T I Y I T S S E L E K H N

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Observed 

Sequence3 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 PjΔ6D 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 MTKTIYITSS ELEKHNKPGD LWISIHGQVY DVSSWAALHP GGIAPLLALA 

 51 GHDVTDAFLA YHPPSTSRLL PPFSTNLLLE KHSVSETSSD YRKLLDSFHK

101 MGMFRARGHT AYATFVIMIL MLVSSVTGVL CSENPWVHLV CGAAMGFAWI 

151 QCGWIGHDSG HYRIMTDRKW NRFAQILSSN CLQGISIGWW KWNHNAHHIA 

201 CNSLEYDPDL QYIPLLVVSP KFFNSLTSRF YDKKLNFDGV SRFLVQYQHW

251 SFYPVMCVAR LNMLAQSFIL LFSRREVANR VQEILGLAVF WLWFPLLLSC 

301 LPNWGERIMF LLASYSVTGI QHVQFSLNHF SSDVYVGPPV GNDWFKKQTA 

351 GTLNISCPAW MDWFHGGLQF QVEHHLFPRM PRGQFRKISP FVRDLCKKHN

401 LTYNIASFTK ANVLTLETLR NTAIEARDLS NPIPKNMVWE AVKNVG 

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87769-Produced PjΔ6D 

Protein 

The amino acid sequence of the MON 87769 PjΔ6D 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 amino acids) of the expected protein sequence was 

identified. 

 
Monsanto Company 09-SY-200U Page 225 of 395 

C.3.3. PjΔ6D Protein Immuno-reactivity 

A western blot analysis using goat anti-PjΔ6D antibody was conducted to further confirm 

the identity of the MON 87769-produced PjΔ6D protein. As demonstrated in Figure C-2, 

an immuno-reactive band was observed at the molecular weight of approximately 46 

kDa. As expected, the immuno-reactive signal increased with increasing amount of 

protein loaded. 

The immuno-reactive profiles shown in Figure 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 electrophoresis (i.e., the samples were not heated to prevent further 

aggregation), it is reasonable to assume that the diffuse signal observed in these blots 

consisted mainly of PjΔ6D protein as it is immuno-reactive with anti-PjΔ6D antibody. It 

is also possible that the diffuse signal observed in high molecular area originates from 

endogenous proteins cross-reacting with PjΔ6D antibody proteins. The PjΔ6D-specific 

antibody also recognized a band of approximately 15 kDa. This fragment is likely the 

result of proteolytic degradation of the PjΔ6D protein generated during the purification 

procedure or originates from endogenous cross-reacting proteins co-purified with the 

PjΔ6D protein. 

Based on the above analysis, the identity of the MON 87769-produced PjΔ6D protein 

was confirmed. 

C.3.4. PjΔ6D Protein Molecular Weight 

The molecular weight of the MON 87769-produced PjΔ6D protein was determined using 

SDS-PAGE (Figure C-1). The MON 87769-produced PjΔ6D protein migrated with an 

apparent molecular weight of 46 kDa. The average purity of the full length PjΔ6D 

protein was estimated to be 47%. 

C.3.5. PjΔ6D 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 required for protein glycosylation. There are 

two forms of glycosylation. The first occurs on asparagine side chains, 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. PjΔ6D is an integral membrane protein of 

higher plant origin (Primula juliae) which contains two putative N-glycosylation sites 

Asn-Xxx-Ser/Thr (Marshall, 1972). 

To test whether the MON 87769-produced PjΔ6D protein was glycosylated, the purified 

protein was analyzed for the presence of covalently bound carbohydrate moieties using a 
Monsanto Company 09-SY-200U Page 226 of 395 

Glycoprotein Detection Module (GE Healthcare, 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 PjΔ6D 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 concentration-dependent manner (Figure C-4, Lanes 2-5). No 

detectable signal was observed at the expected molecular weight of approximately 46 

kDa for the PjΔ6D 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 consideration that the purity of PjΔ6D protein is 

approximately 47% and that no signal was observed at the expected MW for PjΔ6D 

protein (~46 kDa), it was concluded that the faint signals observed on the blot are not 

derived from the PjΔ6D protein. Most likely, the observed faint bands originated from 

plant proteins that co-purified during the preparation of the PjΔ6D protein. Therefore, 

the above data demonstrate that the MON 87769-produced PjΔ6D is not glycosylated. 

 
Monsanto Company 09-SY-200U Page 227 of 395 

Figure C-4. Glycosylation Analysis of the MON 87769-Produced PjΔ6D Protein 

Aliquots of transferrin (positive control) and MON 87769-produced PjΔ6D protein were 

separated by SDS-PAGE and transferred to a PVDF membrane. Carbohydrate moieties 

were labeled with biotin-hydrazide and detected using a Streptavidin-horseradish 

peroxidase (Streptavidin-HRP) antibody. Chemiluminescent activity of the HRP tag was 

detected using Hyperfilm ECL. Approximate molecular weights indicated in kDa 

correspond to protein standard markers (lane 6 and 9). The arrow indicates the expected 

position of PjΔ6D 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 PjΔ6D 0.500 

8 MON 87769-produced PjΔ6D 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 PjΔ6D 

Protein 

A panel of analytical techniques was used to characterize the purified MON 87769-

produced PjΔ6D protein. The identity of the MON 87769-produced PjΔ6D protein was 

confirmed by its recognition with anti-PjΔ6D 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% overall coverage of the expected protein 

sequence. The apparent molecular weight of the MON 87769-produced PjΔ6D protein 

was estimated to be 46 kDa. The MON 87769-produced PjΔ6D protein was confirmed to 

be a non-glycosylated protein. Taken together, these data confirm the identity of the 

PjΔ6D protein isolated from MON 87769. 

C.4. Characterization of the MON 87769-Produced NcΔ15D Protein 

To characterize the purified NcΔ15D protein produced in MON 87769 a number of 

analyses were performed as described in Section C.2. The analyses employed for the 

characterization of MON 87769-produced NcΔ15D protein were the same as the analyses 

utilized to characterize PjΔ6D protein and included: 

1. N-terminal sequence analysis. 

2. MALDI-TOF mass spectrometry to generate a tryptic peptide map. 

3. Immunoblot analysis to establish protein identity through immunoreactivity with 

NcΔ15D –specific antibody. 

4. SDS-PAGE to assess the apparent molecular weight of the protein. 

5. Glycosylation analysis to evaluate potential 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 stained SDS-polyacrylamide gel (Figure C-5) 

resulted in the sequence expected for the NcΔ15D 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 aminopeptidase, is a common modification that 

occurs co-translationally before completion of the nascent protein chain and has no effect 

on protein structure or activity (Arfin and Bradshaw, 1988; Polevoda and Sherman, 2000; 

Schmidt et al., 1992) and is common in many organisms. 

The N-terminal sequence information, therefore, confirms the identity of the NcΔ15D 

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 NcΔ15D Protein 

Aliquots of the MON 87769-produced NcΔ15D protein were separated by SDS-PAGE, 

followed by Coomassie blue staining. Approximate molecular weights (kDa) are shown 

on the left and correspond to the marker loaded in lanes 2 and 9. Arrow indicates 

NcΔ15D protein. 

Lane Sample 

Amount loaded 

(μg) 

1 Empty 

2 BioRad Broad Range Marker 0.2 

3 MON 87769-produced NcΔ15D protein 1 

4 MON 87769-produced NcΔ15D protein 1 

5 MON 87769-produced NcΔ15D protein 2 

6 MON 87769-produced NcΔ15D protein 2 

7 MON 87769-produced NcΔ15D protein 3 

8 MON 87769-produced NcΔ15D protein 3 

9 BioRad Broad Range Marker 0.2 

10 Empty 

 
Monsanto Company 09-SY-200U Page 230 of 395 

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

NcΔ15D Protein 

Amino acid 

residue # from 

the Nterminus

→ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Predicted 

NcΔ15D 

Sequence1,2

→ M A V T T R S H K A A A A T E P 

 │ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Observed 

Sequence3 → 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 NcΔ15D 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 NcΔ15D protein was further confirmed by 

tryptic peptide mass mapping analysis using MALDI-TOF MS. In general, protein 

identification made by peptide mapping is considered to be reliable if the measured 

coverage of the sequence is 15% or higher with a minimum of five matched peptides 

(Jensen et al., 1997). Observed tryptic peptides were considered a match 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, digested with trypsin, 

guanidinated, and the masses of the tryptic peptides were measured. 

There were 15 unique protein fragments identified that matched the expected masses of 

the NcΔ15D trypsin-digested peptides. The identified masses were used to assemble a 

coverage map indicating the matched peptide sequences for the entire NcΔ15D 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 NcΔ15D 

protein. 

 
Monsanto Company 09-SY-200U Page 231 of 395 

Figure C-6. MALDI-TOF MS Coverage Map of the MON 87769-Produced NcΔ15D 

Protein 

The amino acid sequence of the MON 87769 NcΔ15D 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 amino acids) of the expected protein sequence was 

identified. 

C.4.3. NcΔ15D Protein Immuno-reactivity 

A western blot analysis using goat anti-NcΔ15D antibody was conducted to further 

confirm the identity of the MON 87769-produced NcΔ15D protein. As demonstrated on 

Figure C-7, a predominant immuno-reactive band was observed at the expected 

molecular weight of approximately 46 kDa (Figure C-7, lanes 2-6). As expected, the 

immuno-reactive signal increased with increasing amount of protein loaded. 

The immuno-reactive profiles shown in Figure 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 taken during electrophoresis (i.e., the samples 

were not heated to prevent further aggregation), it is reasonable to assume that the diffuse 

signal observed in these blots consisted mainly of the NcΔ15D protein as it is immunoreactive with anti-NcΔ15D antibody. 

In addition, two minor bands with molecular 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 NcΔ15D protein that occurs during the 

purification procedure or originate from endogenous cross-reacting proteins co-purified 

with the NcΔ15D protein. 

Based on the above analysis, the identity of the MON 87769-produced NcΔ15D protein 

was confirmed. 

 
Monsanto Company 09-SY-200U Page 232 of 395 

Figure C-7. Western Blot Analysis of MON 87769-Produced NcΔ15D Protein 

Aliquots of the purified, MON 87769-produced NcΔ15D protein were separated by SDSPAGE, and electro-transferred to a PVDF membrane. The membrane was probed with 

goat anti-NcΔ15D antibody and developed using an ECL system (GE Healthcare, 

Piscataway, NJ ). Approximate molecular weights (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 NcΔ15D protein 2.0 

3 MON 87769-produced NcΔ15D protein 4.0 

4 MON 87769-produced NcΔ15D protein 6.0 

5 MON 87769-produced NcΔ15D protein 8.0 

6 MON 87769-produced NcΔ15D protein 10.0 

7 Empty 

8 Empty 

9 Empty 

10 Empty 
Monsanto Company 09-SY-200U Page 233 of 395 

C.4.4. NcΔ15D Protein Molecular Weight 

The molecular weight of the MON 87769-produced NcΔ15D protein was determined 

using SDS-PAGE (Figure C-5). The MON 87769-produced NcΔ15D protein migrated 

with an apparent molecular weight of 46 kDa. The average purity of the full length 

NcΔ15D protein was estimated to be 74%. 

C.4.5. NcΔ15D 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 required for protein glycosylation. There are 

two forms of glycosylation. The first occurs on asparagine side chains, 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. NcΔ15D is an integral membrane protein of 

microbial origin, which contains two putative N-glycosylation sites Asn-Xxx-Ser/Thr 

(Marshall, 1972). 

To test whether the MON 87769-produced NcΔ15D protein was glycosylated, the 

purified protein was analyzed for the presence of covalently bound carbohydrate moieties 

using a Glycoprotein Detection Module (GE Healthcare, 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 NcΔ15D 

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-8). The positive control, transferrin, was detected at the expected 

molecular weights of ~75 kDa in a concentration-dependent manner (Figure C-8, lanes 2-

5). No detectable signal was observed at the expected MW of approximately 46 kDa for 

the NcΔ15D protein (Figure C-8, lanes 7-8). 

Two faint bands were detected at ~70 kDa in lanes 7-8 containing purified NcΔ15D 

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 

NcΔ15D protein is only 74% and that no signal was observed at the expected MW for 

NcΔ15D protein ~46 kDa, it was concluded that the faint signals observed on the blot are 

not derived from the NcΔ15D protein. Most likely, the observed faint bands originate 

from plant proteins that co-purified during the preparation of the NcΔ15D protein. 

Therefore, the above data demonstrate that the MON 87769-produced NcΔ15D is not 

glycosylated. 

 
Monsanto Company 09-SY-200U Page 234 of 395 

Figure C-8. Glycosylation Analysis of the MON 87769-Produced NcΔ15D Protein 

Aliquots of transferrin (positive control) and MON 87769-produced NcΔ15D 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 HRP tag was detected using Hyperfilm ECL. 

Approximate molecular weights indicated in kDa correspond to protein standard markers 

(lane 6 and 9). The arrow indicates the expected position of NcΔ15D protein. 

 

Lane Sample Amount loaded (μg) 

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 NcΔ15D protein 0.50 

8 MON 87769-produced NcΔ15D protein 1.00 

9 BioRad Precision Plus MWT Marker 0.20 

10 Empty 
Monsanto Company 09-SY-200U Page 235 of 395 

C.4.6. Conclusions on the Characterization of the MON 87769-Produced NcΔ15D 

Protein

A panel of analytical techniques was used to characterize the purified MON 87769-

produced NcΔ15D protein. The identity of the purified NcΔ15D protein was confirmed 

by its recognition with anti-NcΔ15D 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 coverage of the expected protein sequence. The 

apparent molecular weight of the NcΔ15D protein was estimated to be 46 kDa. In 

addition, the MON 87769-produced NcΔ15D was confirmed to be a non-glycosylated 

protein. Taken together, these data confirm the identity of the NcΔ15D protein isolated 

from MON 87769. 

References 

Aebersold, R. 1993. Mass spectrometry of proteins and peptides in biotechnology. Curr. 

Opin. Biotechnol. 4:412-419. 

Arfin, S.M. and R.A. Bradshaw. 1988. Cotranslational 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. Database analysis of O-glycosylation 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 accuracy peptide mass mapping and database 

searching. Analytical Chemistry 69:4741-4750. 

Marshall, R.D. 1972. Glycoproteins. Annual 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 detergents designed for the structural 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-resistant 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. Subramanian. 1992. Purification and characterization 

of 7 chloroplast ribosomal-proteins: Evidence that organelle 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 practical 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 and 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 sites were shipped to the Monsanto Sample 

Processing facility. Processed tissue samples were stored in a -80 °C freezer until 

shipped on dry ice to Monsanto’s analytical facility. All processed tissue samples were 

stored in a -80 °C freezer during the study. 

The PjΔ6D and NcΔ15D proteins were extracted from soybean tissue by shaking tubes 

mounted in a Harbil mixer for two 3.5 minute cycles. 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 inhibitors [Roche Diagnostics, Indianapolis, IN]) 

and a tissue-to-buffer ratio of 1:10 for root, leaf, and forage 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. PjΔ6D Antibodies 

Goat polyclonal antibodies (lot # 7580971) were raised against a peptide fragment of the 

PjΔ6D protein (EARDLSNPIPKN), which is located close to the C terminus of the 

PjΔ6D protein. The antibodies were affinity purified by Invitrogen Life Sciences 

(Carlsbad, CA). The concentration of the purified IgG was determined to be 2.2 mg/mL 

by spectrophotometric methods. The purified 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. NcΔ15D Antibodies

Goat polyclonal antibodies (lot # 7580965) were raised against a peptide fragment of the 

NcΔ15D protein (TEADRNQRKLANLYMDKET) located in the middle region of the 

NcΔ15D protein. The antibodies were affinity purified by Invitrogen Life Sciences. The 

concentration of the purified IgG was determined to be 2.6 mg/mL by spectrophotometric 

methods. The purified antibody was stored in Borate Buffered Saline. 

D.4. PjΔ6D 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 Laemmli 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. The reference standards ranged from 20 ng to 1 

ng. The nontransgenic extracts used for the reference standards for each tissue type were 

pooled from all sites. Samples were loaded in triplicate and at least two were used for 

densitometry and quantitation. Additionally, the Precision Plus molecular weight marker 
Monsanto Company 09-SY-200U Page 238 of 395 

(Bio-Rad) was loaded on the gel to demonstrate 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 western blot analysis 

were conducted according to the current versions of SOPs BR-ME-0924 and BR-ME0392, respectively. Proteins separated by SDS-PAGE were electrophoretically 

transferred to 0.45 μm Criterion Nitrocellulose membrane (Bio-Rad) using 1X Trisglycine transfer buffer (Bio-Rad) 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 PjΔ6D protein with a 1:2500 dilution 

of purified goat antibodies, anti-PjΔ6D 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 according to the manufacturers’ instructions. 

The membrane was exposed to Hyperfilm ECL (Amersham, Piscataway, NJ) to record an 

image of the immunoreactive bands. 

D.5. NcΔ15D 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 appropriate reference standards. The reference 

standards were prepared in tissue-specific nontransgenic extracts for each tissue type and 

diluted in 2X Laemmli buffer. The reference standards ranged from 75 ng to 1 ng. The 

nontransgenic extracts used for the reference standards for each tissue type were pooled 

from all sites. Samples were loaded in triplicate and at least two were used for 

densitometry and quantitation. Additionally, the Precision Plus molecular weight marker 

(Bio-Rad) was loaded on the gel to demonstrate 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 blot analysis were conducted 

according to the current version of SOPs BR-ME-0924 and BR-ME-0392, respectively. 

Proteins separated by SDS-PAGE 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 Saline 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 NcΔ15D protein with a 1:2500 dilution 

of purified goat antibodies, anti-NcΔ15D 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-goat IgG antibody conjugated to HRP (Pierce, 

Rockford, IL) in 1X TBST with 5% (w/v) NFDM. Unbound anti-goat IgG-HRP 

antibodies were removed by rinsing the membrane briefly and then washing four times 

of 10 minutes each in 1X TBST. The SuperSignal West Pico Chemiluminescent 

Substrate (Pierce, Rockford, IL) was added 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 Instrument 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 samples of a given tissue type grown at a 

given site. These pools were prepared for all tissue types in this study. The average 

percent moisture for each TSSP was calculated from triplicate analyses. A TSSP Dry 

Weight Conversion Factor (DWCF) was calculated as follows: 

 DWCF = 1 – [Mean % TSSP Moisture / 100] 

The DWCF was used to convert protein levels assessed on a μg/g fresh weight (FW) 

basis into levels reported on a μg/g dry weight (DW) basis using the following 

calculation: 

 

The protein levels that were reported to be less than or equal to the LOD or less than the 

LOQ on a fresh weight basis were not reported on a dry weight basis. 

D.7. Data Analyses 

Quantitation of the bands was performed according to the current version of BR-ME0932. At least three standards were present on the film and used to generate the standard 

curve for sample quantitation. For the tissues that protein expression was not detected, 

only the standard curve was generated. For the 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

Protein Level FreshWeight Protein Level in Dry Weight =
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 control (A3525) and conventional reference 

soybean varieties were grown at five U.S. locations in 2006. MON 87769 and the 

conventional soybean control were grown 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 addition, 10 conventional soybean varieties produced alongside of 

MON 87769 were included for the generation of a 99% tolerance interval. The varieties, 

locations, and seed lot numbers 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 prior 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 were 
Monsanto Company 09-SY-200U Page 241 of 395 

further characterized by an event-specific PCR analysis 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 conventional 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 soybean 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 harvested 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. Each sub-sample was ground, stored in a -20 °C 

freezer located at Monsanto Company (St. Louis, MO), and then shipped overnight, on 

dry ice to Covance Laboratories Inc. (Madison, WI) for compositional analyses. The 

labels on the samples shipped to Covance Laboratories Inc. listed the composition study 

number, tissue type, material name, storage conditions, and a unique sample ID number. 

E.4. Analytical Method Summaries and Reference 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, carbohydrate, and ash. An acetone wash removed the 

fats and pigments. The lignocellulose fraction was collected on the frit and determined 

gravimetrically. The limit of quantitation 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 (including asparagine) 

Total threonine 

Total serine 

Total glutamic acid (including glutamine) 

Total proline 

Total glycine 

Total alanine 

Total valine 

Total isoleucine 

Total leucine 
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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. The sulfur-containing amino acids required an 

oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the 

samples for the remaining amino acids was accomplished through direct acid hydrolysis 

with hydrochloric acid. Once hydrolyzed, the individual amino acids were then 

quantitated using an automated amino acid analyzer. The limit of quantitation was 0.100 

mg/g. 

Reference Standards 

• Beckman K18, 2.5 μmol/mL per constituent except cystine (1.25 μmol/mL), Lot 

Number S702114 

• Sigma, L-Tryptophan, 100%, Lot Number 076K50075 

• Fluka, L-Cysteic Acid Monohydrate, 100%, Lot Number 1157629 

• Sigma, L-Methionine Sulfone, 100%, Lot Number 012H3349 

Reference 

AOAC. 2005. Method 982.30 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD.

E.4.3. Ash 

The sample was placed in an electric furnace at 550°C and ignited to drive off all volatile 

organic matter. The nonvolatile matter remaining was quantitated gravimetrically and 

calculated to determine percent ash. The limit of quantitation was 0.100%. 

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 using 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. Agriculture Handbook No. 74. United States 

Department of Agriculture, Washington, D.C. 

E.4.5. Fat by Acid Hydrolysis 

The sample was hydrolyzed with hydrochloric acid at an elevated temperature. The fat 

was extracted with ether and hexane. The extract was evaporated on a steambath, redissolved in hexane and filtered through a sodium sulfate column. The hexane extract 

was then evaporated again on a steambath under nitrogen, dried, and weighed. The limit 

of quantitation was 0.100%. 

Reference 

AOAC. 2005. Methods 922.06 and 954.02 in Official Methods of Analysis of AOAC 

International, 18th ed. AOAC International, Gaithersburg, MD.

E.4.6. Fat by Soxhlet Extraction 

The sample was weighed into a cellulose thimble containing sodium sulfate and dried to 

remove excess moisture. Pentane was dripped through the sample to remove the fat. The 

extract was then evaporated, dried, and weighed. The limit of quantitation was 0.100%. 

Reference 

AOAC. 2005. Method 960.39 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD.

E.4.7. Fatty Acid Profile with Trans Fat by GC 

The lipid was extracted, saponified with 0.5N methanolic sodium hydroxide, and 

methylated with 14% BF3-methanol. The resulting methyl esters of the fatty acids were 

extracted with heptane. An internal standard was added prior to the lipid extraction. The 

methyl esters of the fatty acids were analyzed by gas chromatography using external 

standards for quantitation. The limit of quantitation was 0.0200%. 

Reference Standards 

• Nu Chek Prep GLC Reference Standard 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 Standard Hazelton No. 3, Lot Number N28-Q 

• Nu Chek Prep GLC Reference Standard 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 Number 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, used as 100%, Lot Number N-6M-S1-Q 

• Nu Chek Prep Methyl Erucate, used as 100%, Lot Number U-79M-AU3-Q 

• Nu Chek Prep Methyl Lignocerate, used as 100%, Lot Number N-24M-A5-R 
Monsanto 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 Methyl Ester, used as 100%, Lot Number 

171721-172473 

• Nu Chek Prep Methyl Elaidate, used as 100%, Lot Number U-47M-JA18-R 

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-MA27-R 

References 

AOAC. 2005. Method 996.06 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD. 

AOCS. 1997. Method Ce 2c-66 and Ce 1C-89 in Official Methods and Recommended Practices 

of the AOCS, 5th ed. American Oil Chemists' Society, Champaign, IL. 

AOAC. 2005. Method 983.23 in Official Methods of Analysis of AOAC International, 

18th ed. AOAC International, Gaithersburg, MD 

E.4.8. Isoflavones Analysis 

The sample was extracted using a solution of hydrochloric acid and reagent alcohol 

heated on steam baths or hot plates. The extract was brought to volume, diluted, and 

centrifuged. An aliquot of the supernatant was placed onto a C18 solid-phase extraction 

column. Unwanted components of the matrix were rinsed off with 20% methanol and 

then the isoflavones were eluted with 80% methanol. The sample was analyzed on a 

high-performance liquid chromatography system with ultraviolet detection and was 

compared to an external standard curve of known standards for quantitation. 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 Chemistry, 32(3):530-533, (1984). 

Pettersson, H., and K.H. Kiessling. 1984. Liquid Chromatographic Determination of the 

Plant Estrogens Coumestrol and Isoflavones in Animal Feed. Association of Official 

Analytical Chemists Journal, 67(3):503-506. 

E.4.9. Lectin 

The sample was suspended in phosphate buffered saline (PBS), shaken, and filtered. An 
Monsanto Company 09-SY-200U Page 245 of 395 

aliquot of the resulting extract was serially diluted in 10 cuvettes containing PBS. A 10% 

hematocrit of lyophilized rabbit blood in PBS was added to each dilution. After 2.5 

hours, the absorbance of each dilution of the sample and lectin control was measured on a 

spectrophotometer at 620 nm, using PBS to zero the instrument. One hemagglutinating 

unit (H.U.) was defined as the level that caused 50% of the standard cell suspension to 

sediment in 2.5 hours. The limit of quantitation was 0.10 H.U./mg. 

Reference Standard 

Sigma-Aldrich, Red Blood Cells, Rabbit, Product #R1629, Lot Number 105K6042 

References 

Klurfeld, D. M. and Kritchevsky, D., “Isolation and Quantitation of Lectins from 

Vegetable Oils,” Lipids, 22:667-668, (1987). 

Klurfeld, D. M., Personal communication. 

Liener, I. E., “The Photometric Determination of the Hemagglutinating Activity of Soyin 

and Crude Soybean Extracts,” Archives of Biochemistry 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 and converted to percent moisture. The limit 

of quantitation for this study was 0.100%. 

Reference 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods 926.08 and 

925.09, AOAC INTERNATIONAL: Gaithersburg, Maryland, (2005). 

E.4.11. Neutral Detergent Fiber, Enzyme Method 

The sample was placed in a fritted vessel and washed with a neutral boiling detergent 

solution that dissolved the protein, carbohydrate, enzyme, and ash. An acetone wash 

removed the fats and pigments. Hemicellulose, cellulose, and lignin fractions were 

collected on the frit and determined gravimetrically. The limit of quantitation was 

0.100%. 

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-performance liquid chromatography column PRP-1, 

5μm (150 x 4.1mm) with a refractive index detector. 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 Quantitation of Phytic Acid and Some Inositol 

Phosphates in Foods: Problem and Solutions,” Journal of Agricultural and Food 

Chemistry, 42:2726-2731, (1994). 

Lehrfeld, Jacob, “High-Performance Liquid Chromatography Analysis of Phytic Acid on 

a pH-Stable, Macroporous Polymer Column,” Cereal Chemistry, 66(6):510-515, (1989). 

E.4.13. Protein 

Nitrogenous compounds in the sample were reduced in the presence of boiling sulfuric 

acid and a mercury catalyst mixture to form ammonia. The acid digest was made alkaline. 

The ammonia was distilled and then titrated with a previously standardized acid. The 

percent nitrogen was calculated and converted to equivalent protein using the factor 6.25. 

The limit of quantitation was 0.100%. 

References 

Official Methods of Analysis of AOAC INTERNATIONAL, 18th Ed., Methods 955.04 and 

979.09, AOAC INTERNATIONAL, Gaithersburg, Maryland, (2005). 

Bradstreet, R. B., The Kjeldahl Method for Organic Nitrogen, Academic Press: New 

York, New York, (1965). 

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 deionized water and the extract treated with a 

hydroxylamine hydrochloride solution in pyridine, containing phenyl-β-D-glucoside as 

an internal standard. The resulting oximes were converted to silyl derivatives by 

treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas 

chromatography using a flame ionization detector. The quantitation limit was 0.0500%. 

Reference Standards 

• Sigma, Raffinose Pentahydrate, 99% (84% after correction for degree of hydration), 

Lot Number 035K1371 

• Sigma, Stachyose, 98% (96.4% after correction for degree of hydration), Lot Number 

065K3775 

References 

Brobst, K. M. 1972. Gas-Liquid Chromatography of Trimethylsilyl Derivatives in 

Methods in Carbohydrate Chemistry, Vol. 6. Academic Press, New York. 

Mason, B.S., and H.T. Slover. 1971. A Gas Chromatographic Method for the 

Determination of Sugars in Foods. Journal of Agricultural and Food Chemistry, 

19(3):551-554 

E.4.15. Trypsin Inhibitor 

The sample was ground and defatted with petroleum ether. A sample of matrix was 

extracted with 0.01N sodium hydroxide. Varying aliquots of the sample suspension were 

exposed to a known amount of trypsin and benzoy1-DL-arginine~p~nitroanilide 

hydrochloride. The sample was allowed to react for 10 minutes at 37 °C. After 10 

minutes, the reaction was halted by the addition of acetic acid. The solution was 

centrifuged, then the absorbance was determined at 410 nm. Trypsin inhibitor activity 

was determined by photometrically measuring the inhibition of trypsin’s reaction with 

benzoyl-DL-arginine~p~nitroanilide hydrochloride. The limit of quantitation was 1.00 

Trypsin Inhibitor Units (TIU)/mg. 

Reference 

AOCS. 1997. Method Ba 12-75 in Official Methods 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 high-performance liquid 

chromatography using a silica column. The limit of quantitation was 0.500 mg/100 g. 

Reference Standard 

USP, Alpha Tocopherol, 100%, Lot Number M 
Monsanto 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. Schreurs. 1985. Vitamin E composition of some seed 

oils as determined by high-performance liquid chromatography with fluorometric 

quantitation. Journal of Food Science, 50(1):121-124. 

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 Company. The data were reviewed, formatted, 

and sent to Certus International, Inc. for statistical analysis. A statistical sub-report was 

generated by Certus and sent to Monsanto Company. 

In all, 75 different analytical components were measured. Of these evaluated 

components, 26 had more than 50% of observations below the assay limit of quantitation 

(LOQ) and, as a result, were excluded from the statistical analysis. The following 

analytes were excluded from statistical analysis: 8:0 caprylic acid, 10:0 capric acid, 12:0 

lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 

pentadecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic 

acid, 18:1 total trans-octadecenoic acid, 18:2 isolinoleic acid, 18:2 total trans-linoleic acid, 

18:3 9c,12c,15t trans-ALA, 18:3 gamma linolenic acid, 18:4 6c,9c,12c,15t trans-SDA, 

18:4 stearidonic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, 20:4 arachidonic 

acid, 20:5 eicosapentaenoic acid, 22:1 erucic 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 soybean 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 acids 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 eicosenoic 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 studentized 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 conventional 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 analyzed 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: 

 Yij = U + Ti + Bj + eij , 

where Yij = unique individual observation, U = overall mean, Ti = variety effect, Bj = 

random block effect, and eij = residual error. 

Combined-site analyses used the model: 

Yijk = U + Ti + Lj + B(L)jk + LTij + eijk , 

where Yijk = unique individual observation, U = overall mean, Ti = variety effect, Lj = 

random location effect, B(L)jk = random block within location effect, LTij = random 

location by variety interaction effect, and eijk = 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 reference soybean varieties was determined for each 

analytical component. Additionally, the reference soybean varieties’ data were used to 

develop population tolerance intervals. A tolerance interval is an interval that one can 

claim, with a specified degree of confidence, contains at least a specified proportion, p, of 

an entire sampled population for the parameter measured. For each compositional 

component, 99% tolerance intervals were calculated that are expected to contain, with 

95% confidence, 99% of the quantities expressed in the population of conventional 

reference soybean varieties (George et al., 2004; Ridley et al., 2002). Each tolerance 

interval estimate was based upon one observation per unique reference variety. Since 

individual variety with multiple observations would first be summarized across replicates 

within each site and then summarized across sites to obtain a single value for inclusion in 

tolerance interval calculations, a single replicate from each unique reference variety was 

analyzed for inclusion in tolerance interval calculations. Because negative quantities are 

not possible, calculated negative lower tolerance bounds were set to zero. SAS 

programming was used to generate all summary statistics and perform all analyses (SAS 

Software Release 9.1, 2002-2003). Report tables present p-values from SAS as either 

<0.001 or the actual value truncated to three decimals. 
Monsanto Company 09-SY-200U 

Individual-Site Compositional Comparison of MON 87769 vs. the Conventional Soybean Control for For

Table E-1. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control FoSite for Fiber and Proximates 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Fiber (% DW) 

Acid Detergent Fiber 32.11 (1.36) 31.48 (1.36) 0.63 (1.35) -5.20, 6.46 0.688 

 [31.12 - 32.90] [28.48 - 34.85] [-1.95 - 2.64] Neutral Detergent Fiber 33.00 (2.18) 29.47 (2.18) 3.53 (0.83) -0.052, 7.11 0.051  [30.50 - 36.34] [25.48 - 34.20] [2.14 - 5.02] Proximate (% DW, unless noted) Ash 6.26 (0.13) 6.03 (0.13) 0.23 (0.19) -0.57, 1.03 0.342  [6.06 - 6.46] [5.74 - 6.18] [-0.12 - 0.53] Carbohydrates 64.75 (0.58) 66.01 (0.58) -1.26 (0.82) -4.79, 2.28 0.265  [64.27 - 65.07] [65.13 - 67.57] [-3.30 - -0.055] Moisture (% FW) 74.67 (0.57) 73.07 (0.57) 1.60 (0.80) -1.85, 5.05 0.183  [73.20 - 75.90] [72.80 - 73.30] [0.10 - 3.10] Protein 23.17 (0.50) 22.22 (0.50) 0.95 (0.70) -2.06, 3.97 0.306  [22.13 - 23.82] [21.36 - 22.96] [-0.22 - 2.21] Total Fat 5.82 (0.32) 5.74 (0.32) 0.075 (0.26) -1.04, 1.19 0.800  [5.22 - 6.34] [5.33 - 6.36] [-0.32 - 0.56] 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 t
Monsanto Company 09-SY-200U 

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Alanine 1.78 (0.015) 1.72 (0.015) 0.057 (0.021) -0.033, 0.15 0.112  [1.77 - 1.78] [1.68 - 1.75] [0.033 - 0.10] Arginine 3.20 (0.056) 2.94 (0.056) 0.26 (0.079) -0.083, 0.60 0.082  [3.12 - 3.25] [2.81 - 3.04] [0.14 - 0.44] Aspartic Acid 4.57 (0.055) 4.39 (0.055) 0.18 (0.065) -0.097, 0.46 0.106  [4.51 - 4.63] [4.27 - 4.51] [0.12 - 0.31] Cystine 0.64 (0.0098) 0.61 (0.0098) 0.026 (0.0081) -0.0086, 0.061 0.083  [0.63 - 0.65] [0.60 - 0.64] [0.010 - 0.036] Glutamic Acid 7.66 (0.079) 7.29 (0.079) 0.36 (0.11) -0.11, 0.84 0.081  [7.59 - 7.70] [7.10 - 7.46] [0.22 - 0.60] Glycine 1.80 (0.012) 1.72 (0.012) 0.075 (0.017) 0.0027, 0.15 0.046  [1.79 - 1.80] [1.69 - 1.75] [0.047 - 0.11] Histidine 1.09 (0.010) 1.05 (0.010) 0.040 (0.014) -0.023, 0.10 0.111  [1.09 - 1.10] [1.03 - 1.08] [0.013 - 0.068] Isoleucine 1.88 (0.026) 1.79 (0.026) 0.090 (0.036) -0.067, 0.25 0.132  [1.88 - 1.88] [1.72 - 1.84] [0.037 - 0.16] 
Monsanto Company 09-SY-200U 

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Leucine 3.19 (0.022) 3.08 (0.022) 0.11 (0.027) -0.00097, 0.23 0.050  [3.19 - 3.20] [3.04 - 3.14] [0.061 - 0.15] Lysine 2.70 (0.020) 2.62 (0.020) 0.077 (0.024) -0.027, 0.18 0.085  [2.68 - 2.71] [2.59 - 2.67] [0.039 - 0.12] Methionine 0.60 (0.0064) 0.58 (0.0064) 0.026 (0.0085) -0.010, 0.063 0.089 

 [0.60 - 0.61] [0.57 - 0.59] [0.0098 - 0.038] Phenylalanine 2.13 (0.025) 2.03 (0.025) 0.10 (0.017) 0.034, 0.18 0.024  [2.08 - 2.16] [1.99 - 2.07] [0.086 - 0.14] Proline 2.12 (0.020) 2.00 (0.020) 0.12 (0.024) 0.014, 0.22 0.039  [2.11 - 2.13] [1.97 - 2.06] [0.070 - 0.15] Serine 2.21 (0.035) 2.13 (0.035) 0.073 (0.049) -0.14, 0.28 0.278 

 [2.16 - 2.24] [2.05 - 2.19] [-0.023 - 0.19] Threonine 1.60 (0.026) 1.57 (0.026) 0.039 (0.029) -0.084, 0.16 0.304  [1.57 - 1.64] [1.51 - 1.61] [-0.0089 - 0.090] Tryptophan 0.47 (0.011) 0.45 (0.011) 0.019 (0.016) -0.051, 0.088 0.363  [0.45 - 0.50] [0.44 - 0.46] [-0.0067 - 0.059] 
Monsanto Company 09-SY-200U 

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoSite IA for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Tyrosine 1.40 (0.056) 1.34 (0.056) 0.064 (0.079) -0.28, 0.41 0.504  [1.27 - 1.47] [1.26 - 1.41] [-0.072 - 0.21] Valine 1.99 (0.027) 1.89 (0.027) 0.097 (0.038) -0.066, 0.26 0.124  [1.98 - 2.00] [1.82 - 1.94] [0.037 - 0.18] 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  [12.27 - 12.54] [12.05 - 12.06] [0.21 - 0.48] 18:0 Stearic 4.00 (0.027) 4.04 (0.027) -0.043 (0.015) -0.11, 0.019 0.096  [3.96 - 4.06] [4.01 - 4.08] [-0.064 - -0.015] 18:1 Total 18:1 13.42 (0.14) 18.45 (0.14) -5.04 (0.20) -5.92, -4.15 0.001  [13.14 - 13.80] [18.35 - 18.52] [-5.38 - -4.55] 18:2 9c,12c Linoleic 18.46 (0.11) 54.90 (0.11) -36.45 (0.078) -36.78, -36.11 <0.001  [18.24 - 18.68] [54.80 - 55.07] [-36.60 - -36.35] 18:3 Linolenic 11.11 (0.019) 9.85 (0.019) 1.27 (0.0091) 1.23, 1.30 <0.001  [11.08 - 11.13] [9.80 - 9.88] [1.25 - 1.28] 20:0 Arachidic 0.33 (0.0038) 0.30 (0.0038) 0.032 (0.0024) 0.021, 0.042 0.005  [0.32 - 0.34] [0.29 - 0.30] [0.029 - 0.037] 
Monsanto Company 09-SY-200U 

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoSite IA-1 for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

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  [0.075 - 0.15] [0.082 - 0.089] [-0.013 - 0.063] 22:0 Behenic 0.29 (0.014) 0.31 (0.014) -0.028 (0.0039) -0.045, -0.011 0.018  [0.26 - 0.31] [0.30 - 0.34] [-0.035 - -0.022] Fiber (% DW) Acid Detergent Fiber 15.21 (0.50) 16.19 (0.50) -0.98 (0.70) -4.01, 2.05 0.298 

 [14.38 - 16.48] [15.70 - 16.70] [-1.91 - 0.77] Neutral Detergent Fiber 15.79 (0.80) 15.80 (0.80) -0.0054 (1.13) -4.87, 4.86 0.996 

 [15.46 - 16.05] [14.43 - 18.01] [-2.55 - 1.44] Proximate (% DW, unless noted) Ash 5.74 (0.079) 5.72 (0.079) 0.025 (0.11) -0.46, 0.51 0.843  [5.66 - 5.82] [5.60 - 5.92] [-0.17 - 0.21] Carbohydrates 37.57 (0.89) 40.99 (0.89) -3.41 (1.25) -8.81, 1.98 0.112  [35.70 - 39.54] [39.93 - 41.94] [-6.24 - -0.39] Moisture (% FW) 7.55 (0.21) 7.37 (0.21) 0.18 (0.29) -1.08, 1.44 0.607  [7.14 - 8.01] [7.17 - 7.66] [-0.52 - 0.84] Protein 41.93 (0.32) 40.09 (0.32) 1.84 (0.45) -0.080, 3.77 0.054  [41.35 - 42.27] [39.43 - 40.55] [1.07 - 2.75] 
Monsanto Company 09-SY-200U 

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-1Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Proximate 

Total Fat 14.75 (0.65) 13.21 (0.65) 1.54 (0.92) -2.42, 5.50 0.235  [13.35 - 16.31] [12.73 - 13.86] [-0.51 - 3.27] Vitamin Vitamin E (mg/100g DW) 1.02 (0.11) 1.10 (0.11) -0.082 (0.11) -0.57, 0.40 0.543  [0.86 - 1.30] [0.97 - 1.19] [-0.28 - 0.11] 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  [1.52 - 8.07] [0.75 - 3.23] [0.014 - 5.13] Phytic Acid 1.28 (0.048) 1.17 (0.048) 0.11 (0.023) 0.011, 0.21 0.041  [1.22 - 1.34] [1.06 - 1.26] [0.079 - 0.16] Raffinose 0.35 (0.019) 0.31 (0.019) 0.039 (0.022) -0.055, 0.13 0.215  [0.32 - 0.40] [0.29 - 0.32] [0.0029 - 0.079] Stachyose 3.05 (0.087) 2.78 (0.087) 0.27 (0.076) -0.055, 0.60 0.070  [2.93 - 3.27] [2.69 - 2.86] [0.16 - 0.41] Trypsin Inhibitor (TIU/mg DW) 45.27 (5.77) 35.70 (5.77) 9.57 (8.16) -25.56, 44.70 0.361  [33.06 - 54.80] [25.67 - 41.69] [-6.68 - 29.14] Isoflavone (μg/g DW) Daidzein 995.39 (13.83) 1550.96 (13.83) -555.58 (19.56) -639.75, -471.40 0.001  [978.37 - 1026.28] [1531.49 - 1572.77] [-594.40 - -522.35] 
Monsanto Company 09-SY-200U 

Table E-2. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-1 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Isoflavone (μg/g DW) 

Genistein 594.53 (7.52) 973.04 (7.52) -378.51 (4.09) -396.09, -360.93 <0.001 

 [584.75 - 612.91] [966.28 - 983.61] [-384.49 - -370.70] Glycitein 87.40 (9.32) 103.25 (9.32) -15.86 (6.20) -42.55, 10.83 0.124  [73.40 - 106.72] [86.17 - 113.71] [-27.80 - -6.99] 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 t
Monsanto Company 09-SY-200U 

Table E-3. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control FSite for Fiber and Proximates

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Fiber (% DW) 

Acid Detergent Fiber 26.08 (0.67) 25.76 (0.67) 0.33 (0.23) -0.66, 1.32 0.290 

 [24.91 - 26.79] [24.39 - 26.92] [-0.13 - 0.59] Neutral Detergent Fiber 30.83 (1.42) 32.11 (1.42) -1.28 (2.00) -9.90, 7.35 0.589  [28.06 - 34.06] [30.71 - 33.99] [-5.94 - 2.44] Proximate (% DW, unless noted) Ash 7.19 (0.13) 7.45 (0.13) -0.26 (0.16) -0.95, 0.43 0.244  [7.00 - 7.30] [7.25 - 7.75] [-0.49 - 0.048] Carbohydrates 64.72 (0.85) 62.33 (0.85) 2.39 (1.07) -2.19, 6.98 0.154  [62.88 - 65.63] [61.18 - 63.79] [0.88 - 4.45] Moisture (% FW) 72.93 (0.94) 74.03 (0.94) -1.10 (0.67) -3.96, 1.76 0.240  [70.70 - 74.80] [72.90 - 74.70] [-2.20 - 0.10] Protein 23.23 (0.76) 23.95 (0.76) -0.73 (0.64) -3.50, 2.05 0.376  [22.18 - 24.53] [22.33 - 24.98] [-2.01 - -0.017] Total Fat 4.87 (0.20) 6.27 (0.20) -1.40 (0.28) -2.60, -0.20 0.037  [4.40 - 5.28] [6.13 - 6.49] [-2.09 - -0.92] 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 t
Monsanto Company 09-SY-200U 

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Alanine 1.79 (0.018) 1.78 (0.018) 0.0095 (0.022) -0.083, 0.10 0.703  [1.76 - 1.81] [1.74 - 1.81] [-0.018 - 0.052] Arginine 3.42 (0.037) 3.11 (0.037) 0.31 (0.020) 0.23, 0.40 0.003  [3.36 - 3.50] [3.05 - 3.15] [0.28 - 0.35] Aspartic Acid 4.56 (0.053) 4.50 (0.053) 0.060 (0.072) -0.25, 0.37 0.490  [4.51 - 4.61] [4.36 - 4.60] [-0.034 - 0.20] Cystine 0.62 (0.0028) 0.60 (0.0028) 0.024 (0.0019) 0.016, 0.032 0.005  [0.62 - 0.63] [0.59 - 0.61] [0.021 - 0.027] Glutamic Acid 7.73 (0.088) 7.56 (0.088) 0.17 (0.11) -0.32, 0.66 0.283  [7.62 - 7.83] [7.36 - 7.71] [-0.0052 - 0.38] Glycine 1.81 (0.018) 1.78 (0.018) 0.031 (0.022) -0.062, 0.12 0.284  [1.78 - 1.83] [1.74 - 1.81] [0.0039 - 0.074] Histidine 1.10 (0.012) 1.08 (0.012) 0.015 (0.016) -0.056, 0.086 0.455  [1.08 - 1.11] [1.05 - 1.10] [-0.013 - 0.044] Isoleucine 1.89 (0.027) 1.81 (0.027) 0.082 (0.038) -0.081, 0.25 0.162  [1.84 - 1.94] [1.78 - 1.86] [-0.018 - 0.16] 
Monsanto Company 09-SY-200U 

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Leucine 3.21 (0.028) 3.16 (0.028) 0.047 (0.032) -0.092, 0.19 0.279  [3.17 - 3.26] [3.10 - 3.19] [-0.015 - 0.094] Lysine 2.70 (0.029) 2.69 (0.029) 0.014 (0.041) -0.16, 0.19 0.760  [2.65 - 2.73] [2.62 - 2.73] [-0.059 - 0.094] Methionine 0.60 (0.0088) 0.58 (0.0088) 0.025 (0.0099) -0.017, 0.067 0.126 

 [0.59 - 0.61] [0.56 - 0.60] [0.0057 - 0.038] Phenylalanine 2.14 (0.018) 2.11 (0.018) 0.032 (0.025) -0.075, 0.14 0.330  [2.13 - 2.15] [2.07 - 2.15] [-0.016 - 0.084] Proline 2.12 (0.021) 2.06 (0.021) 0.061 (0.0073) 0.029, 0.092 0.014  [2.09 - 2.15] [2.01 - 2.09] [0.048 - 0.073] Serine 2.19 (0.028) 2.22 (0.028) -0.030 (0.040) -0.20, 0.14 0.529 

 [2.18 - 2.21] [2.17 - 2.29] [-0.11 - 0.041] Threonine 1.58 (0.015) 1.60 (0.015) -0.013 (0.017) -0.086, 0.061 0.542  [1.58 - 1.60] [1.56 - 1.62] [-0.039 - 0.020] Tryptophan 0.46 (0.0039) 0.47 (0.0039) -0.0056 (0.0055) -0.029, 0.018 0.417  [0.46 - 0.46] [0.46 - 0.48] [-0.019 - 0.0016] 
Monsanto Company 09-SY-200U 

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Tyrosine 1.41 (0.022) 1.32 (0.022) 0.089 (0.031) -0.043, 0.22 0.100  [1.35 - 1.45] [1.30 - 1.34] [0.015 - 0.14] Valine 2.01 (0.031) 1.92 (0.031) 0.097 (0.043) -0.089, 0.28 0.154  [1.95 - 2.07] [1.89 - 1.96] [-0.017 - 0.18] 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  [11.53 - 11.90] [11.14 - 11.52] [0.0098 - 0.75] 18:0 Stearic 4.21 (0.058) 4.11 (0.058) 0.097 (0.056) -0.14, 0.34 0.224  [4.15 - 4.27] [3.96 - 4.21] [-0.0058 - 0.19] 18:1 Total 18:1 13.56 (0.31) 18.53 (0.31) -4.97 (0.31) -6.32, -3.63 0.003  [12.93 - 14.36] [18.29 - 18.72] [-5.36 - -4.36] 18:2 9c,12c Linoleic 21.19 (0.57) 55.33 (0.57) -34.14 (0.80) -37.60, -30.68 <0.001  [20.36 - 22.78] [55.21 - 55.58] [-35.15 - -32.43] 18:3 Linolenic 11.14 (0.12) 9.96 (0.12) 1.18 (0.16) 0.47, 1.89 0.018  [11.10 - 11.18] [9.73 - 10.28] [0.82 - 1.45] 20:0 Arachidic 0.34 (0.0072) 0.30 (0.0072) 0.036 (0.010) -0.0077, 0.080 0.071  [0.33 - 0.34] [0.28 - 0.32] [0.017 - 0.058] 
Monsanto Company 09-SY-200U 

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

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  [0.086 - 0.15] [0.075 - 0.092] [-0.0061 - 0.072] 22:0 Behenic 0.27 (0.019) 0.32 (0.019) -0.051 (0.027) -0.17, 0.067 0.206  [0.27 - 0.27] [0.28 - 0.37] [-0.11 - -0.019] Fiber (% DW) Acid Detergent Fiber 17.23 (0.90) 16.16 (0.90) 1.07 (0.85) -2.57, 4.71 0.333  [16.45 - 17.62] [13.80 - 17.85] [-0.24 - 2.65] Neutral Detergent Fiber 17.34 (0.97) 17.64 (0.97) -0.31 (1.33) -6.03, 5.42 0.839  [15.47 - 19.15] [16.73 - 19.37] [-1.98 - 2.32] Proximate (% DW, unless noted) Ash 5.52 (0.091) 5.41 (0.091) 0.11 (0.065) -0.17, 0.39 0.229  [5.46 - 5.63] [5.24 - 5.64] [-0.0074 - 0.22] Carbohydrates 36.95 (0.79) 40.25 (0.79) -3.30 (1.12) -8.12, 1.52 0.098  [35.58 - 39.06] [39.89 - 40.93] [-4.71 - -0.88] Moisture (% FW) 8.09 (0.066) 7.98 (0.066) 0.11 (0.093) -0.29, 0.51 0.348  [7.99 - 8.21] [7.88 - 8.11] [-0.12 - 0.26] Protein 42.80 (0.78) 40.42 (0.78) 2.37 (0.95) -1.73, 6.48 0.130  [42.50 - 43.36] [38.31 - 41.58] [0.96 - 4.19] 
Monsanto Company 09-SY-200U 

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Proximate 

Total Fat 14.73 (0.76) 13.91 (0.76) 0.81 (0.74) -2.37, 4.00 0.386  [12.95 - 15.65] [13.24 - 15.13] [-0.30 - 2.22] Vitamin Vitamin E (mg/100g DW) 1.05 (0.11) 1.18 (0.11) -0.13 (0.15) -0.79, 0.53 0.489  [0.92 - 1.22] [0.94 - 1.37] [-0.35 - 0.27] 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  [2.07 - 4.28] [0.71 - 2.71] [0.43 - 1.57] Phytic Acid 0.97 (0.059) 1.01 (0.059) -0.047 (0.057) -0.29, 0.20 0.495  [0.86 - 1.11] [0.94 - 1.06] [-0.13 - 0.064] Raffinose 0.38 (0.024) 0.34 (0.024) 0.042 (0.034) -0.10, 0.19 0.344  [0.35 - 0.45] [0.31 - 0.37] [-0.019 - 0.099] Stachyose 2.87 (0.19) 2.74 (0.19) 0.13 (0.26) -0.99, 1.26 0.659  [2.53 - 3.24] [2.43 - 3.00] [-0.46 - 0.44] Trypsin Inhibitor (TIU/mg DW) 28.51 (1.63) 29.19 (1.63) -0.69 (1.71) -8.02, 6.65 0.726  [27.39 - 29.81] [24.81 - 31.50] [-3.18 - 2.58] Isoflavone (μg/g DW) Daidzein 1076.04 (29.87) 1583.00 (29.87) -506.96 (25.06) -614.77, -399.15 0.002  [999.02 - 1130.31] [1553.50 - 1599.74] [-554.48 - -469.43] 
Monsanto Company 09-SY-200U 

Table E-4. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIA-2 Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Isoflavone (μg/g DW) 

Genistein 656.80 (21.40) 1044.68 (21.40) -387.88 (16.63) -459.42, -316.33 0.001  [612.27 - 687.55] [1023.36 - 1084.46] [-411.09 - -355.65] Glycitein 75.18 (6.78) 76.19 (6.78) -1.01 (1.36) -6.84, 4.82 0.533  [67.00 - 87.16] [65.51 - 90.33] [-3.16 - 1.49] 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 t
Monsanto Company 09-SY-200U 

Table E-5. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control F

for Fiber and Proximates 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Fiber (% DW) 

Acid Detergent Fiber 31.77 (1.82) 31.12 (1.82) 0.65 (1.56) -6.06, 7.37 0.715  [30.24 - 34.02] [27.30 - 35.23] [-1.21 - 3.76] Neutral Detergent Fiber 36.52 (3.76) 37.15 (3.76) -0.64 (4.89) -21.69, 20.42 0.908  [29.02 - 42.44] [30.49 - 42.62] [-9.33 - 7.60] Proximate (% DW, unless noted) Ash 6.79 (0.18) 6.72 (0.18) 0.068 (0.18) -0.70, 0.84 0.741  [6.70 - 6.92] [6.44 - 7.20] [-0.28 - 0.31] Carbohydrates 69.55 (0.72) 69.94 (0.72) -0.39 (0.36) -1.95, 1.17 0.396  [68.50 - 70.34] [68.21 - 70.84] [-0.95 - 0.29] Moisture (% FW) 71.63 (0.62) 71.27 (0.62) 0.37 (0.88) -3.41, 4.14 0.716  [70.60 - 72.90] [70.20 - 72.10] [-0.90 - 2.70] Protein 18.60 (0.40) 18.57 (0.40) 0.028 (0.39) -1.67, 1.72 0.949  [17.76 - 19.23] [18.09 - 19.25] [-0.63 - 0.73] Total Fat 5.06 (0.28) 4.76 (0.28) 0.29 (0.33) -1.14, 1.73 0.470  [4.61 - 5.35] [4.25 - 5.34] [-0.085 - 0.96] 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 t
Monsanto Company 09-SY-200U 

Table E-6. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Amino Acid (% DW) 

Alanine 1.80 (0.020) 1.74 (0.020) 0.052 (0.024) -0.049, 0.15 0.158  [1.76 - 1.84] [1.71 - 1.76] [0.0060 - 0.084] Arginine 3.07 (0.049) 2.90 (0.049) 0.17 (0.070) -0.13, 0.47 0.129  [3.00 - 3.14] [2.80 - 3.00] [0.0027 - 0.27] Aspartic Acid 4.55 (0.063) 4.35 (0.063) 0.20 (0.085) -0.17, 0.56 0.146  [4.45 - 4.70] [4.26 - 4.42] [0.031 - 0.31] Cystine 0.63 (0.0056) 0.61 (0.0056) 0.020 (0.0020) 0.011, 0.028 0.010  [0.62 - 0.64] [0.60 - 0.62] [0.016 - 0.022] Glutamic Acid 7.65 (0.11) 7.26 (0.11) 0.39 (0.14) -0.24, 1.01 0.116  [7.46 - 7.90] [7.08 - 7.36] [0.098 - 0.56] Glycine 1.82 (0.019) 1.75 (0.019) 0.070 (0.023) -0.028, 0.17 0.091  [1.78 - 1.87] [1.72 - 1.76] [0.028 - 0.11] Histidine 1.10 (0.015) 1.06 (0.015) 0.044 (0.020) -0.042, 0.13 0.158  [1.08 - 1.14] [1.04 - 1.07] [0.0068 - 0.075] Isoleucine 1.91 (0.025) 1.82 (0.025) 0.087 (0.036) -0.066, 0.24 0.134  [1.87 - 1.97] [1.79 - 1.84] [0.022 - 0.18] 
Monsanto Company 09-SY-200U 

Table E-6. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Amino Acid (% DW) 

Leucine 3.21 (0.048) 3.10 (0.048) 0.11 (0.059) -0.14, 0.36 0.196  [3.14 - 3.32] [3.03 - 3.14] [0.0023 - 0.20] Lysine 2.68 (0.031) 2.59 (0.031) 0.085 (0.040) -0.087, 0.26 0.168  [2.63 - 2.75] [2.55 - 2.62] [0.014 - 0.15] Methionine 0.61 (0.0070) 0.59 (0.0070) 0.028 (0.0068) -0.0014, 0.057 0.054 

 [0.61 - 0.62] [0.57 - 0.60] [0.015 - 0.038] Phenylalanine 2.12 (0.026) 2.07 (0.026) 0.051 (0.025) -0.058, 0.16 0.182  [2.08 - 2.18] [2.03 - 2.10] [0.0051 - 0.092] Proline 2.09 (0.030) 1.99 (0.030) 0.094 (0.043) -0.090, 0.28 0.159  [2.05 - 2.15] [1.95 - 2.04] [0.016 - 0.17] Serine 2.20 (0.044) 2.11 (0.044) 0.094 (0.059) -0.16, 0.35 0.253  [2.14 - 2.25] [2.00 - 2.18] [0.0050 - 0.21] Threonine 1.58 (0.028) 1.56 (0.028) 0.027 (0.040) -0.14, 0.20 0.564  [1.54 - 1.63] [1.50 - 1.60] [-0.058 - 0.087] Tryptophan 0.50 (0.0096) 0.48 (0.0096) 0.013 (0.010) -0.030, 0.056 0.322  [0.49 - 0.50] [0.46 - 0.50] [-0.0014 - 0.032] 
Monsanto Company 09-SY-200U 

Table E-6. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Amino Acid (% DW) 

Tyrosine 1.38 (0.032) 1.37 (0.032) 0.014 (0.042) -0.17, 0.19 0.773  [1.34 - 1.41] [1.33 - 1.44] [-0.058 - 0.087] Valine 2.01 (0.029) 1.92 (0.029) 0.098 (0.040) -0.076, 0.27 0.136  [1.96 - 2.08] [1.88 - 1.95] [0.011 - 0.20] 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  [12.24 - 12.39] [11.89 - 12.08] [0.21 - 0.35] 18:0 Stearic 4.42 (0.027) 4.33 (0.027) 0.090 (0.036) -0.064, 0.24 0.128  [4.37 - 4.45] [4.28 - 4.37] [0.035 - 0.16] 18:1 Total 18:1 17.89 (0.22) 20.89 (0.22) -3.00 (0.31) -4.31, -1.68 0.010  [17.52 - 18.18] [20.42 - 21.17] [-3.65 - -2.46] 18:2 9c,12c Linoleic 30.48 (0.20) 54.33 (0.20) -23.86 (0.087) -24.23, -23.48 <0.001 

 [30.26 - 30.81] [54.05 - 54.77] [-23.96 - -23.68] 18:3 Linolenic 10.27 (0.090) 7.59 (0.090) 2.68 (0.13) 2.13, 3.22 0.002  [10.20 - 10.38] [7.42 - 7.81] [2.42 - 2.82] 20:0 Arachidic 0.36 (0.0038) 0.34 (0.0038) 0.027 (0.0045) 0.0083, 0.047 0.025  [0.36 - 0.37] [0.33 - 0.34] [0.020 - 0.036] 
Monsanto Company 09-SY-200U 

Table E-6. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

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  [0.20 - 0.20] [0.19 - 0.19] [0.0027 - 0.0098] 22:0 Behenic 0.31 (0.0039) 0.33 (0.0039) -0.022 (0.0049) -0.043, -0.00089 0.046 

 [0.30 - 0.31] [0.32 - 0.34] [-0.029 - -0.013] Fiber (% DW) Acid Detergent Fiber 16.16 (0.20) 17.76 (0.20) -1.60 (0.12) -2.12, -1.08 0.005  [15.91 - 16.61] [17.47 - 18.03] [-1.83 - -1.41] Neutral Detergent Fiber 17.06 (0.12) 17.87 (0.12) -0.81 (0.17) -1.56, -0.060 0.043  [16.72 - 17.25] [17.78 - 17.92] [-1.20 - -0.53] Proximate (% DW, unless noted) Ash 6.06 (0.13) 5.78 (0.13) 0.28 (0.17) -0.44, 1.01 0.234  [5.90 - 6.17] [5.51 - 6.07] [0.099 - 0.62] Carbohydrates 34.20 (0.77) 36.20 (0.77) -2.00 (0.55) -4.38, 0.37 0.068  [33.23 - 36.11] [35.30 - 37.13] [-2.92 - -1.01] Moisture (% FW) 6.98 (0.11) 7.28 (0.11) -0.29 (0.16) -0.98, 0.40 0.209  [6.71 - 7.24] [7.21 - 7.37] [-0.66 - 0.030] Protein 41.64 (0.45) 39.58 (0.45) 2.06 (0.63) -0.64, 4.76 0.081  [41.05 - 42.26] [38.97 - 40.65] [0.97 - 3.14] 
Monsanto Company 09-SY-200U 

Table E-6. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Proximate 

Total Fat 18.10 (0.45) 18.44 (0.45) -0.34 (0.49) -2.44, 1.75 0.554 

 [16.94 - 19.03] [18.14 - 18.64] [-1.20 - 0.49] Vitamin Vitamin E (mg/100g DW) 2.42 (0.070) 2.10 (0.070) 0.31 (0.099) -0.11, 0.74 0.087  [2.27 - 2.54] [2.01 - 2.21] [0.059 - 0.45] 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  [3.69 - 7.02] [4.88 - 11.32] [-7.01 - 2.00] Phytic Acid 1.07 (0.030) 1.04 (0.030) 0.023 (0.043) -0.16, 0.21 0.641  [1.02 - 1.10] [1.00 - 1.11] [-0.094 - 0.10] Raffinose 0.43 (0.015) 0.43 (0.015) 0.00042 (0.021) -0.091, 0.092 0.985  [0.40 - 0.44] [0.40 - 0.45] [-0.049 - 0.044] Stachyose 3.06 (0.088) 3.07 (0.088) -0.0061 (0.12) -0.54, 0.53 0.965  [2.91 - 3.17] [2.88 - 3.21] [-0.30 - 0.29] Trypsin Inhibitor (TIU/mg DW) 38.06 (1.03) 35.27 (1.03) 2.80 (1.46) -3.49, 9.09 0.195  [36.23 - 40.86] [34.82 - 35.95] [0.28 - 5.83] Isoflavone (μg/g DW) Daidzein 1079.87 (87.82) 1484.63 (87.82) -404.76 (120.69) -924.03, 114.51 0.078  [957.23 - 1193.55] [1380.05 - 1691.99] [-603.16 - -186.51] 
Monsanto Company 09-SY-200U 

Table E-6. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoIL Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Isoflavone (μg/g DW) 

Genistein 655.90 (47.11) 837.93 (47.11) -182.04 (52.30) -407.05, 42.97 0.073  [576.70 - 716.13] [770.81 - 940.83] [-265.97 - -86.03] Glycitein 84.86 (5.58) 120.49 (5.58) -35.63 (7.78) -69.10, -2.17 0.044  [83.23 - 87.74] [106.01 - 132.56] [-49.33 - -22.40] 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 t
Monsanto Company 09-SY-200U 

Table E-7. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control FSite for Fiber and Proximates

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Fiber (% DW) 

Acid Detergent Fiber 33.02 (2.24) 33.97 (2.24) -0.95 (3.17) -14.61, 12.71 0.793 

 [27.92 - 36.39] [30.72 - 37.06] [-9.14 - 5.67] Neutral Detergent Fiber 36.16 (2.10) 36.13 (2.10) 0.029 (2.96) -12.72, 12.78 0.993  [31.87 - 40.66] [33.13 - 38.13] [-5.27 - 7.53] Proximate (% DW, unless noted) Ash 5.99 (0.33) 6.00 (0.33) -0.014 (0.077) -0.35, 0.32 0.875  [5.29 - 6.36] [5.38 - 6.39] [-0.091 - 0.14] Carbohydrates 70.54 (1.12) 70.07 (1.12) 0.47 (1.26) -4.94, 5.87 0.745  [68.27 - 72.79] [68.44 - 71.51] [-2.00 - 2.12] Moisture (% FW) 71.30 (0.93) 71.37 (0.93) -0.067 (1.32) -5.73, 5.60 0.964  [69.50 - 72.80] [70.10 - 73.10] [-1.50 - 2.70] Protein 18.80 (0.73) 18.54 (0.73) 0.26 (1.02) -4.11, 4.63 0.822  [17.05 - 20.44] [18.23 - 19.14] [-1.20 - 2.21] Total Fat 4.67 (0.68) 5.39 (0.68) -0.71 (0.54) -3.05, 1.62 0.318 

 [3.80 - 5.99] [4.02 - 6.12] [-1.80 - -0.13] 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 t
Monsanto Company 09-SY-200U 

Table E-8. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Amino Acid (% DW) 

Alanine 1.79 (0.021) 1.72 (0.021) 0.068 (0.030) -0.062, 0.20 0.152  [1.76 - 1.83] [1.69 - 1.76] [0.0015 - 0.11] Arginine 3.32 (0.10) 2.94 (0.10) 0.39 (0.14) -0.23, 1.01 0.114  [3.16 - 3.61] [2.91 - 2.98] [0.19 - 0.68] Aspartic Acid 4.57 (0.064) 4.29 (0.064) 0.28 (0.090) -0.10, 0.67 0.087  [4.48 - 4.73] [4.21 - 4.37] [0.11 - 0.43] Cystine 0.58 (0.011) 0.57 (0.011) 0.012 (0.015) -0.054, 0.077 0.528  [0.56 - 0.60] [0.56 - 0.58] [-0.024 - 0.037] Glutamic Acid 7.62 (0.11) 7.16 (0.11) 0.46 (0.15) -0.20, 1.12 0.094  [7.47 - 7.87] [7.03 - 7.32] [0.15 - 0.76] Glycine 1.78 (0.019) 1.70 (0.019) 0.086 (0.027) -0.032, 0.20 0.088  [1.76 - 1.83] [1.67 - 1.72] [0.034 - 0.14] Histidine 1.09 (0.017) 1.04 (0.017) 0.048 (0.024) -0.053, 0.15 0.176  [1.07 - 1.13] [1.02 - 1.07] [0.0020 - 0.090] Isoleucine 1.82 (0.048) 1.75 (0.048) 0.068 (0.068) -0.23, 0.36 0.422  [1.75 - 1.94] [1.70 - 1.80] [-0.052 - 0.24] 
Monsanto Company 09-SY-200U 

Table E-8. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Amino Acid (% DW) 

Leucine 3.19 (0.047) 3.06 (0.047) 0.13 (0.066) -0.15, 0.41 0.189  [3.13 - 3.28] [3.01 - 3.15] [-0.019 - 0.26] Lysine 2.67 (0.033) 2.56 (0.033) 0.11 (0.047) -0.092, 0.31 0.142  [2.62 - 2.75] [2.51 - 2.61] [0.013 - 0.19] Methionine 0.56 (0.011) 0.56 (0.011) 0.00074 (0.016) -0.068, 0.069 0.967  [0.54 - 0.59] [0.56 - 0.57] [-0.030 - 0.033] Phenylalanine 2.16 (0.032) 2.05 (0.032) 0.11 (0.046) -0.086, 0.31 0.135  [2.11 - 2.24] [2.02 - 2.10] [0.013 - 0.20] Proline 2.09 (0.039) 1.94 (0.039) 0.14 (0.055) -0.091, 0.38 0.119  [2.03 - 2.19] [1.91 - 1.98] [0.056 - 0.25] Serine 2.23 (0.022) 2.12 (0.022) 0.11 (0.030) -0.020, 0.23 0.067 

 [2.23 - 2.23] [2.06 - 2.17] [0.067 - 0.17] Threonine 1.63 (0.023) 1.55 (0.023) 0.082 (0.032) -0.057, 0.22 0.125  [1.60 - 1.65] [1.49 - 1.58] [0.023 - 0.15] Tryptophan 0.48 (0.014) 0.46 (0.014) 0.018 (0.011) -0.030, 0.065 0.253  [0.45 - 0.51] [0.44 - 0.47] [-0.00067 - 0.037] 
Monsanto Company 09-SY-200U 

Table E-8. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Amino Acid (% DW) 

Tyrosine 1.42 (0.049) 1.35 (0.049) 0.072 (0.048) -0.14, 0.28 0.275  [1.29 - 1.50] [1.32 - 1.40] [-0.020 - 0.14] Valine 1.92 (0.054) 1.86 (0.054) 0.065 (0.076) -0.26, 0.39 0.485  [1.84 - 2.06] [1.79 - 1.91] [-0.063 - 0.26] 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  [12.03 - 12.19] [11.70 - 11.92] [0.26 - 0.40] 18:0 Stearic 3.87 (0.054) 3.90 (0.054) -0.022 (0.070) -0.32, 0.28 0.779 

 [3.73 - 3.98] [3.85 - 3.94] [-0.16 - 0.059] 18:1 Total 18:1 12.92 (0.13) 17.44 (0.13) -4.53 (0.097) -4.94, -4.11 <0.001  [12.66 - 13.16] [17.24 - 17.59] [-4.66 - -4.34] 18:2 9c,12c Linoleic 18.40 (0.71) 55.60 (0.71) -37.21 (0.90) -41.08, -33.33 <0.001 

 [16.46 - 19.58] [55.36 - 56.04] [-38.90 - -35.83] 18:3 Linolenic 11.76 (0.029) 10.59 (0.029) 1.16 (0.038) 1.00, 1.33 0.001  [11.72 - 11.80] [10.54 - 10.66] [1.09 - 1.22] 20:0 Arachidic 0.32 (0.0037) 0.29 (0.0037) 0.030 (0.0052) 0.0074, 0.052 0.029  [0.31 - 0.32] [0.28 - 0.29] [0.019 - 0.042] 
Monsanto Company 09-SY-200U 

Table E-8. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

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  [0.077 - 0.084] [0.069 - 0.14] [-0.058 - 0.013] 22:0 Behenic 0.30 (0.0096) 0.29 (0.0096) 0.0060 (0.012) -0.044, 0.056 0.656 

 [0.27 - 0.31] [0.29 - 0.30] [-0.016 - 0.022] Fiber (% DW) Acid Detergent Fiber 17.27 (0.32) 16.77 (0.32) 0.50 (0.21) -0.42, 1.43 0.143  [16.91 - 17.59] [15.98 - 17.35] [0.23 - 0.93] Neutral Detergent Fiber 17.30 (0.55) 17.34 (0.55) -0.038 (0.78) -3.38, 3.30 0.965 

 [16.15 - 18.17] [16.42 - 18.11] [-1.34 - 1.75] Proximate (% DW, unless noted) Ash 5.46 (0.10) 5.51 (0.10) -0.051 (0.15) -0.68, 0.58 0.763  [5.23 - 5.66] [5.35 - 5.61] [-0.34 - 0.31] Carbohydrates 39.11 (1.09) 39.75 (1.09) -0.63 (1.54) -7.27, 6.00 0.720  [38.34 - 39.93] [37.70 - 42.60] [-4.26 - 2.23] Moisture (% FW) 7.15 (0.13) 7.15 (0.13) 0 (0.13) -0.55, 0.55 1.000  [7.01 - 7.31] [6.84 - 7.39] [-0.25 - 0.17] Protein 41.86 (0.67) 39.10 (0.67) 2.76 (0.95) -1.33, 6.86 0.100  [41.08 - 43.29] [38.22 - 40.31] [0.90 - 5.07] 
Monsanto Company 09-SY-200U 

Table E-8. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Proximate 

Total Fat 13.57 (0.77) 15.65 (0.77) -2.08 (0.81) -5.56, 1.40 0.123 

 [13.14 - 14.20] [13.61 - 16.96] [-3.01 - -0.47] Vitamin Vitamin E (mg/100g DW) 1.26 (0.087) 0.86 (0.087) 0.40 (0.12) -0.13, 0.93 0.083 

 [1.22 - 1.34] [0.70 - 1.09] [0.14 - 0.55] Antinutrient Lectin (H.U./mg DW) 1.49 (0.63) 3.44 (0.63) -1.95 (0.28) -3.14, -0.77 0.019  [0.55 - 2.14] [1.95 - 4.39] [-2.25 - -1.40] Phytic Acid (% DW) 0.86 (0.063) 0.88 (0.063) -0.017 (0.061) -0.28, 0.25 0.811  [0.81 - 0.92] [0.75 - 1.03] [-0.10 - 0.10] Raffinose (% DW) 0.34 (0.0085) 0.32 (0.0085) 0.021 (0.012) -0.031, 0.073 0.225  [0.32 - 0.36] [0.32 - 0.33] [-0.0059 - 0.044] Stachyose (% DW) 2.43 (0.066) 2.52 (0.066) -0.093 (0.094) -0.50, 0.31 0.425  [2.28 - 2.53] [2.45 - 2.63] [-0.35 - 0.080] Trypsin Inhibitor (TIU/mg DW) 25.92 (2.94) 27.17 (2.94) -1.24 (4.16) -19.15, 16.67 0.793  [24.30 - 27.94] [21.34 - 34.89] [-10.58 - 6.60] Isoflavone (μg/g DW) Daidzein 1662.22 (99.21) 2750.13 (99.21) -1087.92 (140.30) -1691.59, -484.25 0.016  [1389.19 - 1838.91] [2705.02 - 2775.08] [-1385.89 - -866.12] 
Monsanto Company 09-SY-200U 

Table E-8. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SoMI Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[9

Isoflavone (μg/g DW) 

Genistein 1000.90 (59.25) 1683.74 (59.25) -682.83 (70.98) -988.25, -377.41 0.010  [841.05 - 1118.40] [1662.89 - 1706.74] [-821.84 - -588.34] Glycitein 83.59 (17.61) 114.34 (17.61) -30.75 (24.91) -137.92, 76.41 0.342  [65.37 - 101.09] [84.16 - 158.73] [-93.36 - 0.95] 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 t
Monsanto Company 09-SY-200U 

Table E-9. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control FSite for Fiber and Proximates 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Fiber (% DW) 

Acid Detergent Fiber 32.29 (0.91) 28.31 (0.91) 3.99 (0.40) 2.28, 5.70 0.009  [30.74 - 33.95] [26.53 - 29.41] [3.21 - 4.54] Neutral Detergent Fiber 32.93 (1.50) 31.22 (1.50) 1.70 (1.80) -6.04, 9.45 0.443  [29.20 - 35.58] [29.79 - 32.90] [-1.78 - 4.22] Proximate (% DW, unless noted) Ash 6.67 (0.24) 6.59 (0.24) 0.078 (0.25) -0.99, 1.14 0.782  [6.05 - 7.07] [6.44 - 6.88] [-0.40 - 0.44] Carbohydrates 67.50 (0.78) 66.87 (0.78) 0.64 (0.90) -3.26, 4.53 0.553  [66.33 - 69.34] [65.73 - 67.76] [-0.91 - 2.22] Moisture (% FW) 70.87 (0.56) 71.27 (0.56) -0.40 (0.80) -3.82, 3.02 0.664  [69.90 - 72.40] [71.00 - 71.60] [-1.70 - 1.40] Protein 19.77 (0.58) 20.08 (0.58) -0.32 (0.81) -3.82, 3.19 0.734  [18.74 - 20.87] [19.07 - 20.87] [-1.58 - 1.80] Total Fat 6.06 (0.34) 6.46 (0.34) -0.40 (0.48) -2.45, 1.65 0.490  [5.40 - 6.90] [6.13 - 6.72] [-1.33 - 0.37] 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 t
Monsanto Company 09-SY-200U 

Table E-10. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control S

OH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Alanine 1.77 (0.0068) 1.74 (0.0068) 0.031 (0.0096) -0.010, 0.073 0.083  [1.76 - 1.78] [1.72 - 1.75] [0.0084 - 0.043] Arginine 3.11 (0.020) 2.86 (0.020) 0.25 (0.028) 0.13, 0.37 0.012  [3.07 - 3.15] [2.82 - 2.89] [0.21 - 0.32] Aspartic Acid 4.43 (0.017) 4.27 (0.017) 0.16 (0.024) 0.056, 0.26 0.021  [4.41 - 4.47] [4.25 - 4.29] [0.12 - 0.22] Cystine 0.60 (0.0030) 0.59 (0.0030) 0.015 (0.0043) -0.0037, 0.033 0.075  [0.60 - 0.60] [0.58 - 0.59] [0.0058 - 0.021] Glutamic Acid 7.51 (0.034) 7.17 (0.034) 0.33 (0.048) 0.13, 0.54 0.020  [7.42 - 7.59] [7.16 - 7.19] [0.23 - 0.43] Glycine 1.76 (0.0055) 1.71 (0.0055) 0.049 (0.0078) 0.015, 0.083 0.024  [1.76 - 1.77] [1.70 - 1.73] [0.030 - 0.064] Histidine 1.07 (0.0021) 1.03 (0.0021) 0.033 (0.0021) 0.024, 0.042 0.003  [1.06 - 1.07] [1.03 - 1.04] [0.031 - 0.037] Isoleucine 1.87 (0.024) 1.75 (0.024) 0.11 (0.018) 0.035, 0.19 0.025  [1.84 - 1.90] [1.70 - 1.80] [0.095 - 0.15] 
Monsanto Company 09-SY-200U 

Table E-10. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control S

OH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Leucine 3.16 (0.015) 3.05 (0.015) 0.10 (0.014) 0.046, 0.16 0.016  [3.14 - 3.18] [3.02 - 3.07] [0.080 - 0.13] Lysine 2.62 (0.0092) 2.56 (0.0092) 0.063 (0.0091) 0.024, 0.10 0.020  [2.61 - 2.63] [2.54 - 2.58] [0.045 - 0.074] Methionine 0.59 (0.0038) 0.59 (0.0038) 0.0064 (0.0027) -0.0052, 0.018 0.141  [0.59 - 0.60] [0.58 - 0.59] [0.0028 - 0.012] Phenylalanine 2.12 (0.0068) 2.05 (0.0068) 0.076 (0.0096) 0.034, 0.12 0.015  [2.12 - 2.13] [2.04 - 2.06] [0.053 - 0.099] Proline 2.05 (0.0089) 1.95 (0.0089) 0.097 (0.010) 0.052, 0.14 0.011  [2.03 - 2.06] [1.94 - 1.96] [0.085 - 0.12] Serine 2.16 (0.028) 2.13 (0.028) 0.029 (0.028) -0.092, 0.15 0.412  [2.08 - 2.21] [2.11 - 2.14] [-0.023 - 0.075] Threonine 1.57 (0.0094) 1.56 (0.0094) 0.013 (0.013) -0.043, 0.068 0.432  [1.56 - 1.58] [1.54 - 1.58] [-0.012 - 0.032] Tryptophan 0.46 (0.0059) 0.45 (0.0059) 0.0084 (0.0084) -0.028, 0.045 0.421  [0.45 - 0.47] [0.45 - 0.46] [-0.011 - 0.026] 
Monsanto Company 09-SY-200U 

Table E-10. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control S

OH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Co

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Amino Acid (% DW) 

Tyrosine 1.40 (0.032) 1.34 (0.032) 0.059 (0.044) -0.13, 0.25 0.312  [1.36 - 1.44] [1.28 - 1.41] [-0.024 - 0.13] Valine 1.96 (0.030) 1.84 (0.030) 0.12 (0.021) 0.033, 0.21 0.027  [1.94 - 2.01] [1.78 - 1.90] [0.096 - 0.16] 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  [11.56 - 12.02] [11.58 - 11.71] [-0.080 - 0.44] 18:0 Stearic 4.46 (0.056) 4.37 (0.056) 0.088 (0.048) -0.12, 0.29 0.208 

 [4.32 - 4.53] [4.32 - 4.44] [0.0019 - 0.17] 18:1 Total 18:1 18.10 (0.49) 20.65 (0.49) -2.54 (0.57) -5.00, -0.084 0.046  [16.73 - 18.80] [20.41 - 20.79] [-3.68 - -1.95] Linoleic 25.37 (0.16) 54.50 (0.16) -29.13 (0.22) -30.07, -28.19 <0.001  [25.06 - 25.75] [54.38 - 54.67] [-29.61 - -28.70] 18:3 Linolenic 11.63 (0.11) 8.02 (0.11) 3.61 (0.15) 2.95, 4.28 0.001  [11.41 - 11.75] [7.85 - 8.21] [3.19 - 3.89] 20:0 Arachidic 0.36 (0.0043) 0.33 (0.0043) 0.028 (0.0031) 0.015, 0.041 0.011  [0.35 - 0.36] [0.32 - 0.33] [0.024 - 0.034] 
Monsanto Company 09-SY-200U 

Table E-10. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

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  [0.17 - 0.19] [0.17 - 0.18] [0.0019 - 0.013] 22:0 Behenic 0.29 (0.015) 0.33 (0.015) -0.037 (0.018) -0.11, 0.039 0.172  [0.28 - 0.30] [0.31 - 0.37] [-0.071 - -0.014] Fiber (% DW) Acid Detergent Fiber 17.96 (0.36) 17.65 (0.36) 0.31 (0.51) -1.88, 2.51 0.601  [17.78 - 18.31] [16.69 - 18.15] [-0.35 - 1.62] Neutral Detergent Fiber 16.70 (0.75) 17.25 (0.75) -0.55 (1.06) -5.13, 4.02 0.655  [15.06 - 18.13] [16.10 - 17.88] [-2.82 - 2.03] Proximate (% DW, unless noted) Ash 5.83 (0.040) 5.71 (0.040) 0.12 (0.019) 0.041, 0.20 0.022  [5.78 - 5.93] [5.68 - 5.77] [0.099 - 0.16] Carbohydrates 34.40 (0.53) 36.24 (0.53) -1.83 (0.70) -4.86, 1.20 0.121  [33.55 - 35.89] [36.17 - 36.33] [-2.67 - -0.43] Moisture (% FW) 7.59 (0.15) 7.27 (0.15) 0.32 (0.18) -0.47, 1.11 0.222  [7.20 - 7.87] [7.15 - 7.43] [-0.040 - 0.56] Protein 41.37 (0.24) 39.54 (0.24) 1.83 (0.17) 1.09, 2.57 0.008  [40.92 - 41.70] [39.11 - 39.96] [1.54 - 2.14] 
Monsanto Company 09-SY-200U 

Table E-10. Summary of the Statistical Comparison of MON 87769 vs. the Conventional Control SoybeSite for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (continu

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Proximate 

Total Fat 18.40 (0.42) 18.51 (0.42) -0.12 (0.59) -2.67, 2.44 0.860 

 [17.26 - 18.97] [18.20 - 18.80] [-1.54 - 0.76] Vitamin Vitamin E (mg/100g DW) 2.06 (0.18) 1.90 (0.18) 0.17 (0.092) -0.23, 0.56 0.213 

 [1.68 - 2.33] [1.64 - 2.22] [0.040 - 0.34] Antinutrient Lectin (H.U./mg DW) 4.17 (1.51) 4.15 (1.51) 0.016 (2.13) -9.14, 9.18 0.994  [2.76 - 6.67] [2.08 - 7.56] [-4.80 - 4.60] Phytic Acid (% DW) 1.10 (0.039) 1.01 (0.039) 0.085 (0.033) -0.058, 0.23 0.124  [1.02 - 1.20] [0.97 - 1.04] [0.049 - 0.15] Raffinose (% DW) 0.35 (0.020) 0.35 (0.020) 0.0081 (0.028) -0.11, 0.13 0.801  [0.32 - 0.39] [0.32 - 0.38] [-0.067 - 0.069] Stachyose (% DW) 2.74 (0.10) 2.64 (0.10) 0.099 (0.14) -0.52, 0.72 0.562  [2.61 - 3.01] [2.54 - 2.70] [-0.087 - 0.47] Trypsin Inhibitor (TIU/mg DW) 31.30 (2.41) 28.18 (2.41) 3.12 (3.41) -11.55, 17.79 0.456  [26.11 - 36.42] [25.71 - 31.34] [-5.23 - 8.93] Isoflavone (μg/g DW) Daidzein 1125.54 (39.70) 1668.07 (39.70) -542.53 (26.89) -658.22, -426.83 0.002  [1094.38 - 1183.11] [1583.20 - 1750.03] [-571.84 - -488.82] 
Monsanto Company 09-SY-200U 

Table E-10. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control SOH Site for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients and Isoflavones (Con

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

[

Isoflavone (μg/g DW) 

Genistein 760.07 (20.50) 1143.19 (20.50) -383.12 (21.06) -473.72, -292.52 0.003  [750.00 - 773.91] [1110.39 - 1199.09] [-425.19 - -360.39] Glycitein 82.62 (3.92) 96.62 (3.92) -14.00 (5.55) -37.86, 9.86 0.127  [74.89 - 90.42] [90.63 - 101.88] [-26.98 - -0.22] 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 t
Monsanto Company 09-SY-200U 

Table E-11. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. th

Control (A3525) Forage for Fiber and Proximates 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) 

pValue

Fiber (% DW) 

Acid Detergent Fiber 31.06 (1.36) 30.13 (1.36) 0.93 (0.93) -1.06, 2.92 0.332  [24.91 - 36.39] [24.39 - 37.06] [-9.14 - 5.67] Neutral Detergent Fiber 33.89 (1.37) 33.22 (1.37) 0.67 (1.27) -2.05, 3.39 0.605  [28.06 - 42.44] [25.48 - 42.62] [-9.33 - 7.60] Proximate (% DW, unless noted) Ash 6.58 (0.24) 6.56 (0.24) 0.020 (0.081) -0.20, 0.24 0.814  [5.29 - 7.30] [5.38 - 7.75] [-0.49 - 0.53] Carbohydrates 67.41 (1.32) 67.04 (1.32) 0.37 (0.61) -1.32, 2.06 0.575  [62.88 - 72.79] [61.18 - 71.51] [-3.30 - 4.45] Moisture (% FW) 72.28 (0.64) 72.20 (0.64) 0.080 (0.46) -0.90, 1.06 0.863  [69.50 - 75.90] [70.10 - 74.70] [-2.20 - 3.10] Protein 20.71 (1.05) 20.67 (1.05) 0.040 (0.34) -0.69, 0.77 0.908  [17.05 - 24.53] [18.09 - 24.98] [-2.01 - 2.21] Total Fat 5.30 (0.29) 5.72 (0.29) -0.43 (0.30) -1.26, 0.41 0.226  [3.80 - 6.90] [4.02 - 6.72] [-2.09 - 0.96] 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 t
Monsanto Company 09-SY-200U 

Table E-12. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. th

Control (A3525) Soybean Seed for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrien

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Amino Acid (% DW) 

Alanine 1.78 (0.0084) 1.74 (0.0084) 0.044 (0.011) 0.021, 0.066 0.001  [1.76 - 1.84] [1.68 - 1.81] [-0.018 - 0.11] Arginine 3.23 (0.056) 2.95 (0.056) 0.28 (0.037) 0.20, 0.35 <0.001  [3.00 - 3.61] [2.80 - 3.15] [0.0027 - 0.68] Aspartic Acid 4.54 (0.035) 4.36 (0.035) 0.18 (0.036) 0.077, 0.28 0.007  [4.41 - 4.73] [4.21 - 4.60] [-0.034 - 0.43] Cystine 0.62 (0.0098) 0.60 (0.0098) 0.019 (0.0038) 0.011, 0.027 <0.001  [0.56 - 0.65] [0.56 - 0.64] [-0.024 - 0.037] Glutamic Acid 7.63 (0.059) 7.29 (0.059) 0.34 (0.055) 0.22, 0.46 <0.001  [7.42 - 7.90] [7.03 - 7.71] [-0.0052 - 0.76] Glycine 1.79 (0.012) 1.73 (0.012) 0.062 (0.0098) 0.035, 0.090 0.003  [1.76 - 1.87] [1.67 - 1.81] [0.0039 - 0.14] Histidine 1.09 (0.0073) 1.05 (0.0073) 0.036 (0.0074) 0.021, 0.051 <0.001  [1.06 - 1.14] [1.02 - 1.10] [-0.013 - 0.090] Isoleucine 1.87 (0.018) 1.78 (0.018) 0.088 (0.018) 0.050, 0.13 <0.001  [1.75 - 1.97] [1.70 - 1.86] [-0.052 - 0.24] 
Monsanto Company 09-SY-200U 

Table E-12. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. th

Control (A3525) Soybean Seed for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrien(continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Amino Acid (% DW) 

Leucine 3.19 (0.017) 3.09 (0.017) 0.10 (0.020) 0.059, 0.14 <0.001  [3.13 - 3.32] [3.01 - 3.19] [-0.019 - 0.26] Lysine 2.67 (0.020) 2.60 (0.020) 0.070 (0.016) 0.036, 0.10 <0.001  [2.61 - 2.75] [2.51 - 2.73] [-0.059 - 0.19] Methionine 0.60 (0.0067) 0.58 (0.0067) 0.017 (0.0057) 0.0015, 0.033 0.038  [0.54 - 0.62] [0.56 - 0.60] [-0.030 - 0.038] Phenylalanine 2.14 (0.011) 2.06 (0.011) 0.075 (0.015) 0.038, 0.11 0.002  [2.08 - 2.24] [1.99 - 2.15] [-0.016 - 0.20] Proline 2.09 (0.018) 1.99 (0.018) 0.10 (0.015) 0.070, 0.13 <0.001  [2.03 - 2.19] [1.91 - 2.09] [0.016 - 0.25] Serine 2.20 (0.016) 2.14 (0.016) 0.055 (0.023) 0.0020, 0.11 0.043 

 [2.08 - 2.25] [2.00 - 2.29] [-0.11 - 0.21] Threonine 1.60 (0.0095) 1.57 (0.0095) 0.030 (0.013) 0.0022, 0.057 0.035  [1.54 - 1.65] [1.49 - 1.62] [-0.058 - 0.15] Tryptophan 0.47 (0.0064) 0.46 (0.0064) 0.010 (0.0053) -0.00097, 0.022 0.069  [0.45 - 0.51] [0.44 - 0.50] [-0.019 - 0.059] 
Monsanto Company 09-SY-200U 

Table E-12. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. th

Control (A3525) Soybean Seed for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrien(Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Amino Acid (% DW) 

Tyrosine 1.40 (0.016) 1.34 (0.016) 0.059 (0.021) 0.014, 0.10 0.013  [1.27 - 1.50] [1.26 - 1.44] [-0.072 - 0.21] Valine 1.98 (0.019) 1.88 (0.019) 0.096 (0.021) 0.053, 0.14 <0.001  [1.84 - 2.08] [1.78 - 1.96] [-0.063 - 0.26] 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  [11.53 - 12.54] [11.14 - 12.08] [-0.080 - 0.75] 18:0 Stearic 4.19 (0.10) 4.15 (0.10) 0.042 (0.031) -0.044, 0.13 0.245 

 [3.73 - 4.53] [3.85 - 4.44] [-0.16 - 0.19] 18:1 Oleic 15.18 (0.95) 19.19 (0.95) -4.02 (0.52) -5.46, -2.57 0.001  [12.66 - 18.80] [17.24 - 21.17] [-5.38 - -1.95] 18:2 Linoleic 22.78 (1.64) 54.93 (1.64) -32.16 (2.32) -37.50, -26.81 <0.001 

 [16.46 - 30.81] [54.05 - 56.04] [-38.90 - -23.68] 18:3 Linolenic 11.18 (0.46) 9.20 (0.46) 1.98 (0.50) 0.60, 3.36 0.016  [10.20 - 11.80] [7.42 - 10.66] [0.82 - 3.89] 20:0 Arachidic 0.34 (0.0090) 0.31 (0.0090) 0.031 (0.0027) 0.025, 0.036 <0.001  [0.31 - 0.37] [0.28 - 0.34] [0.017 - 0.058] 
Monsanto Company 09-SY-200U 

Table E-12. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. thControl (A3525) So

ybean Seed for Amino Acids, Fatt

y Acids, Fiber, Proximates, Vitamin E, Antinutrien

(Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

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  [0.075 - 0.20] [0.069 - 0.19] [-0.058 - 0.072] 22:0 Behenic 0.29 (0.0069) 0.32 (0.0069) -0.026 (0.0087) -0.047, -0.0050 0.023  [0.26 - 0.31] [0.28 - 0.37] [-0.11 - 0.022] Fiber (% DW) Acid Detergent Fiber 16.77 (0.42) 16.90 (0.42) -0.14 (0.50) -1.51, 1.24 0.794 

 [14.38 - 18.31] [13.80 - 18.15] [-1.91 - 2.65] Neutral Detergent Fiber 16.84 (0.38) 17.18 (0.38) -0.34 (0.41) -1.19, 0.50 0.411  [15.06 - 19.15] [14.43 - 19.37] [-2.82 - 2.32] 

Proximate (% DW, unless noted) Ash 5.72 (0.092) 5.63 (0.092) 0.098 (0.057) -0.024, 0.22 0.106  [5.23 - 6.17] [5.24 - 6.07] [-0.34 - 0.62] Carbohydrates 36.45 (0.99) 38.68 (0.99) -2.24 (0.53) -3.32, -1.15 <0.001  [33.23 - 39.93] [35.30 - 42.60] [-6.24 - 2.23] Moisture (% FW) 7.47 (0.17) 7.41 (0.17) 0.063 (0.10) -0.22, 0.35 0.572  [6.71 - 8.21] [6.84 - 8.11] [-0.66 - 0.84] Protein 41.92 (0.27) 39.75 (0.27) 2.18 (0.31) 1.51, 2.84 <0.001  [40.92 - 43.36] [38.22 - 41.58] [0.90 - 5.07] 
Monsanto Company 09-SY-200U 

Table E-12. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. th

Control (A3525) Soybean Seed for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrien(Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Proximate 

Total Fat (% DW) 15.91 (1.05) 15.94 (1.05) -0.037 (0.61) -1.74, 1.66 0.955  [12.95 - 19.03] [12.73 - 18.80] [-3.01 - 3.27] Vitamin Vitamin E (mg/100g DW) 1.56 (0.26) 1.43 (0.26) 0.13 (0.10) -0.16, 0.42 0.271  [0.86 - 2.54] [0.70 - 2.22] [-0.35 - 0.55] 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  [0.55 - 8.07] [0.71 - 11.32] [-7.01 - 5.13] Phytic Acid 1.05 (0.059) 1.02 (0.059) 0.031 (0.030) -0.052, 0.11 0.357  [0.81 - 1.34] [0.75 - 1.26] [-0.13 - 0.16] Raffinose 0.37 (0.019) 0.35 (0.019) 0.022 (0.011) -0.00076, 0.045 0.057  [0.32 - 0.45] [0.29 - 0.45] [-0.067 - 0.099] Stachyose 2.83 (0.11) 2.75 (0.11) 0.081 (0.070) -0.064, 0.23 0.259  [2.28 - 3.27] [2.43 - 3.21] [-0.46 - 0.47] Trypsin Inhibitor (TIU/mg DW) 33.81 (2.81) 31.10 (2.81) 2.71 (2.01) -1.44, 6.87 0.190  [24.30 - 54.80] [21.34 - 41.69] [-10.58 - 29.14] 
Monsanto Company 09-SY-200U 

Table E-12. Statistical Summary from the Combined-Site Compositional Analysis of MON 87769 vs. the Control (A3525) Soybean Seed for Amino Acids, Fatty Acids, Fiber, Proximates, Vitamin E, Antinutrients 

(Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] 

Mean (S.E.) 

[Range] 

95% CI 

(Lower, Upper) p-Value

Isoflavone (μg/g DW) Daidzein 1187.81 (188.32) 1807.36 (188.32) -619.55 (120.04) -952.84, -286.26 0.006  [957.23 - 1838.91] [1380.05 - 2775.08] [-1385.89 - -186.51] Genistein 733.64 (114.81) 1136.52 (114.81) -402.88 (80.11) -625.30, -180.45 0.007  [576.70 - 1118.40] [770.81 - 1706.74] [-821.84 - -86.03] Glycitein 82.73 (5.66) 102.18 (5.66) -19.45 (6.25) -32.35, -6.55 0.004  [65.37 - 106.72] [65.51 - 158.73] [-93.36 - 1.49] 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 t
Monsanto Company 09-SY-200U Page 292 of 395 

Table E-13. Trans-ALA, GLA, trans-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; 2S.E. = standard error.

 
Monsanto Company 09-SY-200U Page 293 of 395 

Table E-13. Trans-ALA, GLA, trans-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; 2S.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 Range2

SEED 

Fiber (% DW) 

Acid Detergent Fiber 9.0 – 11.1i

 7.81 – 18.61 

Neutral Detergent Fiber 10.0 – 14.9i

 8.53 – 21.25 

Proximate (% DW, unless noted) 

Ash 4.61 – 5.94b 3.89 – 6.99 

Carbohydrates 29.3 – 41.3c

 29.6 – 50.2 

Moisture (% FW) 5.3 – 8.73c

 4.7 – 34.4 

Protein 36.0 – 48.4e

 33.19 – 45.48 

Total Fat 16.0 – 23.1e 8.10 – 23.56 

19.8 – 26.7d

Amino Acids (% DW) 

Alanine 1.60 – 1.86c 1.513 – 2.104 

Arginine 2.56 – 3.46c 2.285 – 3.400 

Aspartic acid 4.18 – 4.99c 3.808 – 5.122 

Cystine 0.54 – 0.66c 0.370 – 0.808 

Glutamic acid 6.64 – 8.16c 5.843 – 8.201 

Glycine 1.60 – 1.87c 1.458 – 1.997 

Histidine 0.98 – 1.16c 0.878 – 1.175 

Isoleucine 1.65 – 1.95c 1.539 – 2.077 

Leucine 2.81 – 3.37c 2.590 – 3.622 

Lysine 2.47 – 2.84c

 2.285 – 2.839 

Methionine 0.51 – 0.59c 0.431 – 0.681 

Phenylalanine 1.78 – 2.19c 1.632 – 2.346 

Proline 1.86 – 2.23c

 1.687 – 2.284 

Serine 1.96 –2.28c

 1.106 – 2.484 

Threonine 1.51 – 1.73c

 1.139 – 1.862 

Tryptophan 0.56 – 0.63c

 0.356 – 0.502 

Tyrosine 1.35 – 1.59c

 1.016 – 1.613 

Valine 1.71 – 2.02c

 1.597 – 2.204 

Isoflavones (ug/g DW) 

Daidzein 219 – 1190c 60.0 – 2453.5 

Genistein 286 – 1380c

 144.3 – 2837.2 

Glycitein 42.2 – 204a 15.3 – 310.4 

Vitamin (mg/100g DW) 

Vitamin E 1.09 – 2.84g 0.19 – 6.17 
Monsanto 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 Range2

Fatty Acid (% Total FA) 

16:0 Palmitic 10.63 – 11.69c 9.55 – 15.77 

18:0 Stearic 3.85 – 4.55c 2.70 – 5.88 

18:1 Oleic 15.02 – 31.19c 14.3 – 32.2 

18:2 Linoleic 44.03 – 54.96c 42.3 – 58.8 

18:3 Linolenic 5.08 – 10.26c 3.00 – 12.52 

20:0 Arachidic 0.31 – 0.43c 0.163 – 0.482 

22:0 Behenic 0.46 – 0.59c

 0.277 – 0.595 

Antinutrient (% DW, unless noted) 

Phytic acid 1 – 2.74i

 0.634 – 1.960 

Raffinose 0.4 – 1.8h 0.212 – 0.661 

Stachyose 3.08 – 4.13f 1.21 – 3.50 

Trypsin inhibitor (TIU/mg DW) 33.2 – 54.5c 19.59 – 118.68 

Lectin (H.U./mg FW) 0.8 – 2.4c 0.105 – 9.038 

FORAGE 

Fiber (% DW) 

Acid Detergent Fiber 32-38i not available 

Neutral Detergent Fiber 34-40i not available 

Crude Fiber not available 13.58-31.73 

Proximate (% DW, unless noted) 

Ash 8.8-10.5i 6.718-10.782 

Carbohydrates not available 59.8-74.7 

Moisture (% FW) 74-79i

 73.5-81.6 

Protein 11.2-17.3i 14.38-24.71 

Total Fat 3.1-5.1i 1.302-5.132 

1DW = dry weight; DM = dry matter; FW = fresh weight; FA = fatty acid; TIU = trypsin inhibitor 

unit; H.U. = hemaglutinating unit. 

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]. 

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 104

 = μg/g dw; mg/g dw x 103 = mg/kg dw; mg/100g dw x 10 = mg/kg dw; 

g/100g dw x 10 = mg/g. 
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, carbohydrate, and ash. An acetone wash removed the 

fats and pigments. Lignocellulose fraction 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. The sulfur containing amino acids required an 

oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the 

samples for the remaining amino acids was accomplished through direct acid hydrolysis 

with hydrochloric acid. Once hydrolyzed, the individual amino acids were then 

quantitated using an automated amino acid analyzer. The limit of quantitation for each 

amino acid was 0.100 mg/g. 

Reference Standards 

• Thermo Scientific/Pierce, K18, 2.5 μmol/mL per constituent (except cystine 1.25 

μmol/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 Methods 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 nonvolatile matter remaining was quantitated 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. Agriculture 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 thimble containing sodium sulfate and dried to 

remove excess moisture. Pentane was dripped through the sample to remove the fat. The 

extract was then evaporated, dried, and weighed. The limit of quantitation was 0.100%. 

Reference 

AOAC. 2005. Method 96.039 in Official Methods 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% BF3-methanol. The resulting methyl esters of the fatty acids were 

extracted with heptane. An internal standard was added prior to the lipid extraction. The 

methyl esters of the fatty acids were analyzed by gas chromatography using external 

standards for quantitation. The limit of quantitation was 0.005%-0.06% depending 

on the soybean fraction. 

Reference Standards 

• Nu Chek Prep GLC Reference Standard 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 Standard 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, used as 100%, Lot Number N-6M-A25-R 

• Nu Chek Prep Methyl Erucate, used as 100%, Lot Number U-79M-AU3-Q 

• Nu Chek Prep Methyl Lignocerate, used as 100%, Lot Number N-24M-F5-S 

• Nu Chek Prep Methyl Docosapentaenoate, used as 100%, Lot Number U-101M-F18-S 

• Nu Chek Prep Methyl Docosahexaenoate, used as 100%, Lot Number U-84M-AU28-R 
Monsanto 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 Methyl Ester, used as 100%, Lot Number 

182102-186015 

• Nu Chek Prep Methyl Elaidate, used as 100%, Lot Number U-47M-JA18-R 

• Nu Chek Prep Methyl Linoelaidate, used as 100%, Lot Number U-60M-F27-R 

• Nu Chek Prep Methyl 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)-Octadecadienoate, 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 extract was brought to volume, diluted, and 

centrifuged. An aliquot of the supernatant was placed onto a C18 solid-phase extraction 

column. Unwanted components of the matrix were rinsed off with 20% methanol and 

then the isoflavones were eluted with 80% methanol. The sample was analyzed on a 

high-performance liquid chromatography system with ultraviolet spectrophotometric 

quantitation and was compared against an external standard curve of known standards. 

The limit of quantitation for each component was 10.0 μg/g. 

Reference Standards 

Indofine, Daidzein, 99% , Lot Number 020508146 

Indofine, Genistein, >99% (used as 100% in calculations), 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 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 containing PBS. A 10% 

hematocrit of lyophilized rabbit blood in PBS was added to each dilution. After 2.5 

hours, the absorbance of each dilution of the sample and lectin control was read by a 

spectrophotometer at 620 nm, using PBS to zero the instrument. One hemagglutinating 
Monsanto Company 09-SY-200U Page 299 of 395 

unit (H.U.) was defined as the level that caused 50% of the standard cell suspension to 

sediment in 2.5 hours. The limit of quantitation was 0.10 H.U./mg. 

References 

Klurfeld, D.M. and D. Kritchevsky. 1987. Isolation and Quantitation of Lectins from 

Vegetable Oils. Lipids 22:667-668. 

Klurfeld, D. M., Personal communication. 

Liener, I. E. 1955. The photometric determination of the hemagglutinating activity of 

soyin and crude soybean extracts. Archives of Biochemistry and Biophysics, 54:223-231. 

E.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 and converted to percent moisture. The limit 

of quantitation was 0.100%. 

Reference 

AOAC. 2005. Methods 926.08 and 925.09. AOAC International, 18th 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, carbohydrate, enzyme, and ash. An acetone wash 

removed the fats and pigments. Hemicellulose, cellulose, and lignin fractions were 

collected on the frit and determined gravimetrically. The limit of quantitation was 

0.100%. 

References 

AACC. 1998. Methods 32.20 in Approved Methods of the American Association of 

Cereal Chemists, 9th ed. 

USDA. 1970. Forage Fiber Anlyses. Agriculture 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 anion exchange (SAX) column. Sample 

analysis was done on a polymer HPLC column PRP-1, 5μm (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 

077K0693 
Monsanto Company 09-SY-200U Page 300 of 395 

References 

Lehrfeld, J. 1989. High-performance liquid chromatography analysis of phytic acid on a 

ph-stable, macroporous polymer column. Cereal Chemistry 66(6):510-515. 

Lehrfeld, Jacob, “HPLC Separation and Quantitation 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 reduced in the presence of boiling sulfuric 

acid and a mercury catalyst mixture to form ammonia. The acid digest was made alkaline. 

The ammonia was distilled and then titrated with a standard acid. The percent nitrogen 

was calculated and converted to protein using the factor 6.25. The limit of quantitation 

was 0.100%. 

References 

AOAC. 2005. Method 955.04 and 979.09 in Official Methods of Analysis of AOAC 

International, 18th ed. AOAC International, Gaithersburg, MD. 

Bradstreet, R.B. 1965. The Kjeldahl Method for Organic Nitrogen. Academic Press, New 

York. 

Kolthoff, I.M., and E.B. Sandell. 1948. Acidimetry and Alkalimitry in Quantitative 

Inorganic Analysis. The MacMillan Company, New York. 

E.6.13. Phosphatides 

The sample was extracted with a 98% CHCl3 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 quantification. The Limits of Quantitation for 

these assays were as follows: L-alpha-Phosphatidic Acid 0.70%, L-alphaPhosphatidycholine 1.90%, L-alpha-Phosphatidylethanolamine 2.00%, and 

L-alpha-Phosphatidylinositol 1.20%. 

Reference Standard 

• (PA) – Avanti Polar Lipids, L-alpha-Phosphatidic Acid (sodium salt), 100%, Lot 

Number SPA-19 

• (PC) – Avanti Polar Lipids, L-alpha-Phosphatidylcholine, 100%, Lot Number PPC-116 

• (PE) – Avanti Polar Lipids, L-alpha-Phosphatidylethanolamine, 100%, Lot Number 

PPE-132b 

• (PI) – Avanti Polar Lipids, L-alpha-Phosphatidylinositol, 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 deionized water and the extract treated with a 

hydroxylamine hydrochloride solution in pyridine, containing phenyl-β-D-glucoside as 

an internal standard. The resulting oximes were converted to silyl derivatives by 

treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas 

chromatography using a flame ionization detector. The limit of quantitation was 

0.0500%. 

Reference Standards 

• Sigma, Raffinose Pentahydrate, 99% / 84.0% after correction for degree of hydration, 

Lot Number 035K1371 

• Sigma, Stachyose, 98% / 96.4% after correction for degree of hydration, Lot Number 

065K3775 

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 petroleum 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 amount of trypsin and benzoy1-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 acid. The solution was 

centrifuged, then the absorbance was determined at 410 nm. Trypsin inhibitor activity as 

measured by trypsin inhibitor units (TIU) was determined by photometrically measuring 

the inhibition of trypsin’s reaction with benzoyl-DL-arginine-p-nitroanalide 

hydrochloride. The limit of quantitation was 1.00 TIU/mg. 

Reference 

AOCS. 1997. Method Ba12-75 in Official Methods and Recommended Practices of the 

American Oil Chemists’ Society, 5th ed. Chemists’ 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 ether and then quantitated 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. Schreurs. 1985. Vitamin E composition of some seed 

oils as determined by high-performance liquid chromatography with fluorometric 

quantitation. Journal of Food Science, 50(1):121-124. 

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 

Statistical Summary from the Combined-Site Analysis of MON 87769 Processed Fractions 

Table E-15. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soybfrom Combined-Site for Amino Acids, Fiber, Proximates and Antinutrients 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Amino Acid (% DW) Alanine 2.28 (0.025) 2.28 (0.025) 0.0053 (0.013) -0.029, 0.039 0.703 (2.23 

 [2.24 - 2.32] [2.24 - 2.31] [2.0Arginine 4.34 (0.13) 4.17 (0.13) 0.16 (0.067) -0.68, 1.01 0.245 (3.80  [4.18 - 4.51] [4.06 - 4.31] [3.3Aspartic Acid 6.10 (0.030) 6.00 (0.030) 0.10 (0.022) 0.044, 0.16 0.006 (5.80  [6.05 - 6.13] [5.94 - 6.04] [5.2Cystine 0.77 (0.0080) 0.77 (0.0080) 0.0036 (0.0084) -0.10, 0.11 0.740 (0.7 [0.77 - 0.78] [0.75 - 0.79] [0.6Glutamic Acid 9.79 (0.031) 9.58 (0.031) 0.22 (0.043) 0.11, 0.32 0.002 (9.15 

 [9.78 - 9.81] [9.50 - 9.68] [8.21Glycine 2.31 (0.023) 2.28 (0.023) 0.038 (0.015) -0.0017, 0.077 0.057 (2.23 

 [2.28 - 2.34] [2.23 - 2.32] [2.0Histidine 1.42 (0.010) 1.40 (0.010) 0.021 (0.0059) 0.0054, 0.036 0.017 (1.39 

 [1.41 - 1.43] [1.38 - 1.42] [1.2Isoleucine 2.53 (0.050) 2.51 (0.050) 0.023 (0.046) -0.096, 0.14 0.642 (2.47  [2.46 - 2.60] [2.38 - 2.59] [2.1
Monsanto Company 09-SY-200U 

Table E-15. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soyb

from Combined-Site for Amino Acids, Fiber, Proximates and Antinutrients (Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Amino Acid (% DW) Leucine 4.13 (0.037) 4.08 (0.037) 0.047 (0.030) -0.030, 0.12 0.178 (4.04 

 [4.07 - 4.19] [4.01 - 4.15] [3.6Lysine 3.34 (0.026) 3.31 (0.026) 0.027 (0.022) -0.030, 0.084 0.284 (3.20  [3.29 - 3.38] [3.26 - 3.36] [2.9Methionine 0.77 (0.0079) 0.76 (0.0079) 0.0038 (0.011) -0.044, 0.052 0.767 (0.74 

 [0.75 - 0.78] [0.75 - 0.78] [0.6Phenylalanine 2.78 (0.023) 2.74 (0.023) 0.042 (0.020) -0.0099, 0.094 0.091 (2.70 

 [2.75 - 2.81] [2.69 - 2.78] [2.4Proline 2.66 (0.033) 2.62 (0.033) 0.034 (0.047) -0.17, 0.24 0.543 (2.50  [2.64 - 2.68] [2.50 - 2.71] [2.1Serine 2.75 (0.034) 2.68 (0.034) 0.070 (0.048) -0.048, 0.19 0.197 (2.53  [2.71 - 2.80] [2.58 - 2.77] [2.2Threonine 2.00 (0.016) 1.97 (0.016) 0.023 (0.022) -0.035, 0.080 0.361 (1.93  [1.98 - 2.01] [1.91 - 2.01] [1.7Tryptophan 0.68 (0.020) 0.67 (0.020) 0.012 (0.0026) 0.0049, 0.019 0.006 (0.65 

 [0.66 - 0.70] [0.65 - 0.69] [0.5
Monsanto Company 09-SY-200U 

Table E-15. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soybfrom Combined-Site for Amino Acids, Fiber, Proximates and Antinutrients (Continued)

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Amino Acid (% DW) Tyrosine 1.81 (0.016) 1.78 (0.016) 0.027 (0.022) -0.027, 0.081 0.265 (1.65 

 [1.78 - 1.83] [1.73 - 1.82] [1.3Valine 2.67 (0.047) 2.64 (0.047) 0.031 (0.055) -0.11, 0.17 0.596 (2.6 [2.59 - 2.74] [2.50 - 2.71] [2.3Fiber (% DW) Acid Detergent Fiber 5.18 (0.14) 4.52 (0.14) 0.66 (0.19) 0.18, 1.13 0.014 (4.00 

 [4.93 - 5.46] [4.22 - 4.96] [1.7Neutral Detergent Fiber 6.36 (0.13) 5.58 (0.13) 0.78 (0.19) -0.017, 1.58 0.052 (4.4

 [6.19 - 6.56] [5.36 - 5.82] [2.4
Monsanto Company 09-SY-200U 

Table E-15. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soybfrom Combined-Site for Amino Acids, Fiber, Proximates and Antinutrients (Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Proximate (% DW, unless noted) Ash 6.62 (0.27) 6.46 (0.27) 0.16 (0.14) -0.20, 0.52 0.299 (6.37 

 [6.19 - 6.91] [5.96 - 6.83] [5.6Carbohydrates 38.00 (0.29) 40.05 (0.29) -2.06 (0.39) -3.05, -1.06 0.003 (36.26  [37.49 - 38.38] [39.44 - 41.00] [28.9Moisture (% FW) 2.63 (0.56) 2.45 (0.56) 0.18 (0.64) -1.46, 1.81 0.794 (2.36 

 [1.68 - 3.69] [1.76 - 4.12] [0, Protein 54.22 (0.50) 52.64 (0.50) 1.57 (0.70) -7.28, 10.42 0.265 (50.03 

 [53.28 - 55.50] [52.11 - 53.19] [44.7Total Fat 1.17 (0.39) 0.84 (0.39) 0.32 (0.47) -5.70, 6.35 0.619 (0.82 

 [0.50 - 2.00] [0.61 - 1.13] [0, 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 

 [1.19 - 1.52] [1.30 - 1.36] [0.8Trypsin Inhibitor (TIU/mg DW) 2.03 (1.23) 4.82 (1.23) -2.78 (1.75) -7.05, 1.49 0.162 (0.52 

 [0.52 - 3.56] [2.31 - 9.10] [0, 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 zer3 Literature/Historical range references: a Karr-Lilienthal et al., 2004, b Grieshop et al., 2003, c Soybean Meal Information Center, 2001. d Padgette et al., 1996. e Orthoefer, 1978, f OECD, 2001. 
Monsanto Company 09-SY-200U 

Table E-16. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soyb

Combined-Site for Fatty Acids and Vitamin E 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Fatty 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  [0.078 - 0.088] [0.078 - 0.089] [0.0416:0 Palmitic 12.10 (0.050) 11.48 (0.050) 0.61 (0.071) 0.44, 0.79 <0.001 (9.80  [11.98 - 12.23] [11.42 - 11.61] [7.6816:1 Palmitoleic 0.085 (0.0015) 0.091 (0.0015) -0.0065 (0.0021) -0.016, 0.0027 0.093 (0.07 [0.083 - 0.087] [0.088 - 0.095] [0.0417:0 Heptadecanoic 0.10 (0.0094) 0.096 (0.0094) 0.0053 (0.0031) -0.035, 0.045 0.337 (0.08

 [0.090 - 0.11] [0.088 - 0.11] [0.0518:0 Stearic 4.18 (0.019) 4.08 (0.019) 0.098 (0.027) 0.033, 0.16 0.010 (3.9

 [4.13 - 4.20] [4.04 - 4.12] [3.018:1 Oleic 16.02 (0.80) 19.25 (0.80) -3.23 (0.89) -14.51, 8.04 0.170 (19.57 

 [14.49 - 17.34] [19.02 - 19.74] [16.118:2 6c,9c (Isolinoleic Acid) 0.091 (0.0055) 0.075 (0.0055) 0.015 (0.0078) -0.018, 0.049 0.184 (0.030  [0.089 - 0.094] [0.063 - 0.085] [018:2 9c,12c Linoleic 25.66 (3.53) 55.38 (3.53) -29.72 (4.54) -87.36, 27.92 0.096 (54.06  [20.66 - 30.92] [54.82 - 55.87] [50.2
Monsanto Company 09-SY-200U 

Table E-16. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soyb

Combined-Site for Fatty Acids and Vitamin E (Continued)

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Fatty 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 (0.07 [0.47 - 0.54] [0.10 - 0.16] [0, 18:3 Linolenic 10.61 (0.55) 8.31 (0.55) 2.31 (0.47) -3.71, 8.32 0.128 (6.44  [10.34 - 10.95] [7.42 - 9.07] [3.4818:3 (Other 18:3 Trans) 0.064 (0.0091) 0.084 (0.0091) -0.019 (0.013) -0.075, 0.036 0.271 (0.03

 [0.031 - 0.078] [0.069 - 0.098] [0, 20:0 Arachidic 0.35 (0.0072) 0.31 (0.0072) 0.034 (0.0035) -0.011, 0.079 0.065 (0.30 

 [0.34 - 0.35] [0.30 - 0.33] [0.220:1 Eicosenoic 0.18 (0.020) 0.17 (0.020) 0.014 (0.0016) -0.0061, 0.033 0.072 (0.15 

 [0.16 - 0.20] [0.14 - 0.19] [0.0422:0 Behenic 0.32 (0.019) 0.33 (0.019) -0.0086 (0.022) -0.065, 0.048 0.709 (0.32  [0.27 - 0.35] [0.29 - 0.36] [0.224:0 Lignoceric 0.093 (0.015) 0.12 (0.015) -0.030 (0.0024) -0.036, -0.023 <0.001 (0.08 [0.076 - 0.11] [0.10 - 0.14] [0, 
Monsanto Company 09-SY-200U 

Table E-16. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soyb

Combined-Site for Fatty Acids and Vitamin E (Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Vitamin (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 

 [6.56 - 10.90] [5.14 - 9.27] [0, 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 zer3 Literature/Historical range references: a Codex, 2005. 
Monsanto Company 09-SY-200U 

Table E-17. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soybfrom Combined-Site for Amino Acids and Moisture 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Amino Acid (% DW) Alanine 3.70 (0.037) 3.83 (0.037) -0.13 (0.052) -0.36, 0.091 0.124 (3.56  [3.63 - 3.81] [3.77 - 3.90] [3.3Arginine 7.78 (0.11) 7.85 (0.11) -0.072 (0.13) -0.41, 0.27 0.607 (7.54 

 [7.61 - 8.09] [7.61 - 8.01] [6.7Aspartic Acid 10.98 (0.14) 11.07 (0.14) -0.087 (0.20) -0.59, 0.41 0.683 (10.72  [10.70 - 11.34] [10.67 - 11.41] [9.79Cystine 1.14 (0.010) 1.12 (0.010) 0.022 (0.014) -0.013, 0.058 0.173 (1.09  [1.12 - 1.16] [1.09 - 1.15] [0.9Glutamic Acid 18.80 (0.28) 18.83 (0.28) -0.024 (0.37) -0.99, 0.94 0.951 (18.16  [18.42 - 19.65] [18.12 - 19.34] [16.2Glycine 3.93 (0.033) 4.00 (0.033) -0.069 (0.046) -0.18, 0.044 0.184 (3.7 [3.88 - 3.97] [3.90 - 4.06] [3.4Histidine 2.40 (0.021) 2.45 (0.021) -0.048 (0.030) -0.12, 0.025 0.158 (2.33 

 [2.37 - 2.44] [2.39 - 2.51] [2.1Isoleucine 4.43 (0.044) 4.57 (0.044) -0.13 (0.062) -0.40, 0.13 0.161 (4.39 

 [4.33 - 4.50] [4.47 - 4.61] [3.9
Monsanto Company 09-SY-200U 

Table E-17. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soy

Fraction from Combined-Site for Amino Acids and Moisture (Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Amino Acid (% DW) Leucine 7.28 (0.053) 7.47 (0.053) -0.19 (0.075) -0.37, -0.0062 0.044 (7.08 

 [7.13 - 7.37] [7.38 - 7.59] [6.6Lysine 5.78 (0.040) 5.91 (0.040) -0.13 (0.056) -0.27, 0.0028 0.053 (5.68  [5.70 - 5.86] [5.80 - 5.98] [5.4Methionine 1.21 (0.011) 1.22 (0.011) -0.014 (0.015) -0.051, 0.022 0.368 (1.13  [1.20 - 1.23] [1.19 - 1.25] [1.0Phenylalanine 5.00 (0.046) 5.10 (0.046) -0.096 (0.065) -0.25, 0.063 0.189 (4.88  [4.88 - 5.10] [5.02 - 5.19] [4.5Proline 4.89 (0.13) 4.90 (0.13) -0.014 (0.18) -0.46, 0.43 0.939 (4.30  [4.64 - 5.22] [4.57 - 5.08] [3.4Serine 4.96 (0.058) 5.01 (0.058) -0.048 (0.082) -0.40, 0.30 0.614 (4.78  [4.86 - 5.08] [4.82 - 5.11] [4.4Threonine 3.25 (0.052) 3.30 (0.052) -0.053 (0.074) -0.23, 0.13 0.497 (2.96  [3.10 - 3.35] [3.21 - 3.39] [2.6Tryptophan 1.09 (0.011) 1.10 (0.011) -0.014 (0.0070) -0.032, 0.0043 0.107 (1.05  [1.07 - 1.11] [1.09 - 1.12] [0.9
Monsanto Company 09-SY-200U 

Table E-17. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control Soyb

Fraction from Combined-Site for Amino Acids and Moisture (Continued) 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Amino Acid (% DW) Tyrosine 3.43 (0.040) 3.52 (0.040) -0.087 (0.042) -0.19, 0.020 0.091 (3.33  [3.33 - 3.52] [3.44 - 3.58] [3.0Valine 4.48 (0.061) 4.63 (0.061) -0.14 (0.086) -0.51, 0.23 0.236 (4.34 

 [4.44 - 4.56] [4.51 - 4.74] [4.0Proximate Moisture (% FW) 3.22 (0.28) 2.73 (0.28) 0.50 (0.40) -0.47, 1.47 0.256 (1.97 

 [2.56 - 3.64] [2.03 - 3.56] [0.41DW = 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 zer3 Literature/Historical range references: a OECD, 2001, b Mattil, 1974. 
Monsanto Company 09-SY-200U 

Table E-18. Summary of the Compositional Comparison of MON 87769 vs. the Conventional Control So

yb

from Combined-Site for Phosphatides 

 Difference (Test minus Control) 

Analytical Component (Units)1 

MON 87769 

Mean (S.E.) 

[Range] 

A3525 

Mean (S.E.) 

[Range] Mean (S.E.) 

95% CI 

(Lower, Upper) 

pValue

Com

(R

[99% 

Int

Phosphatide (%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 

 [3.16 - 4.54] [2.74 - 4.12] [0.7L-alpha-Phosphatidylcholine 7.16 (1.16) 7.28 (1.16) -0.12 (1.02) -13.06, 12.83 0.926 (5.66 

 [6.37 - 8.16] [5.37 - 10.25] [2.16L-alpha-Phosphatidylethanolamine 5.66 (0.67) 5.48 (0.67) 0.18 (0.72) -9.01, 9.38 0.843 (4.73 

 [5.43 - 6.14] [4.40 - 7.42] [2.9L-alpha-Phosphatidylinositol 4.98 (0.37) 4.67 (0.37) 0.31 (0.53) -1.96, 2.57 0.621 (3.93  [4.44 - 5.47] [4.00 - 5.89] [2.61FW = 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 zer3 Literature/Historical range references: a Szuhaj, 2005. 
Monsanto Company 09-SY-200U Page 314 of 395 

References 

Codex Standard for Named Vegetable 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 nutritional characteristics of United States 

soybeans and soybean meals. J Agric Food Chem 51:7684-7691. 

Guzman, G. J. and Murphy, P. A. 1986. Tocopherols 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, similar yield. Crop Sci. 31:290-292. 

Jacorzynski, B. and N. Barylko-Pikielna. 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-producing 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 Soy 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 compositional considerations for new varieties of 

soybean:key food and feed nutrients and anti-nutrients. Organization for Economic Cooperation and Development, Evironmental Health and Safety Publications. Paris, France. 

ENV/JM/MONO (2001)15. 

Orthoeffer, F.T., 1978. Processing and utilization. Page 239 in Soybean Physiology, 

Agronomy and Utilization. Academic Press, Inc.. New York, NY. 

Padgette, S.R., Taylor, N.B., Nida, D.L., Bailey, M.R., MacDonald, J., Holden, L.R., and 

Fuchs, R.L. 1996. The composition of glyphosate-tolerant 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 nutritional profile of glyphosate-tolerant 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_new/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. MacDonald, A.R. Shariff, and S.R. Padgette. 1999. 

Compositional analysis of glyphosate-tolerant soybeans 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 Assessment 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 decreased the levels of LA in MON 87769. The 

safety and nutritional assessment of MON 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 nutritional impact of SDA, GLA, and the two 

trans-isomers as well as the nutritional impact from the reduced levels of LA in 

MON 87769. 

F.2. Safety and Nutritional Impact 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 sources 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 (GRAS) 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 fatty acids) and the seed oil of 

several plant species, notably 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 history of safe consumption in human foods 

across a range of sources. 

The safety of SDA in MON 87769 is based on: 1) its occurrence as an in vivo

intermediate in the metabolism of ALA to long chain omega-3 fatty acids in mammals, 

2) a long-standing history of safe consumption 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 and 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/day for the rat studies with no adverse 

effects reported. Therefore, SDA is concluded to be safe for human and animal 

consumption. 

F.3. Safety and Nutritional Impact 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 in 
Monsanto 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). Small 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 feeding 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 fed infants will consume 5-10 mg/kg/day of 

GLA, equivalent to an intake of 0.35-0.7 grams/day GLA intake by a 70 kg adult. GLA 

is also found in large concentrations in certain 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, and 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 adverse effects. A number of animal studies 

have also demonstrated the safety of large doses of GLA consumption. There are no 

identified concerns at this time regarding excessive dietary consumption 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 solid 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 consumption of meat and dairy 

products (Chardigny et al., 1996; Ledoux, 2007). The TFA levels in non-hydrogenated 

vegetable oils are generally low by comparison, typically less than 1% of total fatty acids. 

The TFAs arise during oil refining, particularly 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 fatty acids. Total TFAs in MON 87769 are 

well within the range of the total TFA content in samples of commercial soybean oil 

(Chardigny et al., 1996). The contribution of the TFA from MON 87769 to the overall 

human dietary TFA intake will be minimal 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 considered to be an essential 

fatty acid, and is necessary in the human diet to support health. However, the only 
Monsanto 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 species, LA is first converted to GLA and 

subsequently GLA to arachidonic acid (Horrobin, 1992). Daily human LA intake greatly 

exceeds the amount of LA needed to support health. 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 supported by the results of a published 90-day/onegeneration reproductive rat toxicity study in which a no observable 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. Diets 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 significant dose-dependent test article-related adverse 

effects were reported in any of the parameters evaluated, including clinical signs, 

behavior, mortality, body weight, organ weights, 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 laboratory, 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 polyunsaturated fatty acids), they were not considered to be of 

toxicological significance. Therefore, the 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 tested. Additional many 

published studies on SDA and GLA from other 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 conversion 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.html 
Monsanto Company 09-SY-200U Page 319 of 395 

When viewed in its entirety, the scientific evidence presented above does not indicate any 

potential for adverse effects in humans following the consumption of SDA soybean oil 

under the conditions of intended use in foods. Following a critical evaluation of the 

scientific data generally available in the public domain that pertain to the safety of SDA 

soybean oil, a panel of experts, qualified by scientific training and experience to evaluate 

the safety of SDA soybean oil as a component of food, unanimously concluded that the 

proposed uses of SDA soybean oil are safe and suitable and are GRAS, based on 

scientific procedures. Therefore, 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 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, (ii) 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 nutritional 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. Berdeux. 1996. Trans Polyunsaturated Fatty Acids: 

Occurrence and Nutritional Implications. Ann. Appl. Lipid Res. 2:1-33. 

EC. 2008. Commission of the European Communities. Commission Decision of 27 June 

2008 authorising the placing on the market of refined Echium oil as novel food ingredient 

under Regulation (EC) No 258/97 of the European Parliament and of the Council 

(notified under document number C(2008) 3049) (Only the English text 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 Acid- Healthy Canadian Ingredient. 

 

11 Substances Affirmed as Generally Recognized as Safe: 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. 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. 

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. Sebedio. 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 atopic mothers: Comparison with non-atopic 

mothers, and effect of borage 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 acids in breast milk decrease 

the risk of HIV transmission through breastfeeding. Am. J. Clin. 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 Assessment 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 control (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 test and control seed was 

verified by event-specific polymerase chain reaction. The results of these analyses were 

as expected. 

G.2. Field Sites and Experimental Design 

Phenotypic and agronomic data were collected from 21 field locations over two 

consecutive years: 17 locations in 2006 and four locations in 2007. Table VIII-4 

provides a list of the field site locations (including site codes). These locations provided 

a diverse range of environmental and agronomic conditions representative 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 sites was established in a randomized complete 

block design with three 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 variety. 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, NE2, 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 fifth row of each plot. The plots within each 

replicate were separated by approximately 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 established 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 approximately 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 cropping history of the study area at each site 

are listed in Table G-7. Agronomic practices used to prepare and maintain the trial at 

each site were characteristic of the respective 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 characteristics were evaluated at each site in 

2006 and 2007: early stand count, seedling vigor, plant growth stages, 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 designated developmental stages where observations 

occurred are listed in Table VIII-1 of Section VIII.A. 

Analysis of the phenotypic and agronomic data from the 2006 and 2007 field trials were 

conducted separately. An analysis of variance 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 each site (individual-site analysis) and in a 

combined-site analysis, in which the data were 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 test weight, and yield. Growth stage, flower 

color, and plant pubescence data were categorical and were not statistically analyzed. 

For each assessed characteristic, the minimum and maximum means were determined 

from among the reference soybean varieties (reference range) planted at the sites to 

provide values that are representative of commercial soybean varieties. The following is 

a summary of the results from the 2006 and 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 Phenotypic and Agronomic Results 

In the individual-site analysis of the data from the 2006 field trials, a total of 22 

statistically significant differences were detected out of 165 site × characteristic 

comparisons between MON 87769 and the control (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 significant differences were not indicative of 

a consistent plant response associated with the trait and are unlikely to be biologically 

meaningful in terms of increased 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-3). Furthermore, no differences 
Monsanto 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 trials, 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 statistical 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 with the trait and is 

unlikely to be biologically meaningful in terms of increased 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 growth 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 Materials Assessed in the 2006 Field Trials 

Material

Material 

type1 Sites2,3

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 conventional 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 all field sites; the soybean reference varieties were 

planted at specific sites. 

3

 IA1 = Jefferson County, IA; IA2 = Benton County, 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 Materials Assessed in the 2007 Field Trials 

Materials Material type1 Sites2,3

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 conventional 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 all field sites; the reference varieties were planted 

at specific sites. 

3

 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 

Table G-3. Phenotypic and Agronomic Comparison of MON 87769 to the Conventional Control in the IndAnalysis for the 2006 Field Study  Phenotypic Characteristic (units)  Early stand count (#/plot)

Seedling vigor 

(1-9 scale) Days to 50% flowering1 Flower colo

Site

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 

87769 Cont

IA1 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 Purp

IA2 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 Purp

IA3 225.7 (11.6)* 193.7 (9.7) 2.3 (0.3) 2.3 (0.3) 205.0 (0.6) 206.0 (0.0) Purple PurpIA4 106.3 (8.4) 106.0 (14.7) 4.0 (0.6) 4.0 (0.6) 197.0 (0.0)† 197.0 (0.0) Purple Purp

IL1 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 Purp

IL2 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 Purp

IL3 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 Purp

IN1 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 Purp

IN2 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 Purp

KS 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 Purp

MI 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 Purp

MO1 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 Purp

MO2 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 Purp

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 Purp

NE2 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 Purp

OH 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 Purp

WI 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 Purp

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). † No statistical comparisons were made due to lack of variability in the data. 

1 Calendar day number when approximately 50% of the plants in each plot were flowering. 2 Flower color and plant pubescence data were categorical and were not statistically analyzed. 3 Plant pubescence data from the MO2 site was eliminated from the study because the data were collected incorrectly. 
Monsanto Company 09-SY-200U 

Table G-3 (continued). Phenotypic and Agronomic Comparison of MON 87769 to the Conventional ContrSite Analysis for the 2006 Field Study

Plant Characteristic (units)

Plant Height (in)

Lodging 

(0-9 scale) Pod Shattering (0-9 scale) Final Stand Count (

Site 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Co

Mean 

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)* 210IA2 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

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) 204IA4 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) 90IL1 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) 29IL2 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) 263IL3 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) 196IN1 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) 267IN2 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) 136KS 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) 136MI 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) 170MO1 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) 284MO2 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) 163NE 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) 214NE2 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) 143OH 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) 209WI 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

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). † No statistical comparisons were made due to lack of variability in the data.
Monsanto Company 09-SY-200U 

Table G-3 (continued). Phenotypic and Agronomic Comparison of MON 87769 to the Conventional ContrSite Analysis for the 2006 Field Study

Plant Characteristic (unit)

Seed Moisture (%) 100 Seed Weight (g) Test Weight (lb/bu)1 Yield (bu/ac)1

Site 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Co

Mean 

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) 44IA2 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) 57IA3 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) 52IA4 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) 59IL2 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) 68IL3 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) 28IN1 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) 65IN2 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) 65KS 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)* 70MI 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) 54MO1 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) 47MO2 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) 39NE 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) 51NE2 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) 67OH 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) 48WI 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)* 70Note: 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). 1 Test weight and yield data from the IA4 site were excluded from the data analysis because an inaccurate measuring device was used t
Monsanto Company 09-SY-200U 

Table G-4. Plant Growth Stages of MON 87769, the Conventional Control, and Reference Varieties at 17 

Date and Range of Growth Stages Observed2

Site Substance1 Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 OIA1 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 R1 R2 R3 R5 R6 R8 R8  References V2 R1 R2 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. 1 Control = conventional variety A3525. 2 Month-day-year. 

— Information not available.
Monsanto Company 09-SY-200U 

Table G-4 (continued). Plant Growth Stages of MON 87769, the Conventional Control, and Reference Vathe Growing Season at 17 Sites in 2006 

Date and Range of Growth Stages Observed2

Site Substance1 Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 OIL2 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 10/1 MON 87769 V3 V6 R2 R4-R5 R6 R6-R7 R7 R7-R8  Control V3 V6 R2 R4-R5 R6 R6-R7 R7 R7-R8  References V3 V5-V6 R2 R4-R5 R6 R6-R7 R6-R7 R7-R8 IN2 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 08/28/2006 09/11/2006 09/26/2006 — 

 MON 87769 V2 R2 R3 R5 R6 R7 R8 — 

 Control V2 R2 R3 R5 R6 R7 R8 —  References V2 R2 R3 R5 R6 R6-R7 R8 — 

Obs. = Observation number. 1 Control = conventional variety A3525. 2 Month-day-year. 

— Information not available.
Monsanto Company 09-SY-200U 

Table G-4 (continued). Plant Growth Stages of MON 87769, the Control, and Reference Varieties through

Season at 17 Sites in 2006 

Date and Range of Growth Stages Observed2 

Site Substance1 Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 OMI 06/23/2006 07/07/2006 07/21/2006 08/04/2006 08/18/2006 09/04/2006 09/17/2006 09/29/2006 10/1 MON 87769 V2-V3 V5-V6 R1 R3 R5 R6 R6 R7  Control V2-V3 V6 R1 R3 R5 R6 R6 R7  References V2-V3 V6-V7 R1 R3 R5 R6 R6 R6-R8 RMO1 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-R1 R1-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 R2 R5 R6 R6-R7 R8 — 

Obs. = Observation number. 1 Control = conventional variety A3525. 2 Month-day-year. 

— Information not available.
Monsanto Company 09-SY-200U 

Table G-4 (continued). Plant Growth Stages of MON 87769, the Conventional Control, and Reference Vathe Growing Season at 17 Sites in 2006 

Date and Range of Growth Stages Observed2 

Site Substance1 Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 ONE2 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 R2 R3-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 11/0 MON 87769 V2 R1 R2 R3 R5 R5 R6 R8  Control V2-V3 R1 R1-R2 R3 R5 R5-R6 R6 R8  References V2-V3 R1 R1-R2 R3 R5 R5-R6 R6-R7 R8 

Obs. = Observation number. 1 Control = conventional variety A3525. 2 Month-day-year. 

— Information not available.
Monsanto Company 09-SY-200U 

Table G-5. Phenotypic and Agronomic Comparison of MON 87769 to the Conventional Control in the IndAnalysis for the 2007 Field Study Phenotypic Characteristic (units)  Early stand count (#/plot)

Seedling vigor 

(1-9 scale) Days to 50% flowering1 Flower colo

Site

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 

87769 Cont

IL 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 Purp

IN 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 Purp

MI 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 Purp

WI 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 Purp

Plant Characteristic (units)

Plant Height (in)

Lodging 

(0-9 scale) Pod Shattering (0-9 scale) Final Stand Count (

Site 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Co

Mean 

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) 410IN 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

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

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

Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Erro† No statistical comparisons were made due to lack of variability in the data. 1 Calendar day number when approximately 50% of the plants in each plot were flowering. 2 Flower color and plant pubescence data were categorical and were not statistically analyzed. 
Monsanto Company 09-SY-200U 

Table G-5 (continued). Phenotypic and Agronomic Comparison of MON 87769 to the Conventional ContrSite Analysis for the 2007 Field Study

Plant Characteristic (unit)

Seed Moisture (%) 100 Seed Weight (g)1 Test Weight (lb/bu) Yield (bu/ac)

Site 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

MON 87769 

Mean (S.E.) 

Co

Mean 

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 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 

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 

WI 10.7 (0.1) 10.7 (0.0) — — 56.7 (0.3) 57.0 (0.0) 42.4 (0.9) 42.4 Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Erro* 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 w

not have sufficient precision.
Monsanto Company 09-SY-200U 

Table G-6. Plant Growth Stages of MON 87769, the Conventional Control, and Reference Varieties at Fo

Date and Range of Growth Stages Observed2 

Site Substance1 Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 OIL 6/27/07 7/11/07 7/24/07 8/8/07 8/27/07 9/17/07 10/8/07 

⎯

 MON 87769 V2 V3-V4 R1-R2 R3-R4 R5 R7 R7-R8 

⎯

 Control V2 V3-V4 R1-R2 R3-R4 R5 R7 R7-R8 

⎯

 References V2 V3-V4 R1-R2 R3-R4 R5 R7 R7-R8 

⎯

IN 6/25/07 7/16/07 7/24/07 8/17/07 9/12/07 10/17/07 

⎯

⎯

 MON 87769 V3 R1 R1-R2 R3-R4 R5-R6 R8 

⎯

⎯

 Control V2-V3 R1 R1-R2 R3-R4 R6 R8 

⎯

⎯

 References V2-V3 R1 R1-R2 R3-R4 R5-R6 R8 

⎯

⎯

MI 6/20/07 7/5/07 7/18/07 8/1/07 8/14/07 8/29/07 9/12/07 9/26/07 10 MON 87769 V2 V5-V6 R2 R3 R5 R6 R6 R7 

 Control V2 V5-V6 R2 R3 R5 R6 R6 R7 

 References V2 V5-V6 R1-R2 R2-R3 R5 R6 R6 R7 WI 7/3/07 8/3/07 8/16/07 9/4/07 9/26/07 

⎯

⎯

⎯

 MON 87769 V2 R1-R2 R23 R4 R6 

⎯

⎯

⎯

 Control V2 R2 R2 R4 R6 

⎯

⎯

⎯

 References V2 R1-R2 R2-R3 R4 R6 

⎯

⎯

⎯

Obs. = Observation number. 1Control = conventional variety A3525. 2 Month-day-year. 3 This value is representative of two of the three replicates as data for one of the replicates was inadvertently not collected or recorded. 

—Information not available.
Monsanto Company 09-SY-200U 

Table G-7. Field Site Planting Information, Soil Description, and Cropping History 

2006 Field Study 

Site 

Planting 

date1

Planting 

rate 

(seeds/ft) 

Planting 

depth 

(in) 

Plot 

size (ft)2 Rows/plot Soil series, organic matter, pH 

IA1 06/09/2006 9.0 1.5 20 × 30 8 Taintor loam, 3.7%, 6.7 IA2 05/23/2006 9.0 1.5 10 × 20 4 Tama-Muscatine silty clay loam, 3-4%, 6IA3 06/10/2006 9.1 1.0 15 × 20 6 Taintor silty clay loam, 3.5%, 7.7 IA4 05/29/2006 8.0 1.5 15 × 20 6 Carion loam, 4.0%, 6.5 IL1 05/24/2006 9.0 1.5 20 × 30 8 Plano silt loam, 3.3%, 6.7 IL2 05/23/2006 9.0 2.0 10 × 20 4 Tama silt loam, 4.5%, 6.2 IL3 06/08/2006 8.0 1.0 15 × 20 6 Silt loam, 2.8%, 6.6 IN1 06/07/2006 9.0 1.0 10 × 20 4 Reesville silt loam, 2.0%, 7.4 IN2 06/01/2006 9.0 1.5 10 × 20 4 Crosby silty clay loam, 2.9%, 6.4 KS 05/23/2006 9.0 1.5 10 × 20 4 Farnum loam, 1.2%, 7.9 MI 05/26/2006 6.0 1.5 15 × 20 6 Nester loam, 1.9%, 6.2 MO1 06/15/2006 9.0 1.0 10 × 20 4 Gorin silt loam, 4.2%, 5.8 MO2 05/29/2006 9.0 1.5 20 × 30 8 Hammond silt loam, 2.4%, 6.8 NE 05/19/2006 9.0 1.0 20 × 30 8 Hastings silt loam, 3.0%, 6.6 NE2 05/23/2006 7.0 1.0 15 × 20 6 Silty clay loam, 2.7%, 6.5 OH 05/29/2006 8.0 1.0 15 × 20 6 Crosby silt loam, 2.0%, 6.6 WI 05/29/2006 10.5 1.0 15 × 20 6 Radford silt loam, 2-4%, 5.6-7.8 1 Month/day/year. 2 Width × length. 3 This study area was previously used for weed research. 

— Information not available. 
Monsanto Company 09-SY-200U 

Table G-7 (continued). Field Site Planting Information, Soil Description, and Cropping History

2007 Field Study 

Site 

Planting 

date1

Planting 

rate 

(seeds/ft) 

Planting 

depth 

(in) 

Plot 

size 

(ft)2 Rows/plot Soil series, organic matter, pH 

Cr

200

IL 5/31/07 9.0 1.5 20 × 30 8 Plano silt loam, 3.8%, 7.1 CorIN 5/25/07 9.0 1.75 20 × 30 8 Crosby silt loam, 2.9%, 6.4 CorMI 5/23/07 8.9 1.5 20 × 30 8 Nester loam, 2.1%, 6.5 CorWI 6/11/07 9.0 1.0 20 × 30 8 Radford silt loam, 1.9%, 6.7 Win

wheat/t

1 Month/day/year. 2 Width × length. 
Monsanto Company 09-SY-200U Page 338 of 395 

Appendix H. Materials, Methods, and Individual-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 reference soybean varieties produced at the 

Jefferson County, IA; Stark County, IL; and Boone County, IN sites in the 2006 field 

trials (Appendix G). The field trial at each site was established in a randomized complete 

block design with three replications. The seed of MON 87769, the control, and reference 

varieties were harvested from all three replicated plots at each of the three field 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 

Type1 

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. The 

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 absence of MON 87769 in the 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 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 plots and from five out of a total of 36 

reference plots across the three field trials contained between 0 and 1.84% MON 87769. 

In addition, seed 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 varieties were all produced from the same replicated field trials as 

MON 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 plots were low and did not negatively affect the quality of the 

study or interpretation 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 was certified to conduct seed dormancy and 

germination testing consistent with the standards established by the Association of 

Official Seed Analysts (AOSA), an internationally recognized seed testing organization 

(AOSA, 2000; AOSA, 2006). 

Seed materials of MON 87769, the control, and four 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 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 oC. The alternating temperature regimes were maintained at the 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 

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, abnormal 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 oC) was assessed periodically 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 the Monsanto Statistics Technology Center. 

Analysis of variance was conducted according 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 sub-plot was the seed material arranged in a 

completely randomized design. The data were 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 germinated for the AOSA-recommended 20/30 °C 

temperature regime), percent viable hard, percent dead, and percent viable firm-swollen 

seed. The level of statistical significance was set at the 5% probability level (p = 0.05). 

Seed from the three production sites were tested 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 interaction 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 and 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 maximum means were determined from among 

the nine reference varieties (reference range) to provide seed germination values 

representative of commercial soybean grown in the three locations. 

H.5. Individual Site Seed Dormancy and Germination Results 

MON 87769, the control, and reference seed materials were produced at three sites to 

assess germination characteristics of seed grown under various environmental conditions. 

The individual site data in Tables 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 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 regimes 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, 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). 

References 

Association of Official Seed Analysts. 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 soybean seed coat components and their 

relationship with water abosortion. 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. 18:221-224
Monsanto Company 09-SY-200U 

Table H-1. Germination Characteristics of MON 87769, the Conventional Control, and the Reference SoyProduced from each of Three Field Sites and Tested in the AOSA-Recommended Temperature Regime  Mean % (S.E.)1  IA IL Temp. Regime2 

Germination Category3 MON 87769 Control 

Ref. 

Range4 MON 87769 Control 

Ref. 

Range4 MON 87769 

20/30 °C (AOSA) 

Normal Germinated 81.5 (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) Abnormal Germinated 18.0 (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) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0)  Dead 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) 

Viable Firmswollen 0.0 (0.0) 0.0 (0.0) 0.0-0.0 0.0 (0.0) 0.3 (0.3) 0.0-0.0 0.0 (0.0) 

Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed wachambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete block and the 

seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized deseed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional co

1 Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equale

numerical rounding of the means. S.E. = Standard Error. 

2 Temperature Regime. In the alternating 20/30 °C temperature regime, the lower temperature was maintained for 16 hours and the highe

hours.

3 Germinated seed in the AOSA-recommended temperature regime were categorized as either normal germinated or abnormal germinated 

4 Minimum and maximum mean values from among four commercial reference soybean varieties produced from replicated field trials at 
Monsanto Company 09-SY-200U 

Table H-2. Germination Characteristics of MON 87769 and the Conventional Control Seed Produced fro

Field Sites and Tested in the Additional Temperature Regimes 

Temperature Regime Germination Category 

 Mean % (S.E.)1

 IA IL IN

 MON 87769 Control MON 87769 Control MON 87769 

10 °C Germinated 100.0 (0.0) 100.0 (0.0) 99.5 (0.3) 100.0 (0.0) 100.0 (0.0)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

20 °C Germinated 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

30 °C Germinated 99.8 (0.3) 99.3 (0.5) 99.8 (0.3) 99.8 (0.3) 99.5 (0.3) 

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

 Dead 0.3 (0.3) 0.8 (0.5) 0.3 (0.3) 0.3 (0.3) 0.5 (0.3) 

 Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed wachambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete block and the 

seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized deseed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional co

1 Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equale

numerical rounding of the means. S.E. = Standard Error. 
Monsanto Company 09-SY-200U 

Table H-2 (continued). Germination Characteristics of MON 87769 and the Conventional Control Seed P

of Three Field Sites and Tested in the Additional Temperature Regimes 

Temperature Regime1 Germination Category 

Mean % (S.E.)2

 IA IL 

 MON 87769 Control MON 87769 Control MON 8776

10/20 °C Germinated 99.5 (0.5) 99.5 (0.3) 99.8 (0.3) 100.0 (0.0) 100.0 (0.0)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0)  Dead 0.5 (0.5) 0.5 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)  Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 10/30 °C Germinated 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)  Dead 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)  Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

Note: Seed for the germination study were produced in Jefferson County, IA; Stark County, IL; and Boone County, IN in 2006. Seed wachambers in a split-plot design where the whole-plot treatment was seed production site arranged in a randomized complete block and the 

seed material (i.e., MON 87769, conventional control, or commercial soybean reference variety) arranged in a completely randomized deseed material (MON 87769 and control material) interactions were detected; thus, MON 87769 was only compared to the conventional co

1 In the alternating temperature regimes, the lower temperature was maintained for 16 hours and the higher temperature for eight hours. 2 Means based on four replicates (n = 4) of 100 seeds. In some instances, the total percentage of both MON 87769 and the control equale

numerical 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 growth stages. The observed abiotic stressors, 

diseases, and arthropod pests were “natural” (i.e., no artificial infestation 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 symptomology 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 plant 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, arthropod 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 beetle, 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 between slight and severe) 3 41 – 60% 

4 61 – 80% severe (symptoms damaging to plant development) 5 > 80% 

Leafhoppers (e.g., potato leafhopper)

Rating Foliar damage (%) 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 stunting 
Monsanto 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 and the IL, IN, MI, and WI sites in the 2007 

field trials. At these sites, arthropods were collected from the plants in each replicate plot 

three times during the growing season at approximately the R1 – R2, R3 – R5, and R6 – 

R8 growth stages using a beat sheet sampling method (Kogan and Pitre, 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-samples were collected from Rows # 5 and 6 

of each plot (sub-samples 1 and 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 plot. The four sub-samples were combined into 

one container that was either filled with alcohol 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 and six most abundant beneficial arthropods 

were determined for each collection 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 often varied between collections from a site 

and between sites due to differences in temporal activity and geographical distribution of 

the taxa. Results from the 2006 and 2007 arthropod abundance evaluations are 

summarized in Section VIII.D.2.2, while the data are presented in Tables I-7 through 

I-10. 
Monsanto Company 09-SY-200U Page 347 of 395 

Table I-1. Abiotic Stressor Evaluations Using 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 moisture2 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 Using 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 Using 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 blight2 4 4

Charcoal rot 3 3

Downy mildew 15 15

Frogeye leaf spot 14 13*

Fusarium 3 3

Phytophthora3 17 17

Powdery mildew 6 6

Pythium 7 7

Rhizoctonia 5 5

Sclerotinia 4 4

Septoria4 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 (none 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 Using 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 blight2 1 1

Charcoal rot 1 1

Downy mildew 3 3

Frogeye leaf spot 2 2

Phytophthora3 1 1

Powdery mildew 5 5

Pythium 2 2

Rhizoctonia 3 3

Sclerotinia 1 1

Septoria4 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 Using 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 

Aphid2 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 

Leafhopper3 15 15 

Seed corn maggot 1 1 

Soybean stem borer 3 3 

Stink bug4 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 Using 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 

Aphid2 10 10 

Bean leaf beetle 8 8 

Corn rootworm 1 1 

Grasshopper 7 7 

Green cloverworm 3 3 

Japanese beetle 7 7 

Leafhopper3 2 2 

Leafroller 3 3 

Mexican bean beetle 1 1 

Spidermite 1 1 

Stink bug4 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 

Table I-7. Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the Conventional ConReference Varieties in U.S. Field Trials in 2006 

Abundance of Pest Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Aphid IA1 2.0 (1.00) 0.7 (0.33) 0.0 – 1.7 — — — 116.7 (27.95) 

 IL1 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)  MO2 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)  NE — — — 12.7 (10.68) 3.5 (0.50) 2.5 – 10.0 20.7 (6.17) 

Bean leaf beetle 

IA1 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) 

 IL1 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) 

 MO2 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)  NE 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) 

Garden fleahopper 

IA1 — — — — — — 2.0 (0.58) 

 IL1 — — — 0.3 (0.33) 0.0 (0.00) 0.3 – 2.0 0.0 (0.00)† 

 MO2 — — — 1.0 (0.58) 1.0 (0.58) 0.7 – 1.0 0.0 (0.00)  NE — — — 0.0 (0.00) 0.0 (0.00) 0.0 – 0.5 2.3 (0.33) 

* Indicates a statistically significant difference (p

≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, 

R1

−R2, R3 – R5, and R6 – R8 growth stages, respectively. 1 Test and control values represent mean number of arthropods collected ac

S.E. = Standard Error. 2 IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE. † 

were made due to lack of variability in the data. Dash (—) indicates arthropod was not evaluated. 
Monsanto Company 09-SY-200U 

Table I-7 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769

Control, and the Reference Varieties in U.S. Field Trials in 2006 

Abundance of Pest Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Green 

 cloverworm 

IA1 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) 

 IL1 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)* 

 MO2 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)  NE 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) 

Leafhopper 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)  IL1 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)  MO2 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)  NE 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) Looper 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)† 

* Indicates a statistically significant 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. 

1 Test and control values represent mean number of arthropods collected across three replications. S.E. = Standard Error. 2 IA1 = 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 

Table I-7 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769

Control, and the Reference Varieties in U.S. Field Trials in 2006 

Abundance of Pest Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Stink 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) 

 IL1 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)  MO2 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)  NE 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) 

Tarnished plant bug 

IA1 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) 

 IL1 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)  MO2 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)† 

 NE 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) 

Velvetbean caterpillar 

IA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.3 — — — 1.3 (0.88) 

 IL1 0.3 (0.33) 0.0 (0.00) 0.0 – 1.3 — — — —  MO2 — — — — — — 0.0 (0.00)† 

* Indicates a statistically significant difference (p

≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, 

R1

−R2, R3 – R5, and R6 – R8 growth stages, respectively. 1 Test and control values represent mean number of arthropods collected ac

S.E. = Standard Error. 2 IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE. † 

were made due to lack of variability in the data. Dash (—) indicates arthropod was not evaluated. 
Monsanto Company 09-SY-200U 

Table I-8. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, the Coand the Reference Varieties in U.S. Field Trials in 2006 

Abundance of Beneficial Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Big-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)  NE 1.3 (0.67) 0.0 (0.00) 0.0 – 0.5 — — — — Ladybird beetle 

IA1 0.0 (0.00) 0.0 (0.00) 0.0 – 0.7 — — — 0.0 (0.00) 

 IL1 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) 

MO2 0.3 (0.33) 0.0 (0.00) 0.0 – 1.3 — — — 0.7 (0.33) 

NE — — — 0.7 (0.67) 0.5 (0.50) 0.0 – 2.0 0.3 (0.33) 

Micro parasitic hymenoptera 

IA1 — — — 2.0 (1.15) 0.7 (0.67) 0.7 – 1.0 4.0 (0.58) 

IL1 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) 

 MO2 — — — 0.0 (0.00) 1.0 (1.00) 0.0 – 1.7 4.3 (1.45) 

NE 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) 

* Indicates a statistically significant difference (p

≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, 

R

−R2, R3 – R5, and R6 – R8 growth stages, respectively. 1 Test and control values represent mean number of arthropods collected acr

= Standard Error. 2 IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.Dash (—

was not evaluated. 
Monsanto Company 09-SY-200U 

Table I-8 (Continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON Conventional Control, and the Reference Varieties in U.S. Field Trials in 2006 

Abundance of Beneficial Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Nabis IA1 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) 

 IL1 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)*  MO2 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)  NE 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) 

Orius IA1 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) 

 IL1 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)  MO2 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) 

Spider 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) 

 IL1 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)  MO2 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)  NE 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) 

* Indicates a statistically significant difference (p

≤0.05) between MON 87769 and the conventional control (A3525). Collections 1, 2, 

R1

−R2, R3 – R5, and R6 – R8 growth stages, respectively. 1 Test and control values represent mean number of arthropods collected ac

S.E. = Standard Error. 2 IA1 = Jefferson County, IA; IL1 = Stark County, IL; MO2 = Lincoln County, MO; NE = York County, NE.Da

arthropod was not evaluated. 
Monsanto Company 09-SY-200U 

Table I-9. Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769, the Conven

the Reference Varieties in U.S. Field Trials in 2007 

Abundance of Pest Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 8776

Mean (S.E

Aphid IL 7.0 (2.6) 11.0 (5.9) 9.7 – 23.0 — — — 0.0 (0.0) 

 MI 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 leaf beetle 

IL 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) 

 IN 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) 

 MI 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) 

 WI 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) 

Corn flea beetle 

IN — — — — — — 0.3 (0.3) 

Garden fleahopper 

WI — — — 10.3 (4.3) 3.7 (1.5) 7.0 – 12.3 1.3 (0.7) 

* Indicates a statistically significant 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. 

1 Test and control values represent mean number of arthropods collected across three replications. S.E. = Standard Error. 2 Data 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 

Table I-9 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769

Control, and the Reference Varieties in U.S. Field Trials in 2007 

Abundance of Pest Arthropod1

Collection 1 Collection 2 

Arthropod Sites2

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Green cloverworm 

IL 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) 

 IN 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) 

 MI 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)  WI 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)* 

Japanese beetle 

IN 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 statistically significant 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. 

1 Test and control values represent mean number of arthropods collected across three replications. S.E. = Standard Error. 2 Data 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 

Table I-9 (Continued). Abundance of Pest Arthropods in Beat Sheet Samples Collected from MON 87769

Control, and the Reference Varieties in U.S. Field Trials in 2007 

Abundance of Pest Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.)

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Stink 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) 

 IN 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) 

 MI 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)  WI 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) 

Thrips IL 139.3 (61.9) 105.7 (61.8) 135.0 – 174.3 — — — — 

Tarnished plant bug 

IN — — — — — — 0.0 (0.0) 

 MI — — — — — — 11.0 (3.1)  WI 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) 

Western corn rootworm 

IN 1.3 (0.9) 1.7 (1.2) 1.0 – 2.3 — — — — 

* Indicates a statistically significant 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. 

1 Test and control values represent mean number of arthropods collected across three replications. S.E. = Standard Error. 2 Data 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 

Table I-10. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON 87769, the CControl, and the Reference Varieties in U.S. Field Trials in 2007 

Abundance of Beneficial Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Carabid beetle 

IL — — — — — — 1.0 (0.0) 

Lacewing 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)  IN — — — 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) 

WI 5.3 (1.5) 3.0 (2.0) 2.0 – 4.7 0.7 (0.7) 1.3 (0.9) 0.3 – 2.0 — 

Ladybird beetle 

IL — — — 0.0 (0.0) 0.3 (0.3) 0.0 – 3.7 2.0 (0.6) 

IN — — — 0.0 (0.0) 0.3 (0.3) 0.0 – 1.7 0.0 (0.0) 

 MI 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)* 

WI 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) 

* Indicates a statistically significant 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. 

1 Test and control values represent mean number of arthropods collected across three replications. S.E. = Standard Error. 2 Data 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 

Table I-10 (Continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON Conventional Control, and the Reference Varieties in U.S. Field Trials in 2007 

Abundance of Beneficial Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Microparasitic hymenoptera 

IL 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) 

WI 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) 

Nabis IL 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) 

 IN 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)  MI 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)  WI 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) 

Orius IL 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) 

 IN 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)  MI 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)  WI 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) 

* Indicates a statistically significant difference (p

≤0.05) between the test (MON 87769) and the conventional control (A3525). Collecti

at the R1 – R2, R3 – R5, and R6 – R8 growth stages, respectively. 1 Test and control values represent mean number of arthropods colle

replications. S.E. = Standard Error. 2 Data were collected from the following field sites: Stark County, IL; Boone County, IN; Ottawa 

County, WI. Dash (—) indicates arthropod was not evaluated. 
Monsanto Company 09-SY-200U 

Table I-10 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from MON Conventional Control, and the Reference Varieties in U.S. Field Trials in 2007 

Abundance of Beneficial Arthropod1

Collection 1 Collection 2 

Arthropod Sites2 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Control 

Mean (S.E.) 

Reference 

Range 

MON 87769 

Mean (S.E.) 

Spined soldier bug 

IL 0.0 (0.0) 0.3 (0.3) 0.0 – 0.7 — — — — 

 IN — — — — — — 0.7 (0.7) Spider 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) 

 IN 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)  MI 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) 

 WI 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) 

* Indicates a statistically significant 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. 

1 Test and control values represent mean number of arthropods collected across three replications. S.E. = Standard Error. 2 Data 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 (eds.). Springer-Verlag, New York, United 

States of America. 
Monsanto Company 09-SY-200U Page 365 of 395 

Appendix J. Materials and Methods from 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 similar agronomic conditions in a randomized 

complete block design with three replications at a field site in Lincoln County, Missouri. 

Each plot consisted of eight 30 ft long rows spaced approximately 30 inches apart. 

Material 

Type

Material 

Name1 

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 fourth row of each plot. One flower from the 

bottom of the plant, two flowers from the middle, and one flower from the top were 

collected from each of five representative plants per plot. 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, all flowers from a plot were placed into a 

clean, uniquely labeled container. The containers were kept on wet ice for less than eight 

hours until the pollen was extracted 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 pollen into the circle on the slide. The utensil 

was cleaned between extractions of the pollen from the flowers of another plot. 

Approximately 20 μl of Alexander stain (Alexander, 1980) was added to the center of the 

circle containing the pollen. The pollen was stained at ambient temperature for at least 

ten minutes prior to examination. All pollen grains 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 digital color camera. The microscope and 

camera were connected to a computer running Microsoft Windows 2000 Professional 

(© 1981-1999, Microsoft Corp.) and installed with associated camera software [DP 

Controller v1.2.1.108 and DP Manager v1.2.1.107(© 2001-2003, Olympus Optical Co., 

Ltd.)] and imaging software [Image-Pro Plus v4.5.1.27 (© 1993-2002, Media 

Cybernetics, Inc.)]. 

Pollen Viability 

Pollen viability was evaluated for each sample by counting the number of viable and nonviable pollen grains. When exposed to the 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 collapsed in shape, 

depending on the degree of hydration. At least 75 individual pollen grains per plot were 

evaluated for viability from a random field of view under the microscope. Dense clusters 

of pollen or pollen grains adhering to flower parts were not counted because they may not 

have absorbed the stain solution uniformly. 

Pollen Diameter

Micrographs (400X magnification for replication 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 software was used to measure pollen grain 

diameter along two perpendicular axes for each selected pollen grain. Mean pollen 

diameter for each plot was calculated from the 20 total measurements (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) according to a randomized complete block 

design with three replications. Statistical significance was set at the 5% probability level 

(p = 0.05). MON 87769 was compared to the control for percent viable pollen and pollen 

diameter. No statistical comparisons were made between MON 87769 and the reference 

varieties. A reference range for each measured characteristic was determined from the 

minimum and maximum mean values from among the four reference soybean varieties. 

General pollen morphology was qualitative; therefore, no statistical 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 versatile 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 for 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 test and control starting seed was verified 

by event-specific polymerase chain reaction. The results of these analyses were as 

expected. The identity of the reference substances 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 oC. 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, vermiculite, and perlite. 

Four seeds of MON 87769, the control, or reference variety were inoculated with 

approximately 1 x 108 cells of B. japonicum in phosphate-buffered saline and planted in 

each pot. An additional eight replications (for each of two sampling periods) of pots 

were planted with uninoculated control material seed and included in the study to validate 

the test system. Pots were arranged in eight replicated blocks in the greenhouse for 4- 

and 6-week sampling periods using a randomized strip-plot design. 

One week after emergence, all pots were thinned to one seedling per pot and reinoculated with approximately 1 x 108 cells of B. japonicum, with the exception of the 

uninoculated control pots. A solution of nitrogen-free nutrient solution (target of 250 
Monsanto 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 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 enumerated 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 minimum 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 shipping to Agvise Laboratories (Northwood, ND) for 

total nitrogen analysis. Shoot total nitrogen was determined by combustion using a 

nitrogen analyzer. 

K.1.4. Statistical Methods 

An analysis of variance was conducted using a randomized strip-plot design with eight 

replications for each test, control, and reference material at each sampling period. Data 

were analyzed 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 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 weight). The commercial reference varieties provided a range 

of values for each measured endpoint that are representative of commercial soybean 

varieties. A summary of the results is provided 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 nitrogen-deficient test system (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 (A3525) for Nodule Number and Shoot Total 

Nitrogen 

Measurement 

Endpoint 

Sampling 

Period 

A3525 

(Mean)

A3525-

UN 

(Mean) 

Standard 

Error p value Treatments 

SSD1

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 Conventional 
Monsanto Company 09-SY-200U Page 371 of 395 

K.2.2. Characterization of Materials 

The presence or absence of MON 87769 in the test and control starting seed was verified 

by event-specific polymerase chain reaction. The results of these analyses were as 

expected. The identity of the reference substances 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 oC, and a target night-time temperature of 22 oC. At planting, 

eight replications of 6-inch pots were filled with a nitrogen-deficient potting medium 

composed of primarily peat, vermiculite, and perlite. Three seeds of MON 87769, the 

conventional control, negative isoline, or reference soybean variety were inoculated with 

approximately 1 x 107 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 sampling 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 excised at the surface of the potting medium and 

shoot and root plus nodule material were removed from the pots. The shoot material was 

cut into smaller pieces and placed in labeled bags. The plant roots with nodules were 

separated from the potting medium by washing with water. Excess moisture was removed 

using absorbent paper towels and the roots plus nodules were placed in labeled bags. The 

nodules were later removed by hand from the roots of each plant and placed in weighing 

tins. Nodules from each plant were enumerated 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 minimum 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 Laboratory (Fort Wayne, IN) for total nitrogen 

analysis. Shoot total nitrogen was determined by thermal conductance using a nitrogen 

analyzer. 

K.2.4. Statistical Methods 

An analysis of variance was conducted using a randomized complete block design with 

eight replications. Data were analyzed 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 and the negative isoline control for six 

measurement endpoints in separate pairwise comparisons: nodule number, nodule dry wt 

(g), shoot dry wt (g), root dry wt (g), and shoot nitrogen (percent and 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 representative of commercial soybean varieties. A 

summary of the results is provided 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 Environmental Policy Act (NEPA) when making 

the decision whether to grant nonregulated status to MON 87769. MON 87769 has been 

the subject of numerous field trials conducted in the U.S. under APHIS notifications and 

permits since 2003. Information has been developed from these field trials, other tests, 

and the public literature to specifically assess whether the alteration in the soybean oil 

profile through the introduction of two desaturase genes, Primula juliae Δ6 desaturase 

(Pj.D6D) and Neurospora crassa Δ15 desaturase (Nc.Fad3) that encode for the PjΔ6D 

and NcΔ15D proteins, the presence of stearidonic acid (SDA) and/or the plant 

transformation process altered MON 87769 in any way that would impart plant pest 

characteristics or cause significant environmental impacts, including cumulative impacts. 

The purpose of this section is to provide relevant, trait-specific information regarding the 

potential for reasonably foreseeable, significant environmental impacts. 

An analysis of the potential impact of deregulation of MON 87769 on current soybean 

agronomic production systems, and related activities such as soybean processing, food 

and feed uses as well as marketing of soybean and soybean products is presented in this 

section. Factors evaluated as part of the assessment include potential impacts to: 

• land use patterns, non-agricultural lands, farming practices, commodity and 

specialty soybean production, 

• marketability of soybean seed for planting and seed for specialty and commodity 

markets, and 

• public health, impacts to non-target organisms, 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 between 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 conventional 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 the needed intake of 

long chain omega-3 fatty acids. The American Heart Association (AHA) has 

recommended a dietary intake of 1 gram/day of omega-3 fatty acid for heart patients and 
Monsanto Company 09-SY-200U Page 374 of 395 

the incorporation of two servings of fatty fish per week for healthy 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 commercialized as an alternate 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 ALA 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 gram 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 require the action of delta-6 desaturase for 

conversion to EPA. As a result, conversion of SDA to EPA is much more efficient (see 

Appendix F). 

The development of MON 87769 involved the introduction of two desaturase genes, 

Primula juliae Δ6 desaturase (Pj.D6D) and Neurospora crassa Δ15 desaturase (Nc.Fad3) 

that encode for the PjΔ6D and NcΔ15D proteins. The genes and subsequent enzymes 

responsible for production of SDA in MON 87769 are derived from plant and algal 

sources and are not from fish. 

Soybean plants lack the Δ6 desaturase gene, which is a minimal requirement for the 

production of SDA. However, Δ6 desaturase also converts LA to GLA. The addition of 

a Δ15 desaturase with temporal expression similar to the Δ6 desaturase increases the flux 

of ALA to SDA and lowers the substrate pool for GLA production. To produce SDA in 

soybean, the conventional soybean variety A3525 was transformed 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 the developing soybean seed. The oil from 

MON 87769 contains 20% to 30% SDA (weight % of total fatty acids). 

Oil derived from MON 87769 (referred to as SDA soybean oil) is expected to help food 

manufacturers develop foods that can assist consumers in meeting the American Heart 

Association’s (AHA) goals for dietary intake of omega-3 long chain fatty acids and to 

improve heart health. SDA has been utilized in several human clinical studies. For 

example, James et al. (2003) found that by incorporating SDA in the diet at 1500 mg/day, 

EPA levels in red blood cells increased significantly and conversion was several-fold 

more efficient for SDA than for ALA. Harris et al (2007) found that SDA enriched the 

red blood cells and cardiac lipids with EPA in dogs. Likewise, Harris et al. (2008) found 

in a human clinical study using SDA soybean oil over a 16 week period, that there was a 

significant increase in red blood cell EPA levels whereas none was observed for 

participants using only conventional 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 aquaculture 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 proposed deregulation would be relevant to the production of 

an intensively cultivated row crop – soybean. Soybean is grown as a commercial crop in 

over 35 countries. In the United States, it is generally grown on greater than 70 million 

acres in at least 27 states with over a million acres grown in each of the following states: 

IA, IL, MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, NC, KY, and TN (USDANASS, 2006a-b). On the basis of predicted 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 acreage for its meal which is largely used for 

animal feed and for oil that is widely used in commercial processed foods (Sonka et al., 

2004). Approximately 12% of soybean grown 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 linolenic and high protein (ASA, 2009). These 

soybean varieties are specified by buyers and end-users of soybean for production, and 

premiums are paid for delivering a product that meets purity and quality standards 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 (IDP), including closedloop systems, and quality assurance processes (e.g., ISO9001-2000 certification), as well 

as contracts between growers and buyers that specify delivery agreements within product 

specifications. 

This deregulation is being sought in an environment that has rapidly adopted 

biotechnology-derived soybean varieties (James, 2008). Thirteen different 

biotechnology-derived soybean crop products have been deregulated by USDA since 

1994. Biotechnology-derived herbicide-tolerant 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 breeders, 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 systems that allow for the concurrent breeding 

and manufacture of biotechnology-derived and specialty soybean and are capable of 

breeding, manufacturing seed, and producing harvested seed to meet the 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 (Event DP-305423-1) and has granted nonregulated status to 

Laurate canola (Events pCGN3828-212/86 18 & 23) with modified oil characteristics, 

and there are other modified oil soybean products produced by conventional breeding on 

the market. 

In recent years, there has been an increased demand by consumers and food processers 

for soybean and other oilseed crops that have specific physical or chemical characteristics 

to meet specific food or feed needs. Traditional plant breeding has been used to alter the 

fatty acid profiles of oil producing plants such as soybean. For example, soybean 

varieties with lower levels (1-3%) of the polyunsaturated linolenic fatty acid have 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 possibly 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 incorporation into food applications where it is: 1) 

functionally suitable (discussed below); 2) provides acceptable sensory properties 

(reduced off-flavors relative to longer chain omega-3 fatty acids) to foods and feeds; and 

3) gives an appropriate stability profile and shelf life during food and feed production and 

storage. As expected, the SDA soybean oil from MON 87769 contains high levels of 

SDA (approximately 20-30 weight % of total fatty acids), a polyunsaturated fatty acid. 

Vegetable oils containing high levels of polyunsaturated fatty acids, such as oil from 

MON 87769, are known to undergo rapid oxidation 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 categories. 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 products, frozen dairy 

desserts and mixes, grain products and pastas, gravies and sauces, meat products, 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 (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 containing these oils. Through the process of 

hydrogenation, the chemical structure of the fatty acids are changed. In the case of SDA 

soybean oil, hydrogenation would convert SDA (18:4) to other fatty acids present in 

soybean oil (e.g. 18:3, 18:2, and 18:1) and thereby eliminate the benefits 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 suitable 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). These value-added products are typically produced using IDP 

systems that include contracts between all handlers of soybean (seed suppliers, growers, 

handlers, and processors), traceability, product tracking, process verification and separate 

channeling to minimize commingling. Monsanto has introduced the value-added low 

linolenic Vistive® soybean, developed via conventional 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 activities including land use patterns, farming 

practices, commercial activities such as seed production and marketing of soybean and 

impacts to the quality of agricultural products. 

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 acreage in the past five years has been 

relatively stable varying from 64.7 to 75.7 million acres with a 10-year average of 73.3 

million acres. Fluctuations in soybean acreage are due to environmental, agronomic and 

economic factors13, as well as government programs such as the crop reserve program 

(CRP) or ethanol mandates imposed by the U.S. government. Soybean fields are 

typically highly managed agricultural areas that can be expected to be dedicated to crop 

production for many years and cultivation of MON 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 value 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 edible beans, potatoes, canola, proso millet, 

and sugar beets, has remained relatively constant over the past 25 years. From 1983 to 

1995, the average yearly acreage of principal crops was 328 million. This average is 

statistically unchanged at 326 million acres since the introduction of biotechnology-

 

® 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 

6/2009 
Monsanto Company 09-SY-200U Page 378 of 395 

derived crops in 199614. In fact, the total U.S. cropland 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 crops, 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 possibly 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 nonregulated status to MON 87769 is not expected to have a 

significant impact to land use for the following 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 conventional soybean. MON 87769 will be grown on land 

already devoted to soybean production replacing 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 soybean. Based on these reasons, Monsanto 

does not consider that deregulation of MON 87769 will result in significant impacts to 

land use. 

A decision to deregulate MON 87769 similarly is not expected to result in any 

cumulative impacts to land use. It is foreseeable 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, including non-biotechnologyderived, certified seed, organic food-grade, low saturated fat, clear hilum, tofu, natto, 

high sucrose, high oleic, low linolenic and high protein (ASA, 2009). Adoption and 

continued use of these specialty soybean varieties 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 wild in the U.S. (Hymowitz and Singh, 1987; CFIA, 1996; 

OECD, 2000). Soybean does not grow and persist in unmanaged habitats and would not 

be expected to invade and/or persist in the natural environment, including streams, lakes, 

oceans or other aquatic environments. With the exception of production of the PjΔ6D 

and NcΔ15D 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 other commercial soybean varieties currently 

grown in the U.S. including both conventional 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 land 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 applications on MON 87769 that may result in drift 

impacting non-crop or nonagricultural lands would be comparable to those used on 

conventional or currently available soybean varieties. 

L.D.3. Potential Impacts to Agricultural Practices 

MON 87769 has been shown to be no different from conventional soybean in its 

agronomic and ecological characteristics, and has the same levels of resistance to insects 

and diseases as current commercial soybean. 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. Specifically, MON 87769 requires no changes to current cultivation 

practices, including seeding, tillage, fertilizer and pesticide applications and crop rotation 

practices. Growers will utilize best practices associated with production and identity 

preservation (IDP) of harvested soybean similar to those currently used for other 

specialty soybean varieties. 

Potential Impact to Soybean Commodity and Specialty Soybean Production 

Deregulation of MON 87769 would allow for unconfined commercial planting of 

varieties containing MON 87769 and would provide an alternative commercial use of 

soybean oil. It would also provide growers with a higher value crop resulting in greater 

profit potential for U.S. soybean producers. Soybean growers and processors are 

accustomed to accommodating a diversity of specialty soybean varieties in their normal 

handling of commodity soybean. Currently specialty soybean varieties are produced on 

approximately 12% of the U.S. soybean acreage. Soybean growers have 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 segregated from commodity soybean by 

producers as part of their contract and to maintain the value of the crop. Because 

soybean is primarily a self-pollinated crop with minimal gene movement and due to the 

identity preservation production system, no significant impact to commodity or other 

specialty soybean production is anticipated, should APHIS grant nonregulated status to 

MON 87769. 

No cumulative impacts to commodity or other specialty soybean production are expected 

from deregulation. Soybean producers have demonstrated their ongoing ability to 

produce multiple specialty soybean varieties in a diverse marketplace. 
Monsanto Company 09-SY-200U Page 380 of 395 

Potential Impact to Certified Seed Production 

Certified seed production is a carefully managed process. MON 87769 is not expected to 

impact certified seed production practices or production of other certified conventional, 

specialty or organic soybean seed for the reasons described in this section. As discussed 

elsewhere, MON 87769 is expected to be a small acre specialty product grown on less 

than five percent of total soybean acres. The amount of acreage needed to supply seed 

for five percent of the total soybean acreage 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 production could occur within production systems 

already developed by seed producers for certified 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 practices to avoid pollen 

from a biotechnology-derived crop in organic, specialty or conventional soybean seed or 

commodity seed production operations is directly impacted by the nature of soybean 

pollination. Soybean is a highly self-pollinated species that exhibits very low levels of 

outcrossing. When soybean plants were grown 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 were 0.41 and 0.03% at 0.9 and 5.4 m, respectively. Hence, 

certified soybean seed producers can and have effectively implemented practices (e.g., 

isolation distances during the growing season, equipment cleaning during harvest, and 

post-harvest separation of harvested seed) that allow them to maintain commercially 

acceptable levels of varietal purity. 

No cumulative effects are anticipated to certified seed production from deregulation of 

MON 87769. The use of management practices that prevent trait movement and 

comingling of soybean varieties has resulted in production of soybean varieties with 

improved genetics over time. As a result of the implementation of these management 

practices, growers today can choose from numerous varieties of soybean including those 

used to produce commodity and specialty soybean varieties. 

Potential Impacts to Organic Soybean Production 

Organic soybean markets typically enjoy a market premium offsetting the additional 

production and record-keeping costs associated with this production method. Organic 

farming operations as described by the National Organic Program, which is administered 

by USDA’s Agricultural Marketing Service, requires organic production operations to 

have distinct, defined boundaries and buffer zones to prevent unintended contact with 

prohibited substances or products of excluded methods from adjoining land that is not 

under an organic production management plan. Organic production operations must also 

develop and maintain an organic production system plan approved by an accredited 

certifying agent. This plan enables the production operation to achieve and document 

compliance with the National Organic Standards, including the prohibition of the use of 

excluded methods. Excluded methods include a variety of methods used to genetically 

engineer organisms or influence their growth and development by means that are not 
Monsanto 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 excluded method under the National Organic Program 

[7 C.F.R. § 205.2]. 

Organic certification involves oversight by an accredited certifying agent of the materials 

and practices used to produce or handle an organic agricultural product. This oversight 

includes an annual review of the certified operation’s organic system plan and on-site 

inspections of the certified operation and its records. Although the National Organic 

Standards prohibit the use of excluded methods, they do not require testing of inputs or 

products for the presence of excluded methods. The presence of a detectable residue of a 

product of excluded methods alone does not necessarily constitute a violation of the 

National Organic Standards. The unintentional presence of the products of excluded 

methods will not affect the status of an organic product or operation when the operation 

has not used excluded methods and has taken reasonable steps to avoid contact with the 

products of excluded methods as detailed in an approved organic system plan. Organic 

certification certifies that organic production and handling processes have been followed, 

not that the product itself is “free” from any particular substance. 

Production systems designed prior to the introduction of MON 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-preserved specialty soybean with such traits as 

clear hilum or high protein have also been grown and successfully marketed for specific 

food uses in domestic and export markets for many years (Zhanglin et al., 2004). The 

choice to grow biotechnology-derived, organic or conventional soybean depends on 

many factors and the dynamics of the marketplace. The dynamics of the marketplace, 

choice between various varieties of soybean, and the existing production practices will 

not be impacted by the introduction of MON 87769. 

Organic soybean producers utilize production practices designed to specifically avoid the 

presence of both soybean products using conventional herbicide or other pesticide 

treatments, as well as biotechnology-derived crops. These well established practices to 

avoid “excluded methods” will continue with the introduction of MON 87769 varieties. 

They include isolation zones, use of buffer rows surrounding the organic crop, adjusted 

planting dates and varietal selection (www.attra.ncat.org). The implementation of 

management practices to avoid pollen from a biotechnology-derived crop in organic or 

conventional soybean production operations is facilitated by the nature of soybean 

pollination. As noted previously in this petition, soybean is a highly self-pollinated 

species and exhibits very low level of outcrossing. Hence, organic or conventional 

soybean producers can and have effectively implemented practices (e.g., isolation during 

the growing season, equipment cleaning during harvest, and post-harvest separation of 

harvested seed) that allow them to avoid the presence of biotechnology-derived soybean 

and maintain organic or conventional production status. 

Currently, biotechnology-derived herbicide tolerant soybean is planted on 92% of the 

soybean acreage (USDA-ERS, 2008) and the Roundup Ready soybean system – that is, 

planting Roundup Ready soybean and applying glyphosate in crop – has become the most 
Monsanto 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 growers, organic and conventional soybean 

production remains an option for farmers who choose to produce using these production 

practices or varieties of soybean. The decision to grow organic, conventional, or 

biotechnology-derived soybean varieties is typically an economic one based on market 

dynamics. Organic soybean producers and those growing conventional soybean for 

sensitive non-biotechnology markets typically enjoy a market premium offsetting the 

additional production and record-keeping costs. While the widespread adoption of 

Roundup Ready soybean has reduced the number of conventional soybean varieties that 

are available, conventional and organically produced soybean seed is currently available 

from numerous seed suppliers (Table L-1). Additional information on organic seed 

sources is provided though the U.S. Agricultural Marketing Service (AMS) at: 

www.ams.usda.gov. Thus, growers have a choice in the soybean variety they plant, and 

this is not expected to change with the introduction of MON 87769. 

Buyers recognize that where there are biotechnology-derived crop varieties on the market, 

as with soybean, a guarantee that a commodity crop is 100% “free” of 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 where producers utilize production practices 

designed to avoid biotech presence, in some instances buyer allowances between 0.1 to 

5% biotechnology-derived commodity soybean in organic soybean are specified. This 

also is consistent with the USDA National Organic Program allowing for detectable 

residues of excluded methods (including biotechnology-derived crop products) as long as 

the producer has taken steps to avoid those methods (www.ams.usda.gov/nop/Q&A.html). 

Similarly, international regulatory organizations have recognized that testing and 

sampling methodologies limit the ability to confirm that commodity or specialty soybean 

is 100% free of biotechnology-derived material. Thus, they have set allowable tolerances 

for this material in conventional products to support food labeling and traceability laws. 

These tolerances allow from 0.9% (European Union) up to 5% (Japan) of the food to be 

biotechnology-derived in products considered “conventional.” Levels above the 

threshold may trigger special labeling. Thus, de minimis levels of approved 

biotechnology-derived soybean are allowable 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 

Organizations

* 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 susceptibility, and forage and seed composition of 

MON 87769 compared to conventional soybean varieties. 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 voluntary consultation with the FDA, and 

information has been provided to the agency 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., foreign material, damaged grain, etc; see 

www.gipsa.usda.gov for soybean grain standards), the soybean produced from 

MON 87769 varieties is expected to be of comparable quality to soybean commercially 

produced in the U.S. for commodity markets based on the data presented herein. The 

intended use of MON 87769 will be for processing 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 major fractions, the oil and meal, and several 

minor fractions including lecithin and protein isolate. With the exception of the oil 
Monsanto 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 uses as commodity soybean processed fractions. 

The meal, and other non-oil processed fractions are intended to be distributed into the 

commodity stream and will be marketed and sold in the commodity market. Because of 

their substantial equivalence, no significant 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 utilizing 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 that currently use commodity soybean oil. 

Vegetable oils containing high levels of polyunsaturated fatty acids, such as SDA 

soybean oil, are known to undergo rapid oxidation during frying applications that impart 

undesirable taste and odor to the fried foods. For this reason, undiluted SDA soybean oil 

would not be suitable for use 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 other 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 use chain to specify the characteristics of oils, and to 

identify, segregate and test those 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 uses and benefits of 

SDA soybean oil, to discuss and understand the potential impacts on commodity oils and 

processed soybean products from the IDP 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 levels 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 any, 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 nonregulated 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 outside 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 production methods assures the purity 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 food manufacturers routinely manage and handle 

a wide variety of specialty oils derived from oilseed crops (including 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 soybean varieties and would make MON 87769 

available to breeders, certified seed producers, and growers. Like other biotechnologyderived traits, it is expected that breeders and certified seed producers would use 

MON 87769 to supply seed for planned commercial markets in the U.S. Monsanto 

anticipates that commercial use of MON 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 occur through agricultural production, industrial 

operations, or through consumption. Therefore, this health and safety discussion will 

focus on food and feed safety as well as safety of workers exposed to MON 87769. SDA 

soybean oil derived from MON 87769 is intended to be commercialized as an omega-3 

oil that will replace non-hydrogenated soybean oil 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 Act (FFDCA), it is the responsibility of 

food and feed manufacturers to ensure that the products they market are safe and properly 

labeled. Food and feed derived from MON 87769 must be in compliance with all 

applicable legal and regulatory requirements. Biotechnology-derived crops for food and 
Monsanto Company 09-SY-200U Page 386 of 395 

feed use undergo a voluntary consultation process with the FDA prior to release onto the 

market. Although a voluntary process, Monsanto completes a consultation with the FDA 

prior to placing a new biotechnology derived crop product on the market. A list of 

completed consultations on biotechnology-derived crop products is available at the FDA 

website. 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 assessment of the safety of the oil produced by 

MON 87769 and a panel of independent experts 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 available in the public domain that pertain to 

the safety of SDA soybean oil, a panel of experts, qualified by scientific training and 

experience to evaluate the safety of SDA soybean oil as a component of food, 

unanimously concluded that the proposed uses of SDA soybean oil are safe and suitable 

and are GRAS, based on scientific procedures. Therefore, 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. The GRAS assessment has been submitted 

to the FDA for review (GRAS Notice No. GRN 000283). 

Soybean produced by MON 87769 contain approximately 20% to 30% stearidonic acid 

(SDA) and 7% gamma linolenic acid (GLA) (see Appendix F). Oil produced by 

MON 87769 represents a sustainable alternate source of an omega-3 fatty acid 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 marine algae and has a long standing history of 

safe consumption in humans (see Table X-1). The safety of SDA soybean oil is 

supported by the results of a 90-day/one generation reproductive toxicity rat study 

(Hammond et al., 2008). Furthermore, no adverse effects attributable to SDA soybean oil 

were reported in a published human study (Harris et al., 2008). Additional published 

studies on SDA and GLA from other 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 the other primary fatty acids present in SDA soybean oil, namely 

ALA, LA, and palmitic acid. 

As part of the consultation with the FDA 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 products. Monsanto also provided information 

on the potential allergenicity and toxicity of the expressed PjΔ6D and NcΔ15D proteins. 

The development of MON 87769 involves the introduction of two proteins (PjΔ6D and 

NcΔ15D) produced by genes from plant and fungal sources (see Section VI for details). 

These two proteins are not currently 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 dietary exposure to the PjΔ6D and NcΔ15D 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 modification, 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 nonregulated status to MON 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 availability of products that 

contain SDA from SDA soybean oil may increase the intake of omega-3 fatty acids 

thereby providing a health benefit. These effects are not considered significant because 

there are other sources of SDA and other heart 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, growers may be exposed to pesticides during 

application to the crop. Downstream processing and manufacturing 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 87769 will have no significant impact to 

agricultural workers. Similarly, there are no changes to industrial processing of 

MON 87769 relative to the processing of commercial soybean. 

SDA soybean oil derived from MON 87769 is expected to substitute for other oils 

currently used in foods and possibly in feeds. The composition of SDA soybean oil 

contains fatty acids that are already used in the food and feed industries. Since workers 

are already accustomed to handling various oils for formulation into foods, there are no 

significant impacts identified to processors or food formulators either. 

L.D.7. Potential Impacts to Plant, Animal and Microbial Communities Including 

Threatened or Endangered Species and Biodiversity 

In assessing the potential impact to plant and animal communities, the potential for gene 

movement and introgression from MON 87769 was evaluated because movement and 

establishment of the gene and trait to related species could have indirect impacts to plant 

and animal communities that extend beyond the original recipient organism. Monsanto 
Monsanto 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 animal communities is nonexistent if APHIS 

were to grant the petition for nonregulated status. 

Animals 

Soybean production systems in agriculture are host to many animal species. Mammals 

and birds may seasonally use grain, and invertebrates can feed on the plant during the 

entire growing season. Agronomic practices used to produce MON87769 will be 

substantially the same as those used to produce many types of specialty or conventionally 

grown soybean, thus this discussion for MON 87769 on animals will focus on the 

potential effects of the two introduced proteins (PjΔ6D and NcΔ15D) and the modified 

fatty acids in MON 87769. Animals that feed primarily on soybean are seed-feeding 

insects and rodents found in agricultural fields. Rodents, such as mice or squirrels, may 

seasonally feed exclusively on soybean seeds. Thus, these animals may have a diet 

containing significant amounts of soybean seeds. Deer may also browse in soybean 

fields on the forage and on seed left after harvest. 

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 containing MON 87769. As discussed previously, 

there is no meaningful risk to animal or human health from dietary exposure to PjΔ6D 

and NcΔ15D from MON 87769. There are no toxic properties associated with either the 

PjΔ6D or NcΔ15D 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) comparable to that of conventional soybean seed. 

Furthermore, forage produced by MON 87769 is compositionally equivalent to forage 

produced by conventional soybean. This information 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 SDA 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, further 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 near or in soybean fields 

containing MON 87769. 

Plants 

Soybean production systems in agriculture are host to many plant species as well. The 

landscape surrounding a soybean field varies depending on the region. In certain areas, 

soybean fields may be bordered by other soybean, corn (or any other crop); fields may 
Monsanto Company 09-SY-200U Page 389 of 395 

also be surrounded by wooded and/or pasture/grassland areas. Therefore, the types of 

vegetation around a soybean field depend on the area where the soybean is planted. 

If MON 87769 is granted nonregulated status, agricultural 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 exhibit characteristics associated with 

weedy growth and will not compete with plants found outside of agricultural production. 

Weeds within fields of MON 87769 will be managed using mechanical, cultural, and 

chemical control measures, similar to current management practices 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 around fields cultivated with 

MON 87769 will not be impacted and, likewise, no indirect effects are expected to other 

plants that rely on pollinators. Thus, granting nonregulated status to MON 87769 will 

have no significant impact to plant communities. 

Threatened and Endangered Species 

No significant impact to threatened and endangered species is expected due to APHIS 

granting nonregulated status to MON 87769. As previously noted, consumption of the 

PjΔ6D and NcΔ15D 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 are 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 sexually compatible with any federally listed TES or a 

species proposed for listing. The only TES animal listed that occupies habitat that is 

likely to include soybean fields and that might feed on soybean is the federally 

Endangered Delmarva Peninsula Fox Squirrel (Sciurus niger cinereus), found in areas of 

the mid-Atlantic Eastern seaboard. It is known to utilize certain agricultural lands readily, 

but its diet includes acorns, nuts/seeds of hickory, beech, walnut, and loblolly pine; buds 

and flowers of trees, fungi, insects, fruit, and an occasional bird egg. Given all these 

factors and the lack of noted adverse effects 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 organisms in the field, it is concluded that 

MON 87769 will not have an effect on the Delmarva Peninsula Fox Squirrel. 

No significant impact to any threatened or endangered 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 PjΔ6D and 

NcΔ15D proteins, no accumulation of these proteins is expected in soils where 

MON 87769 will be grown. Information has also been presented in the petition 

demonstrating the established presence of SDA in the environment. No adverse effects 
Monsanto 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. Monsanto 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 PjΔ6D and NcΔ15D proteins compared to a conventional soybean 

control. On the basis of these observations 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, that 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 organisms common to agricultural 

ecosystems or threatened or endangered species. Based on this analysis, it is concluded 

that deregulation of soybean varieties containing 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 cumulative 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 biotechnology-derived soybean products have been 

deregulated or are under consideration for deregulation. 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 deregulated biotechnology-derived soybean crop 

products as well as with conventional soybean, creating new improved varieties. APHIS 

has determined that none of the biotechnology-derived individual soybean products it has 

deregulated display increased plant pest characteristics and that any progeny derived 

from crosses of these soybean varieties with other conventional or biotechnology-derived 

soybean are unlikely to exhibit new plant pest properties. 

An assessment of the stability of the genetic insert in MON 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 modified 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 biotechnology-derived traits. For example, in an 
Monsanto Company 09-SY-200U Page 391 of 395 

assessment by McCann et al. (2005), it was shown that during three years of breeding 

into multiple varieties, the composition of glyphosate-tolerant soybean remained 

equivalent to that of conventional soybean. Given that there have been no plant pest 

characteristics associated with MON 87769, or with any of the previously deregulated 

events listed below, no significant impacts are expected to other soybean varieties 

through the use of MON 87769 in breeding programs and in combination with any of the 

previously deregulated soybean crop products. 

All biotechnology-derived soybean products on the market today have satisfactorily 

completed the FDA consultation process expressly 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 events through conventional breeding because the 

deregulated events have a history of safe use and on the basis of knowledge of the type of 

modifications made to each of the deregulated events, the biochemical pathways are not 

likely to unexpectedly interact or result in the production of novel constituents. As 

expected, breeding with other modified oil 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 the single event products in terms of 

phenotype, agronomic characteristics, and the efficacy of the traits. This testing is a part 

of the process to develop any new soybean variety. 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 Products1

Phenotype ID Code(s) Institution Date Deregulated 

Herbicide Tolerant NA2 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 characterized 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 previous biotechnology-derived crops have resulted in 

no unexpected effects on the quality of the human environment as defined under NEPA 

and have provided benefits to growers, consumers and the environment. In this respect, a 

decision to deregulate a new biotechnology-derived soybean product is not precedent 

setting nor are the effects to the quality of the human environment highly uncertain or 

unpredictable. 

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, and has no significant 

impact on threatened or endangered species or biodiversity. Soybean produced from 

MON 87769 has been demonstrated to be safe and wholesome for food and feed purposes. 

MON 87769 will not significantly impact the commercial interests of soybean producers 

or those involved in the marketing and sale of soybean and soybean products. The 
Monsanto Company 09-SY-200U Page 393 of 395 

amount of land devoted to soybean production or crop production in general has not 

changed with the introduction of biotechnology-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 the commercial introduction of MON 87769. 

MON 87769 will utilize common cultivation practices typically employed for production 

of commodity soybean and many types of specialty soybean, and management practices 

typically used for specialty soybean (i.e., IDP production practices). Hence, agricultural 

practices for MON 87769 are not likely to change. 

The introduction and adoption of specialty soybean 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 opportunity for growers to produce a land-based 

omega-3 fatty acid is a benefit from an economic 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 Identity Preserved Value-Added Soybeans. 

American Soybean Association, St. Louis, Missouri. 

Born, H. 2005. Marketing organic grains. ATTRA publication. Available 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 study 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 eicosapentaenoic 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 emerging cardiovascular risk marker. Lipids 

43:805-811. 
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Hymowitz, T., and R.J. Singh. 1987. Taxonomy 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. 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. 

James, C. 2008. Global Status of Commercialized 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 disease: New recommendations from the 

American Heart Association." Arteriosclerosis, 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 testing. Journal of Agriculture 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. Environmental 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 summary, 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 Agricultural Statistics Service, Washington, D.C. 

USDA-NASS. 2008. Crop Values: 2007 Summary. United States Department of 

Agriculture - National Agricultural Statistics 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 soybeans 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 July 1

st, 2011

OECD Unique Identifier: MON-87769-7

Monsanto Petition Number: 09-183-01p
Page 2 of 25

1. Introduction

Monsanto has developed biotechnology-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 long 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 approximately 20 to 30% SDA (wt% of total 

fatty acids) 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 Safe (GRAS) in the United States

and can be used for the production of products such as margarine, shortenings, salad dressings, 

ready-to-eat foods, and other food categories specified in the GRAS notification1. 

MON 87769 is a specialty trait soybean, not developed for the commodity soybean market, and 

it is anticipated that MON 87769 will 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 policy2, 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 „market 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 applications. 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 result in potential 

market and trade impacts.

At the time SDA soybean oil is first introduced to the market, we do not expect that MON 87769

will have approvals in all key soybean export markets with functioning regulatory systems. 

Until such approvals are received, MON 87769 will be grown and handled in a closed loop 

stewardship system (CLSS).

Monsanto will utilize and implement the CLSS throughout the initial product introduction phase,

at least until approvals are received from key soybean export countries with functioning 

regulatory systems. After such approvals are received, Monsanto will, based 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.pdf
Page 3 of 25

and learning gained during the initial product introduction phase under the CLSS, refine and 

update the stewardship system for long-term implementation. This long-term system will 

include measures to preserve the identity of this high value product and mitigation measures to 

minimize any impact resulting from the inadvertent comingling of SDA soybeans or oil with 

commodity or other specialty soybean products. 

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. This document also describes the CLSS

which Monsanto will implement during the initial phase of the product introduction.

2. Intended Uses 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 formulation 

options for food companies and consumers. SDA soybean oil is generally recognized as safe in 

the United States3. 

SDA is a metabolic precursor to the long chain omega-3 fatty acids, EPA and DHA, in humans 

and animals and is found in products 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 consumer 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 habits. 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 dressings, ready-to-eat foods, and other food categories described 

in the GRAS notification4. The use of SDA soybean oil in selected food categories could 

provide a wide range of dietary alternatives for increasing omega-3 fatty acid intake. 

Given the targeted 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.pdf
Page 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 uses 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 levels of SDA (approximately 20-30 

wt% of total fatty acids). Vegetable oils containing high levels of polyunsaturated fatty acids

(PUFUs) like SDA are known to undergo rapid oxidation during frying applications and will 

impart undesirable taste and odor to 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 polyunsaturated fatty acids (LCPUFAs). 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 products, frozen dairy desserts and 

mixes, grain products and pastas, gravies 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 mixes5.

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 omega-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 industry 

between 2008 and 2013 is 13.4% (Strategic Analysis of the North American Marine and Algae 

Oil Omega-3 Ingredients Market, Frost & Sullivan, March 20106). The value added product 

derived from MON 87769 is SDA soybean oil. SDA soybean oil provides increased alternatives 

for food companies to formulate omega-3 fatty acids into a wider range of foods. Because SDA 

soybean oil can be used in wider food categories, Monsanto estimates, for the purposes of this 

analysis that the industry growth of omega-3 oils in functional foods could 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 relative conversion of 

SDA to EPA in humans is estimated at about 5:1 and marine oils in average may contain 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-01-00-00-00
Page 5 of 25

expected that about 7.5 times the volume of SDA 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% industry 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 100,000 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 

purposes of SDA soybean and/or SDA soybean oil exports. The United States is the largest 

producer of soybeans and regularly exports soybean and soybean products to global markets. In 

2010 approximately 75 million acres of soybean crop was grown in the U.S. producing 

approximately 87 million MT of soybeans. 

4. Stewardship of SDA Soybean

Monsanto is committed to product stewardship7and adhere to the BIO Product Launch 

Stewardship Policy8. In compliance with BIO's Product Launch Stewardship Policy, Monsanto 

considered Annex 2 “Special Use Traits in Commodity Crops” to develop launch plans for SDA 

soybean including: (1) identifying relevant stakeholders for the trait and crop and engaging them 

in dialogue and outreach regarding use of SDA soybean, (2) conducting a market and trade 

assessment to evaluate the potential impact from the introduction of MON 87769 to the market 

place, and (3) implementing 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 American 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: American 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 regarding the oil composition, stewardship plan and performance 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 continued

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. At a minimum, the same 

outreach, education, and quality principles will be applied as we further develop SDA soybean

stewardship and commercialization plan. These quality systems are described in the following 

sections. 

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. Approximately 

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 soybean 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 (IDP), including closed-loop stewardship systems, and quality 

assurance 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 potential 

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 producer10. Biotechnology-derived crops are 

subject to regulation in many countries. In order to support free trade in soybean, Monsanto is 

pursuing regulatory approval for MON 87769 and any stacked products intended for 

commercialization in all key soybean export countries with a functioning 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 basis. 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 approval 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 in the 

unlikely event that comingling of SDA soybean or oil in the commodity stream 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.jsp. 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.htm
Page 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 discussed earlier, the SDA soybean and 

oil are for a specialty market and are not meant to be part of the commodity chain. The 

comingling scenarios and impacts are presented 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 

occur could only happen 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 truck containing 100% SDA soybean into the commodity stream at an elevator or 

processor where 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 soybeans 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: the 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 bushels of soybeans. Thus, this 

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 smallest soybean oil 

holding tank at U.S. processing plants is approximately 200,000 lbs. Thus the accidental 

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 soybean 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 to 
Page 8 of 25

human health because the oil has been through the food safety assessment11. 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 elevator. In 

this case, the SDA soybean would be diluted with commodity soybean in a grain storage bin that 

typically holds 100,000 to 500,000 bushels of soybean, resulting in 125X to 625X dilution of 

SDA soybean in commodity soybean. The commodity soybean oil 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. Soybean 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 diverted 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 unintentionally is very small. 

Commodity soybean shipped for export are typically loaded into unit trains (360,000 bushels) or 

barges (50,000 bushels/barge) that eventually are loaded into 2 million bushel ocean going 

vessels. Before a ship loaded with commodity soybean reaches a destination country, the 

inadvertent delivery of a truck containing 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 total 

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 Soybean 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 soybean meal from domestic and international shipping (NOPA,

2006). With the exception of the presence of minor amounts of the Pj 6D and Nc 15D proteins 

and de minimis amounts of residual oil, the soybean meal and other non-oil processed fractions 

used for animal feed and human food applications are unchanged from commodity processed 

soybean fractions. Upon receiving regulatory approval for MON 87769 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.htm
Page 9 of 25

market place. Therefore no trade impact is anticipated from the commingling of SDA soybean 

meal with commodity soybean meal. 

4.2.3 Potential Impact to Commodity Soybean Oil in the Market Place

Monsanto has assessed the impact due to the presence of SDA soybean oil on commodity and 

other vegetable oils. 

Areas of potential impact identified include the following: (1) food ingredient labeling, (2) 

nutritional facts panel labeling (3) functionality of the oil, and (4) sensory evaluation. This 

assessment has been shared with key stakeholders such as NOPA, NAEGA, ASA, QUALISOY, 

USB and GMA. 

Food Ingredient Labeling:

The 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 contains two 

additional fatty acids (SDA and GLA) that are not present in commodity soybean oil. Therefore,

we evaluated the potential impact 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 requires that all ingredients present 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 food12. Residual 

product from a previous soybean oil processing run may occasionally be mixed into a different 

product in a new run. Such residual product would fit the concept of “incidental additive”13 and 

need not be included on the label. Thus, 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 nutrition facts panel of foods.

They are (1) total fat, (2) trans fat, and (3) saturated fat. It is voluntary for a food company to list 

the amounts of monounsaturated fat and polyunsaturated fat 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 fat content of SDA soybean oil is similar to commodity soybean 

oil. Many vegetable oil providers voluntarily list also monounsaturated and polyunsaturated 

fatty acids present in the oil. Since in SDA soybean oil one polyunsaturated fatty acid (LA) is 

replaced with two other (SDA and GLA) polyunsaturated fatty acids; the impact of comingling 

on nutrition facts panel labeling from listing mono and polyunsaturated fatty acid is negligible. 

 

12 21 CFR § 101.100

1321 CFR § 101.100(3)i
Page 10 of 25

Even though bottled SDA soybean oil is not an intended use at this time, a worst case assessment

using 100% commodity soybean oil was considered 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 soybean oil in order to significantly impact the nutrition facts 

panel for major fatty acids on bottled soybean oil. Based on this analysis even 25% of SDA 

soybean oil comingled with commodity soybean oil will not significantly impact the nutritional 

facts panel 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 

facts panel for commodity soybean oil (100 % soybean oil) and a theoretical nutrition facts panel 

for SDA soybean oil. 

Table 1. Impact of Comingling SDA Soybean Oil with Commodity Soybean Oil on 

Nutritional Facts Panel Labeling for Bottled Soybean Oil 

Major Fatty Acids SBO1SDA 

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 labeling is 

voluntary. No change in trans fat.
Page 11 of 25

Figure 1. Nutrition Facts Panel for Commodity Soybean Oil 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 oil exists 

in the database. SDA soybean oil‟s typical fatty acid composition was utilized to develop 

a theoretical nutrition facts panel.

Oil Functionality:

Oxidative Stability Monsanto conducted an assessment of the impact of the oxidative stability of 

commodity soybean oil comingled with SDA soybean oil. Figure 2 demonstrates that 

comingling of SDA soybean oil with commodity soybean oil decreases the Oxidative Stability 

Index (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. The 
Page 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 

oxidative stability of soybean oil is significantly influenced by the proportion of 

monounsaturates to polyunsaturates. 

To assess the impact of comingling on the OSI, the OSI of 21 commercially available soybean 

oil as well as blends of commodity soybean oil with SDA soybean oil were evaluated.

Commodity/SDA soybean oil blends were prepared 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 antioxidants. SDA soybean oil used for preparing the blends contained 22.9% of SDA 

(%wt of total fatty acid). Oil Stability Index measurements were obtained on an Omnion OSI 

instrument at 110oC using AOCS Official Method Cd 12b-92. OSI was run in duplicate for each 

blend sample, and means were averaged to obtain the data 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 these 

oils ranged 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 soybean 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 falls below the range observed for commercial soybean oils. 
Page 13 of 25

Figure 2. Oxidative Stability Index (OSI) of SDA Soybean Oil Blended with Commodity 

Soybean Oil

Heat Treatment Vegetable oils containing high levels of PUFA are known to undergo rapid 

oxidation during frying applications and are known to impart undesirable taste and odor to the 

fried foods. SDA soybean oil contains relatively high levels of PUFAs compared to other 

vegetable oils, and therefore, is not intended for frying applications. However, as part of the 

evaluation of the potential impact of SDA soybean oil on the functional properties of commodity 

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 (Ortheofer 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 

byproducts 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 Oil Meter (Ebro International, Lino 

Lakes, MN). As oil degrades during frying, 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 determine when the oil has reached 24% total polar material (TPM), which 

typically indicates the need to discard the oil. Figure 3 shows the time in hours that it took for 

each SDA soybean oil/commodity soybean oil blend to reach 24% TPM during frying. The 
Page 14 of 25

results indicate within the range of blends tested the level of SDA soybean oil had minimal 

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 sensory assessment of SDA soybean oil was performed to evaluate consumer 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 environment of 

high temperature food frying applications 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, french fries were prepared. The five 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 french 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 evaluation showed 
Page 15 of 25

that up to 15% of SDA soybean oil can be comingled 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 in the unlikely event comingling did occur, 

the impact to labeling and functionality of commodity soybean oil would be minimal. Monsanto

will have an appropriate 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 commercialization 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 materials generated within the closed loop. Stewarded materials include 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 

will continue to utilize the CLSS through the initial product 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 

system for long-term implementation based on the experience developed during the introduction 

phase and in consultation with key stakeholders. 

Under the CLSS, Monsanto will develop and implement best practices and systems consistent 

with the Excellence Through Stewardship14 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 will 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 work with downstream partners that have proven traceability, identity 

preservation, and CLSS production capabilities. Monsanto, its licensees, partners and processors 

will implement an annual auditing process for material under their control. Commercial 

production 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 appropriately. 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 directly 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 CLSS are organized into five relevant sections identifying critical control 

points: (1) Production of SDA Soybean Seed 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 personnel involved with producing 

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 articles 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 to 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 industry.
Page 19 of 25

Production of SDA Soybean Seed for Planting:

Monsanto is a leader in crop biotechnology having successfully introduced numerous 

biotechnology-derived crops to the marketplace globally. Monsanto has developed and 

implemented seed quality standards and practices 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 minimum 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. Monsanto‟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 Standardization15 (ISO) certification 

standards and include internal and external audits (ISO, 2009). By following ISO quality 

standards it is possible to validate that the processes are followed which have been designed to 

generate 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 consistently 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. Monsanto‟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 conventional soybeans 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 specialty soybean such as food grade, organic, Vistive low linolenic or 

other specialty soybeans. 

 

15http://www.iso.org/iso/ims_2009.htm
Page 20 of 25

The income opportunity is achieved by the farmer only when the soybean is delivered to the 

processor within specifications. Therefore, the motivation is a financial incentive for the farmer 

to avoid comingling with commodity soybean, keeping SDA soybean identity 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 comingling that may occur during 

the harvest. If a farmer chooses 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 processors will be responsible to issue production 

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 been issued for SDA soybean

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 involved 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, the 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, provided 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. This is to confirm the soybean contains the SDA 

trait as required by the production contract. Upon confirmation that the SDA soybean

meets specification, the processor or elevator will approve the premium payment to the 

farmer. 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 as for the 

production of biofuel.

Soybean Grain Elevator Grain elevators play an important role in specialty programs with their 

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 typically 
Page 21 of 25

prefer to empty their storage prior to planting of the new crop and prior to temperature warm up 

in the spring. The warm spring and summer weather presents challenges as condensation can 

build up in the bins creating moisture-related issues that make soybean-eating insects more 

active. To avoid this, commercial elevators have expert grain managers on staff to monitor 

soybean quality and keep soybean in good condition in all weather situations throughout the 

year. Commercial grain elevators are also better equipped to ship soybeans to processors during 

times of severe weather even when farmers 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 assure proper routing of SDA soybean through the 

supply chain. The elevator pays 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 preserve the identity of the soybean

as it is delivered to the processor. Every load delivered to the processor by the elevator will be 

analyzed to make this determination. Processors will approve the premium payment to the 

elevator after analysis of the soybean to confirm the SDA trait. 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 as 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 identity preserve SDA soybeans. They 

must demonstrate the ability to clean the equipment associated with the receiving and subsequent 

handling of SDA soybeans. The processor‟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 standards after processing SDA soybeans. Monsanto and the 

processor will enter into an agreement to establish the terms and conditions of SDA soybean

processing services. These agreements will also include terms for distribution of SDA soybean 

co-products resulting from processing as stewarded products within the United 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 transition 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 comingled with commodity soybean oil are handled in an 

appropriate manner and segregated from other oils. This will include all comingled oil before 

and after the collection of SDA soybean oil. Testing methods will be used to aid in identifying 
Page 22 of 25

the appropriate time to start and stop collecting crude SDA soybean oil. Storage tanks approved 

for SDA soybean oil will be assigned for the storage of crude and refined SDA soybean oil. 

Upon the completion of SDA soybean 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 

flushed to an acceptable level, yielding commercially acceptable commodity soybean oil. Offspec, commingled or flush SDA soybean oil will be segregated from commodity soybean oil and 

directed to appropriate use such as for the production of biofuel. 

SDA Soybean Oil Packaging and Storage:

Soybean oil produced under the CLSS will be distributed as a stewarded product within the 

United States until receiving approvals 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 packaging will be conducted in 

an area that is clean, secure, and segregated to eliminate potential comingling with commodity 

soybean and/or other vegetable oils. The containers will be properly labeled using 

predetermined methods that will include chain of custody 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 to 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 use such as for the production of biofuel.

SDA Soybean Oil Distribution to Food Company Supply Chain:

Monsanto, its licensees and/or partners will execute agreements with food companies to establish 

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 batches of the SDA soybean oil as ingredient. These contracts will include stewardship 

obligations and will require compliance including auditing. Third party consultants will be used 

if necessary under the CLSS. These third party consultants will have expertise 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 after verifying that it meets specific customer 
Page 23 of 25

requirements including quality factors. From the time the oil is packaged until it is utilized at the 

specific customer‟s facility there will be proper identification of the oil through labeling and 

manufacturing 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 soybean oil as a food ingredient consistent with the GRAS 

notification16. Each individual food company has in place systems for ingredient (oil) ordering, 

receipt, storage, access and lot identification at specific manufacturing locations, as well as 

finished product (the food which incorporates SDA soybean oil) batch identification, 

manufacturing facility, storage, shipment to distribution centers, customer order picking, 

customer order shipment and receipt. Appropriate procedures are currently in place to ensure 

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 consultants 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 personnel 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 breach of containment of 

stewarded 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. The incident investigation and analysis process will determine the reason for 

the occurrence and develop recommendations 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 all relevant regulatory approvals will be utilized through the initial product 

introduction phase until all relevant global approvals in key soybean export countries with 

functioning regulatory systems are received. Monsanto will refine and update the stewardship 

system for long-term implementation based upon the experience and learning developed during 

the CLSS production phase and through continued dialogue with stakeholders. This dialog will 

 

16 http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000283.pdf
Page 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 derived 

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 alternative that will provide food industry with choices to satisfy a growing 

omega-3 market. The closed loop stewardship system for the production and handling of 

MON 87769 and derived products 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 

in the unlikely event comingling did occur, the impact to labeling and functionality would be 

minimal. Because SDA soybean is generally recognized as safe in the United States17 and the 

defatted meal from 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. Given the 

abundance of vegetable oils on the market and demonstrated ability of the system to adapt to 

consumer preferences incorporating new oils into existing food manufacturing processes, the 

introduction of SDA soybean is expected to be easily managed by existing industry mechanisms 

and identity preservation systems. Monsanto continues to engage stakeholders and educate them 

on the benefits of SDA soybeans and proper stewardship practices.

 

17 http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/GRASListings/ucm185688.htm
Page 25 of 25

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